Accumulation of S-adenosyl-L-homocysteine impairs methylation and development in Saccharomyces cerevisiae and Drosophila melanogaster

Accumulation of S-adenosyl-L-homocysteine impairs methylation and development in Saccharomyces cerevisiae and Drosophila melanogaster | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Accumulation of S -adenosyl- L -homocysteine impairs methylation and development in Saccharomyces cerevisiae and Drosophila melanogaster Markus S. Brunner, Hansjörg Habisch, Jutta Hafner, Maximilian Mack, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5144569/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract S -adenosyl- L -homocysteine (SAH), the product inhibitor of S -adenosyl- L -methionine (SAM)-dependent methyltransferases, and its degradation product homocysteine (Hcy) are evolutionarily conserved master regulators of methylation metabolism. They can affect more than 200 methyltransferases in humans interfering with numerous methylation-dependent processes. Hyperhomocysteinemia (HHcy), characterized by elevated Hcy levels in the blood, is an independent risk factor for atherosclerosis and a strong predictor of cardiovascular mortality, yet, the mechanisms by which elevated Hcy contributes to pathological consequences remain poorly understood. Here we developed a Drosophila dietary HHcy model, as well as a genetic SAH hydrolase (SAHH) in vivo knockdown model and compared them to corresponding yeast models to reveal evolutionarily conserved developmental effects and methylation pattern changes. Feeding Drosophila a Hcy-containing diet or growing yeast on Hcy-supplemented medium similarly to genetically blocking SAH degradation leads to SAH accumulation, developmental delay and growth defects. Furthermore, Hcy supplementation or genetically induced SAH accumulation leads to impaired protein and phospholipid (PL) methylation in both model organisms. While total protein arginine methylation is significantly decreased in wild type yeast grown in presence of Hcy or in the ∆ sah1 yeast mutant, it is unaffected in Drosophila larvae raised on Hcy-supplemented diet. In contrast, histone methylation is affected in Drosophila and yeast, but exhibited differences in responses of particular histone methylation sites. Similarly, PL methylation was reduced in both organisms and resulted in deregulation of lysoPL metabolism suggesting PL remodeling. Functional characterization of evolutionary conserved Hcy/SAH-dependent methylation targets in Drosophila and yeast will reveal mechanisms of SAH toxicity which may be operative in HHcy-associated human pathologies. General Biochemistry S-adenosyl-L-homocysteine homocysteine protein and phospholipid methylation yeast Drosophila Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cardiovascular disease (CVD), the leading cause of death worldwide, is still insufficiently understood 1 – 4 . Hyperhomocysteinemia (HHcy), i.e. elevation of homocysteine (Hcy) concentration in the blood, is an independent risk factor for the development of atherosclerosis and increases CVD risk in combination with hypercholesterolemia 5 . Moreover, it is also associated with cardiac pathology 6 – 10 and increased CVD mortality 11 – 14 . Hcy is particularly highly elevated in chronic kidney disease (CKD), a pathological condition associated with drastically increased CVD mortality 4 , 15 . Hcy is also linked to many further human diseases as well as all-cause mortality 16 – 18 . Elevated Hcy is a common pathological condition that is especially prevalent in the elderly 5 , 19 , 20 and is in most cases associated with deficiency of vitamins required for Hcy degradation 21 as well as with physical inactivity 22 , low skeletal muscle mass 23 , high fat diet 24 and obesity 23 , 25 . Attempts to decrease CVD and neurological outcomes associated with elevated Hcy by lowering plasma Hcy levels were disappointing 26 – 28 indicating necessity to understand the detailed mechanisms downstream of Hcy leading to pathological consequences. In our recent publication we showed that Hcy independently of cholesterol alters aortic wall structure and functionality as well as leads to deregulation of lipoprotein metabolism 29 . Elevation of plasma Hcy levels by intravenous injections of Hcy into rabbits fed a diet blocking Hcy degradation leads to impaired vascular reactivity of the aorta, an accumulation of compromised, morphologically altered cells and deregulation of lipid metabolism in the aortic wall as well as disorganization of aortic collagen and elastin 29 . Furthermore, elastin fragmentation and an accumulation of electron-dense inclusions enriched in nitrogen in media elastin in response to elevated Hcy suggesting elastin degradation indicate how elevated Hcy can contribute to CVD progression 30 . Furthermore, elevation of Hcy levels in rabbits fed a diet blocking Hcy degradation was associated with a drop of total methylated arginines in proteins as well as decreased levels of creatine, which requires methylation for its synthesis, 29 suggesting involvement of deficient methylation in the pathological consequences associated with elevated Hcy. Inhibition of methylation is an understudied mechanism how elevated Hcy can lead to pathological consequences. There are more than 200 putative S -adenosyl- L -methionine (SAM)-dependent methyltransferases in humans 31 . They produce the universal strong product inhibitor of SAM-dependent methylation, S -adenosyl- L -homocysteine (SAH) 32 . SAH has to be quickly degraded to Hcy and adenosine in a reaction catalyzed by SAH hydrolase (SAHH) in order to allow SAM-dependent methylation 32 . Inability to quickly remove excess Hcy formed by SAH degradation reverses the direction of the equilibrium additionally potentiating SAH accumulation 32 . SAHH offers the sole possibility of SAH degradation in mammals and is highly evolutionarily conserved exhibiting over 70% identity between yeast and human orthologs 32 . High evolutionary conservation of SAH hydrolase as well as of methylation metabolism 32 suggest their central role in the regulation of cellular function. Methylation of various macromolecules is an important regulatory mechanism involved in a wide variety of cellular processes 33 , and its dysregulation is increasingly recognized as a risk factor for CVD development 34 – 37 . CVD has been linked to increased levels of SAH 38 . Furthermore, both elevated SAH and Hcy levels are known to inhibit methylation, however, detailed mechanistic understanding of their downstream signaling cascades leading to CVD is largely missing 39 . Further, lowering of elevated plasma Hcy levels in addition to the inability to reduce associated clinical outcomes 26 – 28 does not decrease SAH levels, indicating a central role of SAH in Hcy-associated pathology 40 . Here, we show that Hcy supplementation, similarly to genetically compromising SAH hydrolase, leads to developmental delay, growth defects as well as impaired protein and phospholipid (PL) methylation both in yeast and in Drosophila . While Hcy/SAH led to a decreased ratio of total protein asymmetrically dimethylated arginines to arginine (ADMA/Arg) along with impaired histone and PL methylation in yeast, total protein ADMA/Arg was unaffected in Drosophila , while, histone and PL methylation were sensitive to Hcy and/or SAH accumulation. Furthermore, Drosophila larvae subject to SAH hydrolase gene knockdown show delayed development, poor viability and reduced size. Material & Methods Chemicals and consumables are listed in Supplemental Data 1. Yeast strains, media, and growth conditions Saccharomyces cerevisiae strains used in this study are congenic with BY4741, a derivative of S288C, and are listed in Table 1 . Cells were grown at 30° C in synthetic dextrose medium (SDC) containing 1.4 g/L Difco yeast nitrogen base, 5 g/L ammonium sulfate, 20 g/L glucose and 0.79 g/L complete supplement mixture (CSM) at 180 rpm in the absence or presence of Hcy, as indicated in individual experiments. Media were solidified by the addition of 20 g/L agar. For growth curves, yeast wild type cells were grown overnight in SDC medium and inoculated to OD 600 = 0.05 in fresh SDC medium containing 0 mM, 1 mM, 2 mM or 5 mM Hcy. The Δ sah1 mutant cells were grown overnight in SDC medium and inoculated to OD 600 = 0.05 in fresh SDC medium without Hcy. 300 µL were applied onto honeycomb well plate in 3 technical replicates per condition. Optical density at 600 nm wavelength was measured every 30 min for 40 hours in Bioscreen C (Dynex) with constant shaking between measurements. Before each measurement shaking was stopped for 5 sec. After 40 h samples were taken for microscopy. For microscopic observation, live cells were immobilized using agar sheets 41 . Transmission images were acquired using a Leica SP5 confocal microscope (Leica Microsystems, Inc.), a 488 nm argon laser line and a HC PL APO 63x oil immersion objective. The experiment was independently repeated once (Supplemental Data 4). Drosophila melanogaster lines, fly food and breeding Fly lines used in this study are listed in Table 2 . FlyBase 42 was consulted for experimental planning. Flies were propagated in 68 mL Drosophila containers equipped with mite-tight stoppers on standard fly food containing 15.7 g/L baker’s yeast, 8.7 g/L soy flour, 5.4 g/L agar-agar, 69.6 g/L maize flour, 19.1 g/L beet syrup, 69.6 g/L malt, 5.4mL/L propionic acid and 1.3 g/L methyl-4-hydrobenzoate (dissolved in 4.4 mL EtOH). If not mentioned otherwise stock keeping and fly experiments were done at 25° C and 70% humidity with 12 h light/12 h dark cycle. For Hcy-supplemented food, 5% (v/v; in water) of 400 mM DL-Hcy were added to fly food prior to solidifying. For control food, 5% water was added instead of Hcy. Eggs from Canton-S or w[1118] flies were collected on apple juice agar plates (2.125% (w/v) agar, 20% (v/v) apple juice, 2% (w/v) sugar, 0.12% (w/v; dissolved in EtOH) nipagin) supplemented with baker’s yeast paste. Agar pieces with 100 eggs each was cut out and transferred onto either standard or Hcy supplemented fly food. Vials were kept at 25° C at 70% humidity. Four individual vials were prepared for each food. From each vial, 10 migratory L3 larvae were collected, rinsed with water, dried with blotting paper and weighed individually on a Sartorius MC 5 scale. Larvae were frozen at -80° C in batches of 5 larvae for SAH and SAM extraction, protein extraction and phospholipid analysis. Remaining larvae were kept at 25° C and 70% humidity and number of pupae and eclosed pupae were counted continuously until no further pupae appeared or hatched. The experiment was independently repeated once (Supplemental Data 4). For RNAi crosses, per vial 10 adult virgin female ubiquitous driver flies and 5 male mCherry RNAi (RNAi control) or SAHH RNAi (SAH hydrolase RNAi) flies were transferred into standard food vials and incubated for 24 h at 25° C and 70% humidity. After incubation, adult flies were flipped to new vials and eggs were counted. New vials were incubated again for 24 h before adult flies were removed and eggs were counted. 4 individual vials were prepared for each crossing and were kept at 25° C and 70% humidity. Number of pupae and eclosed pupae were counted continuously in 2 vials per crossing. Experiment was independently repeated once. Larvae from the other two vials were extracted, rinsed and photographed daily for up to 10 days. Photographs were taken with help of M60 binoculars (Leica) on a Google Pixel 6a smartphone. Migratory L3 larvae or 14 days old non-migratory L3 larvae were collected, rinsed with water, dried with blotting paper, weighed in batches of 5 larvae and frozen at -80° C for RT-PCR, SAH- and SAM extraction, protein extraction and phospholipid analysis. The identity of organ-specific driver lines (see Table 2 ) was confirmed by crossing 3 virgin females of the GFP-reporter line (see Table 2 ) to 3 males of the driver lines. F1 migratory L3 larvae were rinsed with and mounted in cold water before imaging using a Leica M165 FC fluorescence stereomicroscope equipped with a GFP emission filter and a Leica DFC3000 G camera. To score survival and developmental delay in response to organ-specific SAHH knockdown (see Table 2 ) compared to mCherry RNAi controls (see Table 2 ) 5 virgin females of each of the effector lines were crossed to 3–4 organ-specific or ubiquitous driver males. Parental flies were flipped daily 3–4 times and egg numbers as well as eclosed adult flies (based on empty pupal cases) were counted to assess hatching rates. To score developmental delay progeny, the adult eclosion time of balancer-carrying (control) vs. non-balancer carrying (fat body-specific driver) progeny of the heterozygous fat body-targeting driver line (see Table 2 ) was assessed daily after day 11 post crossing. The experiment was repeated with independent parental flies and progeny of 3–4 consecutive daily collections was scored (Supplemental Data 4). Drosophila food intake 1% (w/v) brilliant blue was added to the fly food containing either 5% (v/v; in water) 400 mM DL-Hcy or 0.2% (w/v) caffeine and 5% (v/v) water. Fly food with 1% (w/v) brilliant blue and 5% (v/v) water was used as control. Colored fly food was poured into small petri dishes to cover the bottom. 25 male and 25 female adult flies were put into standard food vials and flipped to new vials every 24 h. All vials were kept at 25° C and 70% humidity. Larvae were extracted from food vials one day prior to reaching migratory stage, rinsed and transferred to colored food plates. Plates were incubated for 1 h in darkness at room temperature (RT) before transferring the plates on ice. Larvae were extracted from food plates, rinsed, collected in cohorts of 10 larvae into 2 mL save-seal tubes and weighed on a Sartorius MC 5 scale. After adding 350 µL MeOH and one 5 mm steel ball samples were homogenized in a Retsch MM40 homogenizer at 30 Hz and 4° C for 2 min. Homogenates were centrifuged for 5 min at 14000 rpm and 4° C in a 5430 R centrifuge (Eppendorf) and the supernatant was transferred to new vial and centrifuged again for 5 min at 14000 rpm and 4° C. Duplicates of 100 µL supernatant were aliquoted into a 96-well plate and absorbance was measured at 626 nm in a Spectrostar Nano spectrophotometer (BMG Labtech). For absolute quantification, a 5-point calibration curve using colored fly food (0.5 mg – 7 mg) after extraction with MeOH was prepared as described above. Experiment was independently repeated once (Supplemental Data 4). SAH/SAM extraction and analysis Extraction of SAH and SAM was based on Gellekink et al. 43 with minor changes. Briefly, for yeast, per sample 20 OD 600 units were harvested and resuspended in 250 µL water + 0.1% formic acid. After addition of 250 µL glass beads, cells were lyzed in Homogenizer MM40 (Retsch) at 30 Hz and 4° C three times for 40 s. Homogenates were diluted 1:2. For Drosophila , per sample 5 larvae were put into 500 µL water + 0.1% formic acid and homogenized with a steel ball in Homogenizer MM40 (Retsch) at 30 Hz and 4° C for 2 min. For SAH and SAM extraction 100 mg, 1 mL Bond Elut PBA columns were used. Solid phase extraction was performed after washing the columns with 4 mL 0.1 M formic acid and equilibrating with 4 mL 20 mM ammonium acetate buffer (pH 7.4). Two separate aliquots of 210 µL were prepared for SAH and SAM extraction, respectively. Per aliquot, 60 µL of internal standard was added (either 2 µM SAH-d4 or 5 µM SAM-d3 in water) and 90 µL of the mix were applied onto the column. The columns were subsequently washed with 3 mL 20 mM ammonium acetate butter (pH 7.4). Samples were eluted in 1 mL 0.1 M formic acid and measured directly via HPLC-QQQ tandem mass spectrometry. Per sample two technical replicates were done. SAH and SAM levels were analyzed by 1290 Infinity UHPLC coupled to a 6470 Triple-Quadrupole mass spectrometer (Agilent) using a BEH C18 column (3.0 mm×150 mm; 1.7 µm) with 50° C column temperature, 5 µL injection volume and a constant flow rate of 200 µL/min. H 2 O + 0.1% formic acid (solvent A) and methanol + 0.1% formic acid (solvent B) were used as solvents. 95% solvent A was held for 2 min, followed by a change to 100% solvent B over the next 2 min, which was held for additional 3.5 min. Re-equilibration was carried out by changing to 95% solvent A within 5 seconds, followed by 3 min at 95% solvent A. Total run time was 11 min. For absolute quantification, independent SAH and SAM dilution series were prepared for 8-point calibration curves in the range from 1.56 to 200.0 nM. All analytes were measured in MRM mode with dwell time of 50 ms and cell acceleration voltage of 4 V for all transitions. Fragmentor voltage was optimized for each transition individually and set between 97 and 115 V. The transitions m/z 385.1 − 135.9 for SAH with a collision energy of 15 eV and m/z 389.1–137.9 for SAH-d4 with a collision energy of 21 eV were used as quantifiers. The transitions m/z 385.1–133.9 (collision energy 15 eV) and 87.9 (collision energy 40 eV) were the qualifiers for SAH, and m/z 389.1–91.9 with a collision energy of 40 eV for SAH-d4. The transitions m/z 399.2–250.0 for SAM with a collision energy of 15 eV and m/z 402.2–249.9 for SAM-d3 with a collision energy of 13 eV were used as quantifiers. The transitions m/z 399.2–135.9 (collision energy 27 eV) and 96.9 (collision energy 35 eV) were the qualifiers for SAM, and m/z 402.2–135.8 and 96.9 both with a collision energy of 33 eV for SAM-d3. Total protein asymmetrically dimethylated arginines to total arginines (ADMA/Arg) ratio For total protein ADMA and arginine analysis, yeast (15 OD 600 units) and Drosophila (10 larvae) samples were suspended in 400 µL of ice-cold methanol and 200 µL of Milli-Q H 2 O, and transferred to Precellys tubes with 1.4 mm diameter zirconium oxide beads. This suspension was homogenized two times for 20 s by Precellys 24 tissue homogenizer at 25° C. Afterwards, the homogenized samples were centrifuged at 10,000 rpm for 30 min at 4° C and stored at -20° C for at least 2 hours. Precipitates were further processed for arginine methylation analyses as described in Zhang et al 2021 and Habisch et al 2021 44,45 . Briefly, the precipitates were hydrolyzed with 6 M HCl to obtain amino acids and lyophilized. For NMR analysis, dried samples were re-dissolved in 500 µL NMR buffer (0.08 M Na 2 HPO 4 , 5 mM 3-(trimethylsilyl) propionic acid-2,2,3,3-d4 sodium salt (TSP), 0.04 (w/v) % NaN 3 in D 2 O, pH adjusted to 7.4 with 8 M HCl and 5 M NaOH). NMR experiments were carried out as described by Zhang et al 2021 and Habisch et al 2021 44,45 . 2D JRES ( 1 H homo-nuclear J-resolved spectroscopy) spectra were acquired at 310 K on a Bruker 600 MHz Avance Neo spectrometer equipped with a TXI 600S3 probe head using the jresgpprqf pulse sequence (16 scans, size of fid 16,384 (direct dimension F2)/256 (indirect dimension F1), 10,000.00/78.042 Hz spectral width in F2 (chemical shift axis)/F1 (spin–spin coupling axis), recycle delay 2 s) with presaturation during the relaxation delay to obtain virtually decoupled spectra. Data were processed in Bruker Topspin version 4.3 using the SINE and QSINE window functions (SSB = 0) in F2/F1. Fourier transform was performed with 16,384/256 F2/F1 points of the fid. 2D J-resolved experiments were processed using back prediction implemented in the Bruker au program proc_jres.be [32] The JRES spectra were then projected along F2 and exported as 1D NMR spectra. Quantification of arginine and ADMA was carried out by integration of characteristic peaks as described elsewhere 44 , 45 . Protein extraction For yeast, total proteins were extracted from 15 OD 600 units per sample. Per condition, 4 biological replicates were analyzed. For extraction, pellets were resuspended in 200 µL 1.85 M NaOH with 7.5% (v/v) β-mercaptoethanol and incubated for 10 min on ice. 200 µL 50% TCA were added and samples incubated for further 10 min on ice. Samples were then centrifuged for 20 min at 14000 rpm and 4° C, supernatant removed and samples centrifuged again for 2 min. Pellets were washed twice, centrifuged for 5 min at 14000 rpm and 4° C and supernatant removed. Pellets were then resuspended in 100 µL sample buffer (0.156 M Tris-HCl, 5% SDS, 20% glycerol (87%), 0.01% bromophenol blue, 300 µM DTT) and samples were stored at -20° C. For Drosophila , total proteins were extracted from 5 migratory L3 larvae per sample. Per condition, 4 biological replicates were analyzed. Per sample, 100 µL extraction buffer (9.75 mL PBS (pH 7.4) + 50 µL Triton X-100 + 200 µL 0.1 M (in acetone) PMSF) and one 5 mm steel ball were added, and the sample homogenized in a Retsch MM40 homogenizer at 30 Hz and 4° C for 2 min. Samples were incubated for additional 10 min at 4° C on overhead rotator SB3 (Stuart). Afterwards, homogenate was transferred to a new vial and centrifuged for 10 min at 6500 g and 4° C in a 5430 R centrifuge (Eppendorf). The supernatant was removed and the pellet was resuspended in 100 µL 0.4 M HCl followed by 5 min incubation on ice. Samples were then centrifuged for 10 min at 6500 g and 4° C. Supernatant was transferred to a new vial and pH was set to 7 with 1 M NaOH. Sample was mixed with equal volume of sample buffer (0.3125 M Tris-HCl, 10% SDS, 40% glycerol, 0.02% bromophenol blue, 600 µM DTT) and stored at -20° C. RT-qPCR SAHH primers were selected from Fly RNAi Database ( https://www.flyrnai.org/flyprimerbank ), GAPDH primers were selected according to Beaucher et al 2007 46 and are listed in Table 3 . RNA from migratory L3 larvae ubiquitously expressing either mCherry RNAi or SAHH RNAi was extracted using RNeasy Minikit according to the manufacturer’s instructions for animal tissues. RNA concentration was determined by Nanodrop ND-1000 (Peqlab). 1 µg RNA was treated with DNAse I and reverse transcribed using the Invitrogen superscript III One-Step RT-PCR System according to the manufacturer’s instructions. For qPCR 8 ng cDNA, 0.1 nmol forward- and reverse primers ( Table 3 ), 10 µL Taq Universal SYRB Green Supermix and water to reach a final reaction volume of 20 µL were used and analyzed on StepOne Plus RT-PCR system (Applied Biosystems). GAPDH was used as a housekeeping gene (see Table 3 ) for normalization. Results were calculated in Excel (Office Professional Plus 2021; Microsoft) according to Schmittgen et al 2008 47 and depicted as 2 −ΔCt values. All samples were measured in two biological replicates, each biological replicate in two technical replicates. Western blot Polyacrylamide gel electrophoresis (PAGE) was performed using Invitrogen NuPAGE (12% BT 1.0) gels. Per well, 5 µL of sample were applied and consecutively adjusted to equalize signals of normalization antibody. After PAGE, proteins were blotted onto polyvinylidene fluoride (PVDF) Immobilon-P Transfer Membrane (0.45 µm). Western blot analysis was performed using the following antibodies with given dilutions for yeast and Drosophila samples respectively: GAPDH antibody (1:14000 or 1:5000), MMA antibody (1:1000 or 1:200), ADMA antibody (1:1000 or 1:200), H3 antibody (1:5000 or 1:1000), H3K4Me3 antibody (1:1000 or 1:200), H3K36Me2 antibody (1:1000 or 1:200), H3K36Me3 antibody (1:1000 or 1:200), H3K79Me1 antibody (1:1000 or 1:200), H3K79Me2 antibody (1:1000 or 1:200), H3K79Me3 (1:500 or 1:100) and secondary α-rabbit horseradish peroxidase-conjugated antibody (1:15000). For immunostaining membranes were blocked in 2% (w/v) skim milk in TST buffer (0.05 M Tris-HCl, 0.15 M NaCl, 0.1% (v/v) Tween-20, pH 7.4) for 1 h at RT. Primary antibody was diluted in 1% (w/v) skim milk in TST buffer and membranes were incubated in the primary antibodies for 1 h at RT. Membranes were then washed 3 times for 5 min in TST buffer at RT. Secondary antibody was diluted in 1% (w/v) skim milk in TST buffer and membranes were incubated in secondary antibody for 1 h at RT. Membranes were then washed 3 times for 5 min in TST buffer at RT. Protein signals were detected using Clarity Western ECL Substrate Kit and captured with a ChemiDoc Touch Imaging System (Bio-Rad). Quantification was done in ImageLab Software Version 6.0.1 (Bio-Rad). Between different immunostainings primary/secondary antibodies were stripped off the membranes by incubation in stripping buffer (2% (v/v) SDS, 0.064 M Tris-HCl, 0.7% (v/v) β-mercaptoethanol) for 20 min at 60° C before the next immunostaining. Experiment was independently repeated once (Supplemental Data 4). Full blots are shown in Supplemental Data 5. Phospholipid methylation Lipids were extracted from either five Drosophila L3 larvae or 20 OD 600 units in yeast per sample according to protocol described in Matyash et al. 2008 48 . Briefly, 700 µL MTBE:MeOH (10:3, v/v) and 50 µL internal standard mix (yeast: PC 38:0, PE 34:0, LPC 17:0 and LPE 17:1; Drosophila : PC 38:0, LPC 17:0 and LPE 17:10.2 mg/mL in MTBE:MeOH (10:3; v/v)) were added to larvae or yeast cell pellets in addition to either a 5 mm steel ball to Drosophila larvae or 250 µL glass beads to yeast pellets in 2 mL safe-seal tubes. Samples were homogenized in Homogenizer MM40 (Retsch) at 30 Hz and 4° C for 5 min. 200 µL water (MS-grade) were added and mixed in Homogenizer MM40 (Retsch) at 30 Hz and 4° C for further 5 min. Upper phase was collected and second extraction was performed by adding 700 µL MTBE:MeOH (10:3, v/v), mixing in Homogenizer MM40 (Retsch) at 30 Hz and 4° C for another 5 min and upper phase was pooled with the first extraction. Solvent was removed under nitrogen stream at 38° C. For measurement, samples were resuspended in 1 mL isopropanol with 10 mM ammonium acetate, 0.1% formic acid and 8 µM phosphoric acid. Phospholipids were analyzed by 1290 Infinity UHPLC coupled to a 6470 Triple-Quadrupole mass spectrometer (Agilent) using a BEH C18 column (3.0 mm×150 mm; 1.7 µm) with 50° C column temperature, 5 µL injection volume and a constant flow rate of 200 µL/min. H 2 O + 10 mM ammonium acetate + 0.1% formic acid + 8 µM phosphoric acid (A) and isopropanol + 10 mM ammonium acetate + 0.1% formic acid + 8 µM phosphoric acid (B) were used as solvents. 50% solvent A was held for 0.5 min, followed by a change to 80% solvent B over the next 8.5 min and a change to 100% solvent B over next 13 min. 100% solvent B was held for 2.5 min. Re-equilibration was carried out by changing to 50% solvent A within 0.5 min, which was held for 5 min. Total run time was 30 min. All analytes were measured in dynamic MRM mode with optimized individual retention times, retention window of 4 min and cell acceleration voltage of 5 V for all transitions. Fragmentor voltage and collision energy was individually optimized for each lipid class individually. A full list with all analyzed transitions, including individual retention times, fragmentor voltage and collision energy is shown in Supplemental Data 2. Figure preparations & Statistics All diagrams (bar charts, xy-charts and box plots) were prepared in Prism 8 (GraphPad). All collages (yeast- and Drosophila photographs, western blots and figure blocks) were compiled in PowerPoint (Office Professional Plus 2021; Microsoft). All statistics were calculated in SPSS 27.0 (SPSS Inc.). Data sets were checked for normal distribution by Shapiro Wilk test. If not normally distributed (non-parametric), significances were calculated by Mann-Whitney-U test for single comparisons and Kruskal-Wallis test with Bonferroni correction for multiple testing. If normally distributed (parametric), homogeneity of variance was checked by Levene test. In case of homogenous variances, significances were calculated via Students t -tests for single comparisons and one-way ANOVA with Bonferroni correction for multiple testing or Games-Howell correction for multiple testing for results with non-homogenous variances. All reported p-values are two-sided with an α-level of 0.05. If applicable, parametric significances are marked by asterisks (*) and non-parametric significances are marked by hashtags (#). All results of statistical analyses are shown in Supplemental Data 3. Results 1. Differential response of SAH and SAM to Hcy supplementation or genetic SAH hydrolase inhibition in yeast and Drosophila We have shown previously that Hcy supplementation leads to SAH accumulation in wild type yeast cells (called the yeast Hcy supplementation model in the following). The yeast mutant carrying a deletion of the gene encoding SAHH (∆ sah1 mutant) accumulates SAH independently of Hcy supplementation (termed the yeast SAHH genetic model in the following) 49 . Consistent with these published results, SAH accumulates in both the yeast Hcy supplementation and the SAHH genetic model compared to non-supplemented wild type yeast ( Fig. 1A ). While SAM levels were not increased in Hcy-supplemented wild type yeast, they were significantly elevated in the yeast Δ sah1 mutant in comparison to non-supplemented wild type ( Fig. 1A ). This resulted in significantly decreased SAM/SAH ratio in Hcy-supplemented wild type yeast, while the SAM/SAH ratio remained largely unaltered in the yeast Δ sah1 mutant ( Fig. 1A ). In line with the yeast results, Drosophila wild type larvae (Canton-S) grown on food containing 20 mM Hcy (called the fly Hcy supplementation model in the following) showed over 25-fold increase in SAH levels compared to larvae grown on non-supplemented food ( Fig. 1B). To confirm SAH accumulation to be an universal response of Drosophila larvae to dietary Hcy supplementation we subjected a genetically unrelated w[1118] strain to the same food regimen to find similar 25-fold increase in SAH content compared to the same genotype on regular food (Fig. S2 ). Moreover, to rule out that SAH accumulation is an indirect effect of dietary supplementation due to an orexigenic effect of Hcy, we performed larval food intake measurements to find no significant difference between larvae on food ± Hcy ( Fig. S3 ). This SAH accumulation in the fly Hcy supplementation model was similar to the SAH level increase in Drosophila larvae subject to ubiquitous SAHH gene knockdown mediated by an in vivo RNAi construct (called the fly SAHH genetic model in the following) compared to control larvae expressing an unrelated RNAi construct (mCherry RNAi) ( Fig. 1B ). The SAHH gene knockdown efficiency is over 70% in this model ( Fig. S1). Consistent with the corresponding yeast model, also in the fly Hcy supplementation model, SAM levels were unaffected by the significant increase in SAH ( Fig. 1B ). In contrast to the fly Hcy supplementation model - but in line with the yeast SAHH genetic model – SAM levels were moderately but significantly increased compared to controls in the fly SAH genetic model ( Fig. 1B ). As a result, the SAM/SAH ratio decreased in both dietary and genetic fly models, compared to the respective controls (Fig. 1B ). In addition, while feeding Hcy-containing food to Canton-S larvae did not significantly affect SAM levels, feeding Hcy-containing food to w[1118] resulted in significant decrease of SAM levels and altogether not as strongly decreased SAM/SAH ratio in response to Hcy supplementation in w[1118] as compared to Canton-S ( Fig. S2 and 1B ). Collectively, the SAH accumulation response to Hcy supplementation or SAHH inhibition is very similar in yeast and Drosophila . Differential responses, such as the moderate SAM increase in the fly compared to the yeast genetic model cause different SAH/SAM ratios. These differences can be exploited to correlate phenotypic differences between the organismal models with changes in particular metabolites. Given the role of SAH as competitive inhibitor of SAM-dependent methyltransferases we next compared developmental phenotypes of the fly and yeast models. 2. Growth defects and developmental delay in yeast and Drosophila models of SAH accumulation Tight regulation of SAH levels is essential as witnessed by the embryonic lethality of homozygous SAH hydrolase mutant mice 50 . Similarly, the yeast SAHH genetic model (Δ sah1 ) exhibits a massive growth defect 51 (and Fig. 2A ), which cannot be rescued by methionine supplementation 49 . This suggests that SAH accumulation is causal for the growth defect triggered by the block in SAH hydrolysis. We hypothesized that SAH accumulation driven by Hcy supplementation would similarly affect growth in yeast. Accordingly, we measured growth of the Hcy supplementation yeast model in the absence (0 mM) or in the presence of increasing Hcy concentrations (1 mM, 2 mM or 5 mM) in the medium. Indeed, we observed dose-dependent growth inhibition of the yeast Hcy supplementation model. This growth defect is similar to the severe growth defect of the yeast SAHH genetic model grown without Hcy ( Fig. 2A ). However, while cultures of the SAHH genetic model never reach stationary phase density, the Hcy supplementation model does under all tested Hcy concentrations ( Fig. 2A ). In accordance, microscopic analysis showed no apparent morphological difference between Hcy-supplemented and non-supplemented yeast cells in stationary phase. In contrast, yeast Δ sah1 mutant cells are characterized by multi-budding, altered morphology and aggregation in the same growth phase ( Fig. 2B ), in accordance with previous observations 52 . To compare the developmental progress and success of the fly Hcy supplementation model to controls on regular food, we assessed the following parameters: the wet weight of individuals at migratory L3 larval stage and the time of/survival rate at two developmental transition states: pupariation (larval to pupal) and hatching (pupal to adult). Migratory L3 larvae grown on Hcy-containing food showed significantly lower body weight compared to larvae grown on food without Hcy supplementation ( Fig. 2C ). Moreover, pupariation and hatching of the fly Hcy supplementation model was substantially delayed compared to controls. The first pupae on Hcy food emerged after 232 h after egg laying (AEL) compared to 136 h on standard food ( Fig. 2D ). Additionally, the last larvae pupariated after 401.5 h AEL on Hcy-containing food compared to 232 h on standard food ( Fig. 2D ). This extended pupariation phase in the population might indicate a Hcy-dependent desynchronization of developmental time by individually different growth retardation. Both aspects, developmental delay and extended developmental phase equally manifest when comparing Hcy-fed flies to controls at pupal hatching to adults ( Fig. 2D ). This indicates that metamorphosis is unaffected by Hcy exposure during larval feeding. Of note, despite the developmental delay, Hcy supplementation did not decrease overall survival rates at pupariation or hatching ( Fig. 2D ). Reduced larval body weight and developmental delay combined with unaffected pupariation and hatching rates appears to be a universal signature of Hcy-fed flies. In support of this, the genotypically unrelated w[1118] stock display the same set of phenotypes as Canton-S wild type strain in response to Hcy feeding (compare Fig. S4 to Fig. 2C-D ). In accordance with SAH accumulation playing a central role in growth and development, larvae of the fly SAHH genetic model are severely retarded at pupariation and hatching ( Fig. 2E ). The first pupae of the genetic SAHH model only emerged 192 h AEL compared to 144 h in the case of control larvae with normal SAHH activity ( Fig. 2E ). While the onset of the developmental delay is similar in the fly Hcy supplementation and SAHH genetic models, the pupariation phase at the population level is much more extended in the genetic model (compare Fig. 2D to 2E ). Consistently, not only the growth of larvae subjected to ubiquitous SAHH gene knockdown is retarded but size heterogeneity of larvae of the same chronological age ( Fig. 2F ) also indicated developmental desynchronization. In line with the severe growth phenotype, but unlike the Hcy supplementation model, drastically reduced survival rates by 70% and 85% at pupariation and hatching, respectively, characterize the SAHH genetic model ( Fig. 2E ). These data suggest the growth retardation to result in developmental arrest at larval stages for the majority of individuals in response to global reduction in SAHH activity. We asked next, in which organs SAHH activity is particularly relevant for proper development. To this aim, we targeted the SAHH gene knockdown selectively to different organs and scored for the hatching rate of the respective flies compared to controls subject to organ-specific expression of an mCherry RNAi construct ( Fig. 2G ). SAHH gene knockdown in the tracheal system (insect respiratory system) and in muscle significantly reduced the hatching rate as does the ubiquitous knockdown of the gene as shown above ( Fig. 2G ). In contrast, targeted SAHH gene knockdown in the central nervous system (CNS) neurons, the endocrine cells of the gut or the Malpighian tubules (insect kidneys) did not impact hatching ( Fig. 2G ). Of note, organ-specificity of the SAHH knockdown was confirmed by GFP-reporter gene control crosses ( Fig. S6 ). Interestingly, targeted SAHH impairment in the fat body (insect liver and adipose tissue equivalent), does not significantly affect developmental success, but causes developmental delay and developmental phase extension at the population level ( Fig. 2H ) reminiscent to the fly Hcy supplementation model. Collectively, the fly Hcy supplementation and SAHH genetic models demonstrate that SAH accumulation severely affects Drosophila growth and development. We also present first evidence for organ-selective roles of SAHH in these processes. Characterization of the lethal phase and the disclosure of the underlying mechanisms of developmental delay and arrest deserve future research attention. It is noteworthy that presumable melanotic tumors were frequently observed in larvae subject to ubiquitous SAHH gene knockdown ( Fig. S5 ), which might contribute to death during development. Given the central role of SAH as inhibitor of SAM-dependent methyltransferases, we asked next whether the methylation profiles of proteins and phospholipids were selectively altered in the yeast and fly models. 3. Hcy supplementation similarly to genetic SAH hydrolase inhibition leads to impaired protein methylation in yeast and Drosophila Both elevated SAH and Hcy levels are known to inhibit methylation 39 . To analyze whether Hcy supplementation, which leads to SAH accumulation, results in inhibition of protein methylation, we first analyzed total protein asymmetrically dimethylated arginine versus total arginine (ADMA/Arg) ratio in wild type yeast grown in the presence of Hcy and in the yeast Δ sah1 mutant. Indeed, wild type yeast cultivated in the presence of Hcy, similarly to the Δ sah1 mutant, exhibits a decreased ratio of ADMA/Arg in proteins ( Fig. 3A ). Cultivation of wild type yeast in the presence of Hcy also resulted in altered intensities of distinct protein bands detected by an anti-monomethylarginine (MMA)-specific antibody ( Fig. 3B ). While the signals of two protein bands detected by the MMA antibody were increased, two others were decreased ( Fig. 3B ). In contrast, the total protein ADMA/Arg ratio in the fly Hcy supplementation model was not significantly changed in comparison to non-supplemented larvae ( Fig. 3C ). In the fly Hcy supplementation and SAHH genetic models the MMA antibody detected differentially monomethylated proteins, similarly to observations made in yeast (compare Fig. 3B to 3D ). These observations suggest that the inhibition of protein methylation by SAH may vary depending on specific proteins or residues involved. To directly assess whether different methylation sites are differentially affected, we next used site-specific antibodies to detect distinct methylated residues. First, we analyzed whether Hcy supplementation, which leads to SAH accumulation, leads to inhibition of lysine methylation of histone 3. Indeed, wild type yeast grown in the presence of Hcy exhibited significantly decreased levels of all analyzed tri-methylated sites, H3K4Me3, H3K36Me3, and H3K79Me3, with H3K79Me3 being the most affected one in response to Hcy supplementation ( Fig. 4A ). Similarly, the yeast Δ sah1 mutant shows significant decrease in tri-methylated H3K36Me3 and H3K4Me3, however not in H3K79Me3 ( Fig. 4A ). Methylation of H3K79Me3 in the Δ sah1 mutant is also decreased, however, it is higher than in the Hcy-supplemented wild type yeast ( Fig. 4A ). Hcy supplementation has no effect on methylation of H3K79Me2 and a weak effect on methylation of H3K36Me2 ( Fig. 4A ). In accordance, methylation of H3K79Me2 in the Δ sah1 mutant is unaltered, however methylation of H3K36Me2 is significantly decreased ( Fig. 4A ). Noteworthy, Hcy supplementation also led to significantly increased levels of H3K79Me1 as compared to non-supplemented wild type yeast ( Fig. 4A ). In accordance, a non-significant trend to elevated H3K79Me1 levels was also observed in the Δ sah1 mutant ( Fig. 4A ). Histone 3 methylation levels in the Drosophila Hcy supplementation model were not affected compared to controls in any of the tested histone methylation sites ( Fig. 4B ). In contrast, histone 3 methylation in larvae of the SAHH genetic model was decreased at all the tested sites, with H3K4Me3, H3K36Me2, and H3K79Me1 being significantly decreased, and H3K36Me3 and H3K79Me2 being non-significantly decreased with exception of H3K79Me1, which was unaffected ( Fig. 4B ). We conclude that while SAH inhibits histone 3 lysine methylation both in yeast and Drosophila , the extent of inhibition varies across different methylation sites. This further supports the hypothesis that SAH accumulation selectively inhibits distinct protein methylation processes. 4. Hcy supplementation similarly to genetic SAH hydrolase inhibition leads to impaired phospholipid methylation in yeast and Drosophila Phospholipid (PL) methylation is a major consumer of SAM both in yeast and mammals, and requires three-step methylation via monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE) for the synthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) 32 . Alternatively, PC can be synthesized both in yeast and mammals by the salvage Kennedy pathway using choline 32 . We have shown previously that PL methylation is sensitive to SAH accumulation as well as Hcy supplementation in choline free-medium 49 , 51 . However, the medium we used for the cultivation of yeast cells in the current study contained choline. Thus, next we analyzed whether Hcy supplementation impairs PC synthesis by the methylation pathway also in the presence of choline, and whether it leads to a drop in PC levels and interferes with PL metabolism. For this we analyzed the levels of PE, MMPE, DMPE and PC in wild type yeast grown in the presence or absence of Hcy in the medium and in the yeast Δ sah1 mutant, as well as in larvae of the corresponding Drosophila Hcy supplementation and SAHH genetic models. In yeast, both Hcy-supplemented wild type and the Δ sah1 mutant exhibited decreased total PC levels compared to non-supplemented wild type yeast, although only Hcy-supplemented wild type yeast showed significantly decreased PC ( Fig. 5A ). In accordance, total PE levels were significantly increased only in Hcy-supplemented wild type, but not in the yeast Δ sah1 mutant, in which they were even significantly decreased ( Fig. 5A ). However, both Hcy-supplemented wild type and the Δ sah1 mutant displayed significantly increased relative PE levels as well as decreased relative PC levels ( Fig. S8A ). In accordance, PC/PE ratios were significantly decreased in both Hcy-supplemented wild type as well as in the Δ sah1 mutant compared to non-supplemented wildtype, but the Δ sah1 mutant showed significantly higher PC/PE ratios compared to Hcy-supplemented wild type ( Fig. 5C ). Both Hcy-supplemented wild type and the Δ sah1 mutant displayed significantly decreased total levels of the intermediates of PL methylation, MMPE and DMPE ( Fig. 5A ). It has to be noted, however, that the levels of MMPE and DMPE were stronger decreased in Hcy-supplemented wild type than in the Δ sah1 mutant in line with significantly higher PC/PE ratios in the Δ sah1 mutant compared to Hcy-supplemented wild type yeast ( Fig. 5A and C ). Similarly, larvae of the Drosophila SAHH genetic model exhibited significantly decreased PC, DMPE and MMPE levels compared to controls ( Fig. 5B ). Though PE levels were unaltered, the PC/PE ratio was significantly decreased in this model ( Fig. 5B and D ). Surprisingly, larvae of the fly Hcy supplementation model contained significantly more PC as well as markedly, but non-significantly elevated PE compared to larvae grown in the absence of Hcy ( Fig. 5B ). Even so, larvae grown on Hcy containing food showed a non-significant trend of decreased PC/PE ratios compared to non-supplemented larvae ( Fig. 5D ). Interestingly, while levels of MMPE were significantly increased, the levels of DMPE were significantly decreased in larvae of the Hcy supplementation model compared to control food ( Fig. 5B ). Inhibition of PL methylation alters PC to PE levels and consequently the content of nonbilayer-forming PLs in the membranes which might lead to up-regulation of PL remodeling in response to Hcy/SAH accumulation. Therefore, we analyzed the levels of the lysophospholipids, LPE, LMMPE, LDMPE and LPC, which are central to PL remodeling. Analysis of lysophospholipids in yeast showed that LPE, LMMPE, LDMPE and LPC levels were deregulated following exactly the same pattern as PE, MMPE, DMPE and PC both in Hcy-supplemented wild type and the Δ sah1 mutant ( Fig. S7A ). In particular, LMMPE, LDMPE and LPC were significantly decreased in Hcy-supplemented wild type similarly as MMPE, DMPE and PC ( Fig. S7A ). In contrast, in the Δ sah1 mutant only LPC levels unlike PC levels were significantly decreased, while LPE, LMMPE and LDMPE levels differently to PE, MMPE and DMPE levels were only insignificantly decreased compared to non-supplemented wild type ( Fig. S7A ). LysoPL molecular species analysis of the Drosophila genetic model showed the same pattern as the analyzed PLs except for LPC ( Fig. S7B ). While PC levels are significantly decreased in Drosophila larvae carrying SAHH RNAi, LPC levels are only insignificantly decreased in response to reduced SAH hydrolase activity ( Fig. S7B ). Furthermore, in Drosophila dietary model the levels of analyzed lysoPLs molecular species did not change compared to controls, in contrast to significant elevation of MMPE and PC and significant decrease of DMPE in larvae fed Hcy-containing food compared to larvae on control diet ( Fig. S7B ). It has to be noted that the yeast Δ sah1 mutant exhibited a significant decrease of relative PC levels and Drosophila larvae carrying SAHH RNAi showed significant increase of relative PE and a decrease of relative LPE levels ( Fig. S8A-D). We conclude that Hcy even in the presence of choline leads to inhibition of PL methylation, altered PC/PE ratio and PL remodeling in wild type yeast similarly to the yeast and Drosophila SAHH genetic models., however, the extent of perturbations varies depending on the secondary effects. This further supports the hypothesis that inhibition of PL methylation by SAH accumulation along with inhibition of protein methylation is one of the central mechanisms how SAH leads to pathological consequences. Altogether, developed yeast and Drosophila dietary and genetic models of HHcy-associated SAH accumulation show that methylation inhibition is an important mechanism how elevated Hcy can lead to pathological consequences. Functional characterization of evolutionary conserved Hcy/SAH-dependent methylation targets in Drosophila and yeast will reveal mechanisms of SAH toxicity which may be operative in HHcy-associated human pathologies. Discussion CVD, the leading cause of death worldwide, can only be to 50% explained by established risk factors including cholesterol 1 – 4 . Hcy, a sulfur containing amino acid involved in methylation metabolism, is an independent risk factor for the development of atherosclerosis, increases CVD risk in combination with hypercholesterolemia 5 , is linked to cardiac pathologies 6 – 10 and further human diseases including neurological disorders, fatty liver disease, insulin resistance, and cancer as well as to CVD and all-cause mortality 11–14,16−18 . In accordance with the central role of Hcy, CKD, which is associated with highly elevated Hcy, is also linked to dramatically increased CVD as well as increased all-cause mortality 4 , 15 , 53 . Elevated Hcy is found in 5–10% of the general population, in up to 30% of the elderly and in as many as 70% of men over 80 years of age 5 , 19 , 20 . In most cases it is associated with deficiency of vitamins required for Hcy degradation 21 and is also linked to physical inactivity 22 , low skeletal mass 23 , high fat diet 24 and obesity 23 , 25 . Attempts to decrease CVD and neurological outcomes associated with elevated Hcy by lowering plasma Hcy levels were disappointing 26 – 28 indicating necessity to understand detailed mechanisms downstream Hcy leading to pathological consequences. Methylation of different macromolecules is an important regulatory mechanism involved in a wide variety of cellular processes 33 and its dysregulation is increasingly recognized as a risk factor for CVD development 34 – 37 . CVD has been linked to increased levels of SAH, a universal strong product inhibitor of SAM-dependent methylation 38 . Furthermore, both elevated SAH and Hcy levels are known to inhibit methylation, however, detailed mechanistic understanding of their downstream signaling cascades leading to CVD is largely missing 39 . Further, lowering of elevated plasma Hcy levels in addition to the inability to reduce associated clinical outcomes 26 – 28 does not decrease SAH levels, indicating a central role of SAH in Hcy-associated pathology 40 . In our recent publication we showed that Hcy independently of cholesterol alters aortic wall structure and functionality as well as leads to deregulation of lipoprotein metabolism and interferes with organization of elastin, which was reported to be linked to progressive aortic stiffening and all-cause mortality in CKD patients 30 . Furthermore, elevation of Hcy levels in rabbits fed diet blocking Hcy degradation was associated with a drop of total protein methylated arginine as well as decreased levels of creatine, which requires methylation for its synthesis 29 , suggesting involvement of deficient methylation in pathological consequences associated with elevated Hcy. Inhibition of methylation is an overlooked mechanism how elevated Hcy can lead to pathological consequences. More than 200 SAM-dependent methyltransferases in humans that transfer the methyl group of SAM to numerous cellular acceptors including nucleic acids, proteins (including histones) as well as low molecular weight compounds that require SAM for their synthesis, e.g. phospholipids and creatine are centrally involved in many processes in the organism 32 . An accumulation of SAH in response to elevation of Hcy levels may inhibit respective enzymes and interfere with numerous crucial methylation-dependent processes including epigenetic regulation of gene expression, signaling, lipid and energy metabolism. Understanding sensitivity of methylation-dependent processes to Hcy and SAH accumulation will help to understand pathological consequences associated with elevated Hcy. SAH is degraded to Hcy and adenosine in a reversible reaction catalyzed by SAH hydrolase 32 . SAH hydrolase offers the sole possibility of SAH degradation in mammals and is very highly evolutionary conserved exhibiting over 70% of identity between yeast and human orthologs 32 . High evolutionary conservation of SAH hydrolase as well as of methylation metabolism 32 suggest their central role in the regulation of cellular function. Deletion of SAH hydrolase in yeast is lethal unless a yeast-specific sulfur assimilation pathway is active 51 . Similarly, interference with SAH hydrolase locus in mice is embryonically lethal 50 . Here, we developed a dietary Drosophila model of HHcy and compared it with a genetic Drosophila model of SAH-mediated methylation inhibition and corresponding yeast models to reveal evolutionarily conserved Hcy/SAH-sensitive methylation patterns. Feeding Drosophila larvae a Hcy-containing diet or growing yeast on Hcy-supplemented medium similarly to blocking SAH degradation leads to SAH accumulation, developmental delay and growth defects. Surprisingly, while Hcy-supplementation of wild type yeast resulted only in SAH accumulation, in the yeast Δ sah1 mutant both SAH and SAM accumulated leading to virtually unaltered SAM/SAH ratio. Similarly, in Drosophila carrying SAHH RNAi but not in Drosophila fed Hcy-containing food SAM levels were significantly increased leading altogether to a much smaller drop of SAM/SAH ratio in genetic compared to dietary Drosophila model. Block of cystathionine ß-synthase in CBS mice leads to a massive decrease in glutathione levels 54 suggesting that also deletion of SAH hydrolase in the yeast Δ sah1 mutant or its downregulation in Drosophila larvae carrying SAHH RNAi, which is likely to be associated with a decrease in Hcy levels, may lead to glutathione depletion. CBS is allosterically activated by SAM 55 . Moreover, it was shown that during switch from methionine to Hcy-containing medium, CBS protein levels are drastically decreased and binding of SAM stabilizes CBS against degradation 55 . Thus, it appears likely that both the yeast Δ sah1 mutant and Drosophila larvae carrying SAHH RNAi require (elevated) SAM to maintain redox capacity. As observed previously, interference with SAH hydrolase in yeast leads to altered morphology and impaired growth 52 . Similarly, we observed a massive growth defect of the yeast Δ sah1 mutant as well as its altered morphology. Hcy-supplementation resulted in the gradual inhibition of yeast growth, in accordance with a detrimental role of SAH. Aggregation of the Δ sah1 mutant at the end of cultivation in contrast to Hcy-supplemented wild type yeast cells will be studied in the future. In accordance with the growth defect of wild type yeast cultivated in the presence of Hcy, Drosophila larvae fed Hcy-containing food exhibited developmental delay as shown by delayed pupariation and hatching rates. Moreover, larvae fed Hcy-containing food exhibited significantly lower weight, in accordance with the detrimental impact of SAH on cellular functionality. Noteworthy, despite developmental delay survival of larvae during pupariation as well as survival of pupae during hatching was unaffected similarly to unaffected survival of yeast cells despite gradual inhibition of growth in Hcy-containing medium. In contrast, Drosophila carrying SAHH RNAi exhibited massively reduced survival both at pupariation as well as at hatching in addition to drastically delayed pupariation and hatching rates. Additionally, the size of Drosophila carrying SAHH RNAi was markedly decreased and these larvae in addition to delayed development, poor viability and reduced size exhibited black spots reminiscent of melanotic tumors. Formation of melanized bodies can occur for instance through dysregulated expression of Hox genes 56 . Moreover, it has been reported that misexpression of various Hox genes leads not only to formation of melanized (pseudo-) tumors, but also to pupal lethality in Drosophila 56 , which is in line with our observation of strongly decreased survival rates, especially at pupal stage, in Drosophila larvae expressing SAHH RNAi. Noteworthy, expression of Hox genes is regulated via H3 lysine methylation, which is highly conserved across animal species 57 , 58 . Furthermore, H3K79 methylation is critical for mammalian HOX gene expression 59 . Also, other nuclear co-regulators were shown to be linked to deregulated methylation. Monomethylated Yap by Set7 methyltransferase at lysine 494 controls an evolutionarily conserved Hippo signaling pathway that regulates organ size and function in mice 60 . Of note, the Hippo pathway has been also linked to vascular smooth muscle cell proliferation during vascular remodeling in CVD 61 – 63 . Similarly, arginine and lysine methylation of transcriptional coactivator BRD4 that has a central role in regulating transcription and genome stability was shown to be linked to transcription deregulation and DNA repair 64 as well as negative regulation of genes that are involved in translation and total mRNA translation inhibition in mammalian cells 65 , respectively. Furthermore, arginine methylation of mammalian DEAD-box family RNA helicase, DDX5, was shown to regulate resolution of aberrant transcription-associated RNA:DNA hybrid (R-loop) formation, which often causes catastrophic conflicts during replication, resulting in DNA double-strand breaks and genomic instability 66 . Moreover, SAH hydrolase was shown to be essential for cyclic H3K4 trimethylation, genome-wide recruitment of BMAL1 to chromatin and subsequent circadian transcription promoting rhythmic H3K4 trimethylation and cyclic BMAL1 recruitment to target genes in mice 67 . To understand how Hcy/SAH affect methylation of different molecules we next analyzed the total protein ADMA/Arg ratios by NMR as well as MMA methylation by western blotting. Indeed, both Hcy-supplementation of wild type yeast as well as the yeast Δ sah1 mutant exhibited significantly decreased ADMA/Arg ratios. These findings suggest that SAH inhibits protein methylation. However, our data suggest that not all protein methylations are affected to the same extent: the signals of two specific yeast protein bands detected by an MMA-specific antibody were increased; one can speculate that the methylation reactions mediating monomethylation of the corresponding proteins are not (or less) inhibited by SAH or alternatively decreased levels of dimethylated arginines leads to relative increase of MMRs. Similarly, comparing the two Drosophila models we also observed decreased as well as increased signals of specific (but unidentified) fly proteins detected by an MMA-specific antibody. However, in contrast to yeast, Drosophila fed Hcy-containing food exhibited unaltered ADMA/Arg ratio. Of note, only one of nine protein arginine methyltransferases identified in Drosophila is abundantly expressed in larvae with next two being moderately expressed in this developmental stage 68 . Moreover, we observed different extents of inhibition of different types of lysine methylations both in yeast and Drosophila . In particular, in yeast histone 3 (H3) K36 trimethylation was more inhibited in response to Hcy than dimethylation at the same site. Similarly, H3K79 mono-, di- and trimethylation were differently altered in response to Hcy and SAH both in yeast and Drosophila . This suggests that SAH has different inhibitory capacity for different protein methylation reactions. In line with this observation is a previous report that SAH inhibits protein methylation at much lower concentrations than DNA methylation 69 . Histone methylation is a major consumer of methyl groups particularly in the absence of PL methylation 70 , affects metabolism independently of transcriptional regulation 71 and together with histone acetylation influences cellular metabolism 72 . H3K4 and H3K36 methylations are catalyzed by SET domain lysine methyltransferases, however H3K79 methylation is catalyzed by a lysine methyltransferase without a SET domain (Dot1 in yeast or Grappa in Drosophila ) 73 , 74 . A distributive mechanism and highly diverged catalytic properties reported for Dot1 methyltransferases may explain the different inhibitory potential of Hcy and SAH in our models toward H3K79 compared to histone lysine methylation sites methylated by SET domain containing methyltransferases 75 . Interestingly, H3K36 methylation is regulated by demethylation of PP2A, which activates demethylation of H3K36 through hyperphosphorylation of H3K36 demethylase Rph1 in yeast 76 . Moreover, in accordance with observed elevated SAM levels in the yeast Δ sah1 mutant (as well as in Drosophila SAHH RNAi) yeast PP2A mutants as well as yeast mutant lacking H3K36 demethylase Rph1 spares SAM by limiting histone methylation and exhibit elevated SAM levels 76 . Synthesis of PC via three step methylation of PE is another major consumer of SAM 70 , 77 , 78 . In contrast to mammals the first methylation from PE to MMPE in yeast is catalyzed by Cho2 and further methylations to DMPE and PC are catalyzed by Opi3 70 . Deficiencies of either of these enzymes were reported to lead to accumulation of SAM and increased SAM to SAH ratios in yeast 70 . In accordance with inhibition of Cho2 and Opi3 we observed drastically decreased MMPE, DMPE and PC levels, while PE accumulated, resulting in drastically decreased PC/PE ratios in wild type yeast supplemented with Hcy. Similar effects can be seen in respective lysoPLs, with a significant elevation of LPE and significantly decreased LMMPE, LDMPE and LPC in response to Hcy supplementation in wild type yeast. In contrast, despite highly increased SAH levels, block of SAH hydrolase in the yeast Δ sah1 mutant did not lead to decreased overall SAM to SAH ratio and resulted in less drastic decrease in PE, MMPE, DMPE and PC levels as compared to wild type yeast supplemented with Hcy. This is mirrored in PC/PE ratio, which is lower compared to untreated wild type, but higher compared to wild type yeast supplemented with Hcy. Furthermore, no elevation of LPE and even an increase in LMMPE were observed in wild type yeast supplemented with Hcy, while LDMPE and LPC were decreased, however to a lower degree. This suggests further mechanisms are likely to be operative in the yeast Δ sah1 mutant compared to wild type yeast supplemented with Hcy. In contrast to yeast, in mammals PL methylation is catalyzed by a single enzyme, phosphatidylethanolamine methyltransferase (PEMT), catalyzing all three methylation steps 77 . In Drosophila exact mechanisms of PL methylation are still unknown, but PL methyltransferase activity has been detected in Drosophila in the past 79 . Increased PE and decreased DMPE levels as well as decreased PC/PE ratio in Drosophila larvae fed Hcy-supplemented food further suggest PL methylation in Drosophila. Similarly, Drosophila carrying SAHH RNAi exhibited drastically decreased MMPE, DMPE and PC levels and slightly increased PE levels as well as a massively decreased PC/PE ratio. Unexpectedly, we also observed increased MMPE and PC levels in Drosophila larvae fed Hcy-supplemented food but not in Drosophila carrying SAHH RNAi. Similar to major changes in PL methylation pathway being observed for Drosophila carrying SAHH RNAi, we found a decrease in all lysoPLs with strongest decrease in LMMPE and LDMPE levels in the genetic Drosophila model, while Hcy supplementation of Drosophila larvae did not lead to any change in lysoPLs. Interestingly, it was also reported that PEMT-deficiency in mice results in inability to gain weight even on high fat diet, leading to significantly decreased body weight 80 . In summary, comparison of Drosophila and yeast models of Hcy-associated SAH-mediated methylation inhibition showed similar as well as different Hcy/SAH-sensitive methylation patterns. Hcy supplementation or blocking of SAH degradation in both model organisms lead to impaired protein and PL methylation. While in yeast Hcy/SAH accumulation leads to decreased total protein ADMA/Arg ratio, impaired MMA, histone and PL methylation, in Drosophila fed Hcy-containing food total protein ADMA/Arg ratio was unaffected, while MMA, histone and PL methylation, similarly to yeast, was sensitive to Hcy/SAH accumulation in Drosophila fed Hcy-containing food or carrying SAHH RNAi. Furthermore, both Hcy supplementation as well as genetically triggered SAH accumulation are linked to developmental delay and growth defect both in yeast and Drosophila models. Drosophila larvae carrying SAH hydrolase RNAi in addition to development delay, poor viability and reduced size exhibited black spots reminiscent of melanotic tumors. Employing yeast and Drosophila genetic screens will reveal evolutionary conserved Hcy/SAH-dependent mechanisms with high potential relevance for HHcy-associated human pathologies. Declarations Acknowledgements We would like to thank Fred van Leeuwen for providing H3K79 antibodies. The authors thank Raphael Kühnlein and Lydia Misslinger for excellent technical assistance in the context of organ-specific developmental analysis and fly food preparation, respectively. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537), from the Vienna Drosophila Resource Center (VDRC, www.vdrc.at ) and from the KYOTO Drosophila Stock Center (Kyoto Institute of Technology) were used in this study. Sources of funding This research was funded in whole, or in part, by the Austrian Science Fund (FWF) [Grant-DOIs: 10.55776/P31105 and 10.55776/P33672 to OT and grant DOIs 10.55776/P27996 and 10.55776/DOC50 to BP]. Further, this work was supported by the Doctoral Academy of the University of Graz (Consortium Molecular Metabolism MOBILES), the Integrative Metabolism Research Center Graz, Austrian Infrastructure Program 2016/2017, the BioTechMed-Graz Flagship project DYNIMO, and the Province of Styria (Zukunftsfonds, doc.fund) and the City of Graz. The authors acknowledge the financial support by the University of Graz. Author contributions MSB – Investigation, Data curation, Methodology, Formal analysis, Validation, Visualization, Software, Resources ( Drosophila & yeast experiments, analytics), Writing – original draft, Writing – review and editing; HH & TM – Investigation, Data curation, Methodology, Formal analysis, Validation, Visualization, Software, Resources (NMR of global ArgMet), Writing – review and editing; JH & MM – Methodology, Formal analysis (yeast experiments), Writing – review and editing; ZN – Investigation, Data curation, Methodology, Formal analysis, Validation, Visualization, Software, Resources (establishment of Drosophila dietary model), Writing – review and editing; HW – Methodology, Formal analysis, Visualization, Software, Resources (microscopy), Writing – review and editing; GNR – Data curation, Methodology, Software, Resources (analytic), Writing – review and editing; BR – Conceptualization, Investigation, Methodology, Supervision (yeast subproject: western blots), Writing – review and editing; RPK – Conceptualization, Investigation, Methodology, Supervision, Funding acquisition, Project administration ( Drosophila subproject), Writing – original draft, Writing – review and editing; OT – Conceptualization, Investigation, Methodology, Supervision, Funding acquisition, Project administration (yeast subproject and Hcy-associated models of SAH accumulation), Writing – original draft, Writing – review and editing. Competing interest statements The authors of the manuscript declare no conflicts of interests. Abbreviations Hcy – homocysteine, SAH – S -adenosyl- L -homocysteine, SAM – S -adenosyl- L -methionine, SAHH – SAH hydrolase, HHcy – hyperhomocysteinemia, CVD – cardiovascular disease, CKD – chronic kidney disease, ADMA/Arg – ratio of total protein asymmetrically dimethylated arginines versus arginines, MMA – monomethylarginine, H3 – histone 3, PL – phospholipid, PE – phosphatidylethanolamine, PC – phosphatidylcholine, MMPE – monomethylphosphatidylethanolamine, DMPE –dimethylphosphatidylethanolamine, LPE – lysophosphatidylethanolamine, LPC – lysophosphatidylcholine, LMMPE – lysomonomethylphosphatidylethanolamine, LDMPE – lysodimethylphosphatidylethanolamine, AEL – after egg laying References Lefkowitz RJ, Willerson JT (2001) Prospects for cardiovascular research. JAMA 285:581–587. 10.1001/jama.285.5.581 Morita H, Seidman J, Seidman CE (2005) Genetic causes of human heart failure. 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(2018) Table 2: Drosophila fly lines used in this study Trivial Name Function Genotype Internal Stock No. Source Source Stock No. Canton-S wild type control stock - RKF 1679 - BDSC* 64349 w[1118] white mutant control stock w 1118 ; +/+; +/+; RKF 1084 - VDRC** 6000 Ubiquitous Ubiquitous driver w*; +/+; P{GAL4-da.G32 RKF 908 Wodarz et al. (1995) - RNAi ctrl mCherry RNAi effector y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=VALIUM20-mCherry}attP2 RKF 1645 - BDSC 35785 SAHH RNAi SAH hydrolase RNAi effector w[1118] y[1] fl. Sc[*] fl. V[1] sev[21] fl. ; P{y[+t7.7] v[+t1.8]=TriP.HMS05799}attP40; RKF 2416 This work based on BDSC * based on BDSC 67848 GFP GFP reporter +; P{w[+mC]=UAS-Stinger}2; + / + RKF 1171 Barolo et al. (2000) - CNS Central nervous system-specific driver w*; +/+; P{w[+mW.hs]=GAL4-Nrv2-3} P{w[+m*]UAS-GFP} RKF 211 Sun et al. (1999) - Gut Gut enterocyte-specific driver w[1118]; P{w[+mC]=mex1-GAL4.2.1}10-8 CHF 2396 - BDSC 91368 Tracheal System Tracheal system-specific driver w*; P{UAS-GFP::nLacZ} / CyO float; P{btl-GAL4} / TM3 Sb* Ser* RKF 1912 - KSC*** 109132 Muscle Muscle-specific driver mef2-Gal4 IPF 2174 - BDSC 27390 Malpighian Tubules Malpighian tubules-specific driver +/+; UO-Gal4/CyO float; +/+ JRF 1254 Terhzaz et al. (2010) - Fat body Fat body-specific driver w*; +/+; P{Lpp-GAL4.B}/TM3, P{w[+mC]=ActGFP}JMR2, Ser[1] RKF 1582 This work is based on Brankatschk and Eaton (2010) - * = Bloomington Drosophila Stock Centre, ** = Vienna Drosophila Resource Center, *** = KYOTO Stock Center Table 3: Drosophila RT-qPCR primers Target Forward primer sequence Reverse primer sequence Source SAHH (exon spanning) 5’- AGT ACG GCC CAT CTA AGC C - 3' 5' - CGG CAG CAT TAT CCT GGG T - 3' Fly RNAi Database, identifier PP25254 SAHH (both isoforms) 5' - AGC CCC TGA ACA TGA TCC TG - 3' 5' - CGA CCC TCC TTG AAC ATC TTG T - 3' Fly RNAi Database, identifier PP36991 GAPDH 5' - GTC GGG CTT GTA GGC ATC C - 3' 5' - AGG CAT CCA CTC ACT TGA AGG - 3' Beaucher et al. (2007) Additional Declarations The authors declare no competing interests. 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Kühnlein","email":"data:image/png;base64,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","orcid":"","institution":"University of Graz","correspondingAuthor":true,"prefix":"","firstName":"Ronald","middleName":"P.","lastName":"Kühnlein","suffix":""},{"id":358420691,"identity":"97398fef-b756-4965-be95-7f56b6d58c3f","order_by":10,"name":"Oksana Tehlivets","email":"data:image/png;base64,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","orcid":"","institution":"University of Graz","correspondingAuthor":true,"prefix":"","firstName":"Oksana","middleName":"","lastName":"Tehlivets","suffix":""}],"badges":[],"createdAt":"2024-09-24 11:20:01","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5144569/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5144569/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65198051,"identity":"12e68921-93bf-4075-8242-08022b8a1c1e","added_by":"auto","created_at":"2024-09-24 15:52:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":81682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSAH and SAM accumulation in yeast and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDrosophila\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e dietary and genetic models. \u003c/strong\u003eTotal SAH and SAM levels as well as SAM/SAH ratios in wild type yeast grown in the presence or absence of 5 mM Hcy, and in the yeast ∆\u003cem\u003esah1\u003c/em\u003e mutant grown without Hcy supplementation (n = 4) (A). Total SAH and SAM levels as well as SAM/SAH ratios in migratory L3 Canton-S \u003cem\u003eDrosophila\u003c/em\u003e larvae grown on standard fly food or food containing 20 mM Hcy, and in \u003cem\u003eDrosophila\u003c/em\u003e larvae globally expressing mCherry RNAi or SAHH RNAi grown on standard food (n = 3 - 4) (B). For A, statistics were calculated with Students \u003cem\u003et\u003c/em\u003e-test or Mann-Whitney-U pairwise comparisons with Bonferroni correction for multiple testing. For B, statistics were calculated with Students \u003cem\u003et\u003c/em\u003e-tests (parametric: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, non-parametric: # = p ≤ 0.05, ## = p ≤ 0.01, ### = p ≤ 0.001). All statistics are shown in Supplemental Data 3.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5144569/v1/1ca0b9d85d862c214541b9b1.jpg"},{"id":65198758,"identity":"18393e48-013b-4530-96dc-d12dbe53de49","added_by":"auto","created_at":"2024-09-24 16:00:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":170618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevelopmental delay and growth defects in yeast and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDrosophila\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e dietary and genetic models. \u003c/strong\u003eGrowth curves of wild type yeast grown in the presence or absence of Hcy supplementation as indicated, and of the yeast ∆\u003cem\u003esah1\u003c/em\u003e mutant grown without Hcy supplementation measured over 40 h at OD\u003csub\u003e600 \u003c/sub\u003e(n = 2) (A). Live cell microscopy of wild type yeast grown in the presence or absence of 5 mM Hcy, and of the yeast\u003cem\u003e \u003c/em\u003e∆\u003cem\u003esah1\u003c/em\u003e mutant grown without Hcy supplementation. Cells were analyzed after 40 h of cultivation. Scale bars = 5 µm (B). Migratory L3 larval weight of Canton-S \u003cem\u003eDrosophila\u003c/em\u003e larvae grown on standard fly food or food containing 20 mM Hcy (n = 40) (C). Pupariation and hatching rates of Canton-S \u003cem\u003eDrosophila\u003c/em\u003e larvae grown on standard fly food or food containing 20 mM Hcy (n = 4) (D). Pupariation and hatching rates of \u003cem\u003eDrosophila\u003c/em\u003e larvae globally expressing mCherry RNAi or SAHH RNAi grown standard food (n = 2) (E). Size comparison of \u003cem\u003eDrosophila\u003c/em\u003e larvae globally expressing control RNAi (mCherry RNAi) or SAHH RNAi grown on standard food. Photographs were taken daily, between 3\u003csup\u003erd\u003c/sup\u003e and 10\u003csup\u003eth\u003c/sup\u003e day AEL (F). Hatching rates of \u003cem\u003eDrosophila\u003c/em\u003e flies with organ-specific expression of mCherry RNAi or SAHH RNAi grown on standard food (n = 4) (G). Hatching rates of \u003cem\u003eDrosophila\u003c/em\u003e progeny from the same cross with fat body-targeted or inactive SAHH knockdown construct in fat body grown on standard food. Of note, selective counting started on day 11 AEL and given values represent total hatched flies relative to egg count (n = 3) (H). For C and G, statistics were calculated with Students \u003cem\u003et\u003c/em\u003e-test or Mann-Whitney-U pairwise comparisons (parametric: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, non-parametric: # = p ≤ 0.05, ## = p ≤ 0.01, ### = p ≤ 0.001). All statistics are shown in Supplemental Data 3. AEL = after egg laying.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5144569/v1/84ecb8314be4b2b04365dfba.jpg"},{"id":65198761,"identity":"944475bf-4b1f-47ce-94c0-eb0c26be2706","added_by":"auto","created_at":"2024-09-24 16:00:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTotal protein asymmetrically dimethylated arginine to total arginine ratio in yeast and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDrosophila\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edietary and genetic models. \u003c/strong\u003eNMR analysis of total protein asymmetrically dimethylated arginine to total arginine (ADMA/ Arg) ratios in wild type yeast grown in the presence or absence of 5 mM Hcy supplementation, and in the yeast ∆\u003cem\u003esah1\u003c/em\u003emutant grown without Hcy supplementation (n = 6) (A). Western Blot analysis of monomethylarginine (MMA)-containing proteins in wild type yeast grown in the presence or absence of 5 mM Hcy, and the yeast ∆\u003cem\u003esah1\u003c/em\u003e mutant grown without Hcy supplementation (B). NMR analysis of total protein ADMA/Arg ratios in migratory L3 \u003cem\u003eDrosophila\u003c/em\u003e larvae grown on standard fly food or food containing 20 mM Hcy (n = 5) (C). Western Blot analysis of MMA-containing proteins in migratory L3 \u003cem\u003eDrosophila\u003c/em\u003e larvae grown on standard fly food or food containing 20 mM Hcy, and in L3 \u003cem\u003eDrosophila\u003c/em\u003e larvae globally expressing mCherry RNAi or SAHH RNAi grown on standard food (D). For A, statistics were calculated with Students \u003cem\u003et\u003c/em\u003e-tests with Bonferroni correction for multiple testing. For C, statistics were calculated with Mann-Whitney-U pairwise comparisons (parametric: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, non-parametric: # = p ≤ 0.05, ## = p ≤ 0.01, ### = p ≤ 0.001). All statistics are shown in Supplemental Data 3.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5144569/v1/b43dc9131e4e76485da879da.jpg"},{"id":65198059,"identity":"4a33894a-103c-48fb-a430-fb1e6420aa90","added_by":"auto","created_at":"2024-09-24 15:52:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":136310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH3 lysine methylation in yeast and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDrosophila\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e dietary and genetic models. \u003c/strong\u003eWestern Blot analyses of trimethylated H3K4, di- and trimethylated H3K36, and mono-, di- and trimethylated H3K79 in wild type yeast grown in the presence or absence of 5 mM Hcy, and in the yeast ∆\u003cem\u003esah1\u003c/em\u003e mutant grown without Hcy supplementation. Bar charts represent quantified signals normalized to total H3 (n = 4) (A). Western Blot analyses of trimethylated H3K4, di- and trimethylated H3K36, and mono-, di- and trimethylated H3K79 in migratory L3 \u003cem\u003eDrosophila\u003c/em\u003e larvae grown on standard fly food or food containing 20 mM Hcy, and in L3 \u003cem\u003eDrosophila\u003c/em\u003elarvae globally expressing mCherry RNAi or SAHH RNAi grown on standard food. Bar charts represent quantified signals normalized to total H3 (n = 3) (B). For A, statistics were calculated with Students \u003cem\u003et\u003c/em\u003e-test or Mann-Whitney-U pairwise comparisons with Bonferroni correction for multiple testing. For B, statistics were calculated with Students \u003cem\u003et\u003c/em\u003e-tests (parametric: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, non-parametric: # = p ≤ 0.05, ## = p ≤ 0.01, ### = p ≤ 0.001). All statistics are shown in Supplemental Data 3.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5144569/v1/62a0cd7c81bd99c8796778e5.jpg"},{"id":65198057,"identity":"c09a17bf-2547-46ef-84a4-181929f858e4","added_by":"auto","created_at":"2024-09-24 15:52:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":99254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhospholipid methylation and PC/PE ratios in yeast and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDrosophila\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e dietary and genetic models. \u003c/strong\u003eTotal PE-, MMPE-, DMPE- and PC levels in wild type yeast grown in the presence or absence of 5 mM Hcy, and in the yeast ∆\u003cem\u003esah1\u003c/em\u003e mutant grown without Hcy supplementation. Box plots represent peak integrals relative to internal standard peak integrals (n = 4) (A). Total PE-, MMPE-, DMPE- and PC levels in migratory L3 \u003cem\u003eDrosophila\u003c/em\u003elarvae grown on standard fly food or food containing 20 mM Hcy, and in L3 \u003cem\u003eDrosophila\u003c/em\u003elarvae globally expressing mCherry RNAi or SAHH RNAi grown on standard food. Box plots represent peak integrals relative to internal standard peak integrals and normalized to sample weights (n = 3) (B). PC/PE ratios in wild type yeast grown in the presence of 5 mM Hcy and in the yeast ∆\u003cem\u003esah1\u003c/em\u003e mutant grown without Hcy supplementation relative to non-supplemented wild type yeast PC/PE ratios. Box plot represents ratios of peak integrals normalized to internal standard peak integrals (n = 4) (C). PC/PE ratios in migratory L3 \u003cem\u003eDrosophila\u003c/em\u003elarvae grown food containing 20 mM Hcy relative to larvae grown on standard food and in L3 \u003cem\u003eDrosophila\u003c/em\u003e larvae globally expressing SAHH RNAi relative to larvae globally expressing mCherry RNAi both grown on standard food. Box plot represents ratios of peak integrals normalized to internal standard peak integrals (n = 3) (D). For A and C, statistics were calculated with Students \u003cem\u003et\u003c/em\u003e-test or Mann-Whitney-U pairwise comparisons with Bonferroni or Games-Howell correction for multiple testing. For B and D, statistics were calculated with Students \u003cem\u003et\u003c/em\u003e-test or Mann-Whitney-U pairwise comparisons (parametric: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, non-parametric: # = p ≤ 0.05, ## = p ≤ 0.01, ### = p ≤ 0.001). All statistics are shown in Supplemental Data 3.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5144569/v1/f003cf430f860bf32b95f57c.jpg"},{"id":65199093,"identity":"70ee04ae-3573-46fd-ba62-a70b2ae10293","added_by":"auto","created_at":"2024-09-24 16:08:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1841617,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5144569/v1/de3022ae-b540-4e3c-924f-e8c1251a8fde.pdf"},{"id":65198054,"identity":"417fb198-35a0-4411-a3a4-94ffbbe6199a","added_by":"auto","created_at":"2024-09-24 15:52:04","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":357012,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figures\u003c/p\u003e","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5144569/v1/c5415bc487a68bf3b4c4df78.pdf"},{"id":65198052,"identity":"e3fef7f3-f244-46c6-9cc8-b9270a473b38","added_by":"auto","created_at":"2024-09-24 15:52:04","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14906,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary data 1\u003c/p\u003e","description":"","filename":"SupplementalData1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5144569/v1/00444d2ed60c9b710d5fb483.xlsx"},{"id":65198056,"identity":"f4ab303b-5969-4df6-ae04-19d8c79347c1","added_by":"auto","created_at":"2024-09-24 15:52:04","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":23444,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary data 2\u003c/p\u003e","description":"","filename":"SupplementalData2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5144569/v1/5d4e4f3509a0f2949b393502.xlsx"},{"id":65198760,"identity":"fbbaf63b-c408-47a4-99ff-c4dc1e3d8084","added_by":"auto","created_at":"2024-09-24 16:00:04","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":31335,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary data 3\u003c/p\u003e","description":"","filename":"SupplementalData3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5144569/v1/135ed72053f982576d0062e0.xlsx"},{"id":65198762,"identity":"16bbd33c-4e6b-468f-8d56-3d8ec22c2400","added_by":"auto","created_at":"2024-09-24 16:00:05","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":366053,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary data 4\u003c/p\u003e","description":"","filename":"SupplementalData4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5144569/v1/85f4f71709a05fb268ac0fea.pdf"},{"id":65198060,"identity":"3cfc0286-46c2-4742-8531-3b5dcb529cc4","added_by":"auto","created_at":"2024-09-24 15:52:05","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":757798,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary data 5\u003c/p\u003e","description":"","filename":"SupplementalData5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5144569/v1/4ce13abd3876851bc84513a8.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAccumulation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-adenosyl-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-homocysteine impairs methylation and development in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSaccharomyces cerevisiae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDrosophila melanogaster\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular disease (CVD), the leading cause of death worldwide, is still insufficiently understood \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Hyperhomocysteinemia (HHcy), i.e. elevation of homocysteine (Hcy) concentration in the blood, is an independent risk factor for the development of atherosclerosis and increases CVD risk in combination with hypercholesterolemia \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Moreover, it is also associated with cardiac pathology \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and increased CVD mortality \u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Hcy is particularly highly elevated in chronic kidney disease (CKD), a pathological condition associated with drastically increased CVD mortality \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Hcy is also linked to many further human diseases as well as all-cause mortality \u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Elevated Hcy is a common pathological condition that is especially prevalent in the elderly \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and is in most cases associated with deficiency of vitamins required for Hcy degradation \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e as well as with physical inactivity \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, low skeletal muscle mass \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, high fat diet \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and obesity \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Attempts to decrease CVD and neurological outcomes associated with elevated Hcy by lowering plasma Hcy levels were disappointing \u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e indicating necessity to understand the detailed mechanisms downstream of Hcy leading to pathological consequences.\u003c/p\u003e \u003cp\u003eIn our recent publication we showed that Hcy independently of cholesterol alters aortic wall structure and functionality as well as leads to deregulation of lipoprotein metabolism \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Elevation of plasma Hcy levels by intravenous injections of Hcy into rabbits fed a diet blocking Hcy degradation leads to impaired vascular reactivity of the aorta, an accumulation of compromised, morphologically altered cells and deregulation of lipid metabolism in the aortic wall as well as disorganization of aortic collagen and elastin \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Furthermore, elastin fragmentation and an accumulation of electron-dense inclusions enriched in nitrogen in media elastin in response to elevated Hcy suggesting elastin degradation indicate how elevated Hcy can contribute to CVD progression \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Furthermore, elevation of Hcy levels in rabbits fed a diet blocking Hcy degradation was associated with a drop of total methylated arginines in proteins as well as decreased levels of creatine, which requires methylation for its synthesis, \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e suggesting involvement of deficient methylation in the pathological consequences associated with elevated Hcy.\u003c/p\u003e \u003cp\u003eInhibition of methylation is an understudied mechanism how elevated Hcy can lead to pathological consequences. There are more than 200 putative \u003cem\u003eS\u003c/em\u003e-adenosyl-\u003cem\u003eL\u003c/em\u003e-methionine (SAM)-dependent methyltransferases in humans \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. They produce the universal strong product inhibitor of SAM-dependent methylation, \u003cem\u003eS\u003c/em\u003e-adenosyl-\u003cem\u003eL\u003c/em\u003e-homocysteine (SAH) \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. SAH has to be quickly degraded to Hcy and adenosine in a reaction catalyzed by SAH hydrolase (SAHH) in order to allow SAM-dependent methylation \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Inability to quickly remove excess Hcy formed by SAH degradation reverses the direction of the equilibrium additionally potentiating SAH accumulation \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. SAHH offers the sole possibility of SAH degradation in mammals and is highly evolutionarily conserved exhibiting over 70% identity between yeast and human orthologs \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. High evolutionary conservation of SAH hydrolase as well as of methylation metabolism \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e suggest their central role in the regulation of cellular function.\u003c/p\u003e \u003cp\u003eMethylation of various macromolecules is an important regulatory mechanism involved in a wide variety of cellular processes \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and its dysregulation is increasingly recognized as a risk factor for CVD development\u003csup\u003e\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. CVD has been linked to increased levels of SAH \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Furthermore, both elevated SAH and Hcy levels are known to inhibit methylation, however, detailed mechanistic understanding of their downstream signaling cascades leading to CVD is largely missing \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Further, lowering of elevated plasma Hcy levels in addition to the inability to reduce associated clinical outcomes \u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e does not decrease SAH levels, indicating a central role of SAH in Hcy-associated pathology \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we show that Hcy supplementation, similarly to genetically compromising SAH hydrolase, leads to developmental delay, growth defects as well as impaired protein and phospholipid (PL) methylation both in yeast and in \u003cem\u003eDrosophila\u003c/em\u003e. While Hcy/SAH led to a decreased ratio of total protein asymmetrically dimethylated arginines to arginine (ADMA/Arg) along with impaired histone and PL methylation in yeast, total protein ADMA/Arg was unaffected in \u003cem\u003eDrosophila\u003c/em\u003e, while, histone and PL methylation were sensitive to Hcy and/or SAH accumulation. Furthermore, \u003cem\u003eDrosophila\u003c/em\u003e larvae subject to SAH hydrolase gene knockdown show delayed development, poor viability and reduced size.\u003c/p\u003e"},{"header":"Material \u0026 Methods","content":"\u003cp\u003eChemicals and consumables are listed in Supplemental Data 1.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eYeast strains, media, and growth conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strains used in this study are congenic with BY4741, a derivative of S288C, and are listed in \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e. Cells were grown at 30\u0026deg; C in synthetic dextrose medium (SDC) containing 1.4 g/L Difco yeast nitrogen base, 5 g/L ammonium sulfate, 20 g/L glucose and 0.79 g/L complete supplement mixture (CSM) at 180 rpm in the absence or presence of Hcy, as indicated in individual experiments. Media were solidified by the addition of 20 g/L agar.\u003c/p\u003e \u003cp\u003eFor growth curves, yeast wild type cells were grown overnight in SDC medium and inoculated to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.05 in fresh SDC medium containing 0 mM, 1 mM, 2 mM or 5 mM Hcy. The Δ\u003cem\u003esah1\u003c/em\u003e mutant cells were grown overnight in SDC medium and inoculated to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.05 in fresh SDC medium without Hcy. 300 \u0026micro;L were applied onto honeycomb well plate in 3 technical replicates per condition. Optical density at 600 nm wavelength was measured every 30 min for 40 hours in Bioscreen C (Dynex) with constant shaking between measurements. Before each measurement shaking was stopped for 5 sec. After 40 h samples were taken for microscopy. For microscopic observation, live cells were immobilized using agar sheets \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Transmission images were acquired using a Leica SP5 confocal microscope (Leica Microsystems, Inc.), a 488 nm argon laser line and a HC PL APO 63x oil immersion objective. The experiment was independently repeated once (Supplemental Data 4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDrosophila melanogaster\u003c/b\u003e \u003cb\u003elines, fly food and breeding\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFly lines used in this study are listed in \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e. FlyBase \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e was consulted for experimental planning.\u003c/p\u003e \u003cp\u003eFlies were propagated in 68 mL \u003cem\u003eDrosophila\u003c/em\u003e containers equipped with mite-tight stoppers on standard fly food containing 15.7 g/L baker\u0026rsquo;s yeast, 8.7 g/L soy flour, 5.4 g/L agar-agar, 69.6 g/L maize flour, 19.1 g/L beet syrup, 69.6 g/L malt, 5.4mL/L propionic acid and 1.3 g/L methyl-4-hydrobenzoate (dissolved in 4.4 mL EtOH). If not mentioned otherwise stock keeping and fly experiments were done at 25\u0026deg; C and 70% humidity with 12 h light/12 h dark cycle.\u003c/p\u003e \u003cp\u003eFor Hcy-supplemented food, 5% (v/v; in water) of 400 mM DL-Hcy were added to fly food prior to solidifying. For control food, 5% water was added instead of Hcy. Eggs from Canton-S or \u003cem\u003ew[1118]\u003c/em\u003e flies were collected on apple juice agar plates (2.125% (w/v) agar, 20% (v/v) apple juice, 2% (w/v) sugar, 0.12% (w/v; dissolved in EtOH) nipagin) supplemented with baker\u0026rsquo;s yeast paste. Agar pieces with 100 eggs each was cut out and transferred onto either standard or Hcy supplemented fly food. Vials were kept at 25\u0026deg; C at 70% humidity. Four individual vials were prepared for each food. From each vial, 10 migratory L3 larvae were collected, rinsed with water, dried with blotting paper and weighed individually on a Sartorius MC 5 scale. Larvae were frozen at -80\u0026deg; C in batches of 5 larvae for SAH and SAM extraction, protein extraction and phospholipid analysis. Remaining larvae were kept at 25\u0026deg; C and 70% humidity and number of pupae and eclosed pupae were counted continuously until no further pupae appeared or hatched. The experiment was independently repeated once (Supplemental Data 4).\u003c/p\u003e \u003cp\u003eFor RNAi crosses, per vial 10 adult virgin female ubiquitous driver flies and 5 male mCherry RNAi (RNAi control) or SAHH RNAi (SAH hydrolase RNAi) flies were transferred into standard food vials and incubated for 24 h at 25\u0026deg; C and 70% humidity. After incubation, adult flies were flipped to new vials and eggs were counted. New vials were incubated again for 24 h before adult flies were removed and eggs were counted. 4 individual vials were prepared for each crossing and were kept at 25\u0026deg; C and 70% humidity. Number of pupae and eclosed pupae were counted continuously in 2 vials per crossing. Experiment was independently repeated once. Larvae from the other two vials were extracted, rinsed and photographed daily for up to 10 days. Photographs were taken with help of M60 binoculars (Leica) on a Google Pixel 6a smartphone. Migratory L3 larvae or 14 days old non-migratory L3 larvae were collected, rinsed with water, dried with blotting paper, weighed in batches of 5 larvae and frozen at -80\u0026deg; C for RT-PCR, SAH- and SAM extraction, protein extraction and phospholipid analysis.\u003c/p\u003e \u003cp\u003eThe identity of organ-specific driver lines (see \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e) was confirmed by crossing 3 virgin females of the GFP-reporter line (see \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e) to 3 males of the driver lines. F1 migratory L3 larvae were rinsed with and mounted in cold water before imaging using a Leica M165 FC fluorescence stereomicroscope equipped with a GFP emission filter and a Leica DFC3000 G camera. To score survival and developmental delay in response to organ-specific SAHH knockdown (see \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e) compared to mCherry RNAi controls (see \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e) 5 virgin females of each of the effector lines were crossed to 3\u0026ndash;4 organ-specific or ubiquitous driver males. Parental flies were flipped daily 3\u0026ndash;4 times and egg numbers as well as eclosed adult flies (based on empty pupal cases) were counted to assess hatching rates. To score developmental delay progeny, the adult eclosion time of balancer-carrying (control) vs. non-balancer carrying (fat body-specific driver) progeny of the heterozygous fat body-targeting driver line (see \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e) was assessed daily after day 11 post crossing. The experiment was repeated with independent parental flies and progeny of 3\u0026ndash;4 consecutive daily collections was scored (Supplemental Data 4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDrosophila\u003c/b\u003e \u003cb\u003efood intake\u003c/b\u003e\u003c/p\u003e \u003cp\u003e1% (w/v) brilliant blue was added to the fly food containing either 5% (v/v; in water) 400 mM DL-Hcy or 0.2% (w/v) caffeine and 5% (v/v) water. Fly food with 1% (w/v) brilliant blue and 5% (v/v) water was used as control. Colored fly food was poured into small petri dishes to cover the bottom. 25 male and 25 female adult flies were put into standard food vials and flipped to new vials every 24 h. All vials were kept at 25\u0026deg; C and 70% humidity. Larvae were extracted from food vials one day prior to reaching migratory stage, rinsed and transferred to colored food plates. Plates were incubated for 1 h in darkness at room temperature (RT) before transferring the plates on ice. Larvae were extracted from food plates, rinsed, collected in cohorts of 10 larvae into 2 mL save-seal tubes and weighed on a Sartorius MC 5 scale. After adding 350 \u0026micro;L MeOH and one 5 mm steel ball samples were homogenized in a Retsch MM40 homogenizer at 30 Hz and 4\u0026deg; C for 2 min. Homogenates were centrifuged for 5 min at 14000 rpm and 4\u0026deg; C in a 5430 R centrifuge (Eppendorf) and the supernatant was transferred to new vial and centrifuged again for 5 min at 14000 rpm and 4\u0026deg; C. Duplicates of 100 \u0026micro;L supernatant were aliquoted into a 96-well plate and absorbance was measured at 626 nm in a Spectrostar\u003csup\u003eNano\u003c/sup\u003e spectrophotometer (BMG Labtech). For absolute quantification, a 5-point calibration curve using colored fly food (0.5 mg \u0026ndash; 7 mg) after extraction with MeOH was prepared as described above. Experiment was independently repeated once (Supplemental Data 4).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSAH/SAM extraction and analysis\u003c/h3\u003e\n\u003cp\u003eExtraction of SAH and SAM was based on Gellekink et al. \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e with minor changes. Briefly, for yeast, per sample 20 OD\u003csub\u003e600\u003c/sub\u003e units were harvested and resuspended in 250 \u0026micro;L water\u0026thinsp;+\u0026thinsp;0.1% formic acid. After addition of 250 \u0026micro;L glass beads, cells were lyzed in Homogenizer MM40 (Retsch) at 30 Hz and 4\u0026deg; C three times for 40 s. Homogenates were diluted 1:2. For \u003cem\u003eDrosophila\u003c/em\u003e, per sample 5 larvae were put into 500 \u0026micro;L water\u0026thinsp;+\u0026thinsp;0.1% formic acid and homogenized with a steel ball in Homogenizer MM40 (Retsch) at 30 Hz and 4\u0026deg; C for 2 min. For SAH and SAM extraction 100 mg, 1 mL Bond Elut PBA columns were used. Solid phase extraction was performed after washing the columns with 4 mL 0.1 M formic acid and equilibrating with 4 mL 20 mM ammonium acetate buffer (pH 7.4). Two separate aliquots of 210 \u0026micro;L were prepared for SAH and SAM extraction, respectively. Per aliquot, 60 \u0026micro;L of internal standard was added (either 2 \u0026micro;M SAH-d4 or 5 \u0026micro;M SAM-d3 in water) and 90 \u0026micro;L of the mix were applied onto the column. The columns were subsequently washed with 3 mL 20 mM ammonium acetate butter (pH 7.4). Samples were eluted in 1 mL 0.1 M formic acid and measured directly via HPLC-QQQ tandem mass spectrometry. Per sample two technical replicates were done.\u003c/p\u003e \u003cp\u003eSAH and SAM levels were analyzed by 1290 Infinity UHPLC coupled to a 6470 Triple-Quadrupole mass spectrometer (Agilent) using a BEH C18 column (3.0 mm\u0026times;150 mm; 1.7 \u0026micro;m) with 50\u0026deg; C column temperature, 5 \u0026micro;L injection volume and a constant flow rate of 200 \u0026micro;L/min. H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;0.1% formic acid (solvent A) and methanol\u0026thinsp;+\u0026thinsp;0.1% formic acid (solvent B) were used as solvents. 95% solvent A was held for 2 min, followed by a change to 100% solvent B over the next 2 min, which was held for additional 3.5 min. Re-equilibration was carried out by changing to 95% solvent A within 5 seconds, followed by 3 min at 95% solvent A. Total run time was 11 min. For absolute quantification, independent SAH and SAM dilution series were prepared for 8-point calibration curves in the range from 1.56 to 200.0 nM. All analytes were measured in MRM mode with dwell time of 50 ms and cell acceleration voltage of 4 V for all transitions. Fragmentor voltage was optimized for each transition individually and set between 97 and 115 V. The transitions m/z 385.1 \u0026minus;\u0026thinsp;135.9 for SAH with a collision energy of 15 eV and m/z 389.1\u0026ndash;137.9 for SAH-d4 with a collision energy of 21 eV were used as quantifiers. The transitions m/z 385.1\u0026ndash;133.9 (collision energy 15 eV) and 87.9 (collision energy 40 eV) were the qualifiers for SAH, and m/z 389.1\u0026ndash;91.9 with a collision energy of 40 eV for SAH-d4. The transitions m/z 399.2\u0026ndash;250.0 for SAM with a collision energy of 15 eV and m/z 402.2\u0026ndash;249.9 for SAM-d3 with a collision energy of 13 eV were used as quantifiers. The transitions m/z 399.2\u0026ndash;135.9 (collision energy 27 eV) and 96.9 (collision energy 35 eV) were the qualifiers for SAM, and m/z 402.2\u0026ndash;135.8 and 96.9 both with a collision energy of 33 eV for SAM-d3.\u003c/p\u003e\n\u003ch3\u003eTotal protein asymmetrically dimethylated arginines to total arginines (ADMA/Arg) ratio\u003c/h3\u003e\n\u003cp\u003eFor total protein ADMA and arginine analysis, yeast (15 OD\u003csub\u003e600\u003c/sub\u003e units) and Drosophila (10 larvae) samples were suspended in 400 \u0026micro;L of ice-cold methanol and 200 \u0026micro;L of Milli-Q H\u003csub\u003e2\u003c/sub\u003eO, and transferred to Precellys tubes with 1.4 mm diameter zirconium oxide beads. This suspension was homogenized two times for 20 s by Precellys 24 tissue homogenizer at 25\u0026deg; C. Afterwards, the homogenized samples were centrifuged at 10,000 rpm for 30 min at 4\u0026deg; C and stored at -20\u0026deg; C for at least 2 hours. Precipitates were further processed for arginine methylation analyses as described in Zhang \u003cem\u003eet al\u003c/em\u003e 2021 and Habisch \u003cem\u003eet al\u003c/em\u003e 2021\u003csup\u003e44,45\u003c/sup\u003e. Briefly, the precipitates were hydrolyzed with 6 M HCl to obtain amino acids and lyophilized. For NMR analysis, dried samples were re-dissolved in 500 \u0026micro;L NMR buffer (0.08 M Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 5 mM 3-(trimethylsilyl) propionic acid-2,2,3,3-d4 sodium salt (TSP), 0.04 (w/v) % NaN\u003csub\u003e3\u003c/sub\u003e in D\u003csub\u003e2\u003c/sub\u003eO, pH adjusted to 7.4 with 8 M HCl and 5 M NaOH). NMR experiments were carried out as described by Zhang \u003cem\u003eet al\u003c/em\u003e 2021 and Habisch \u003cem\u003eet al\u003c/em\u003e 2021\u003csup\u003e44,45\u003c/sup\u003e. 2D JRES (\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH homo-nuclear J-resolved spectroscopy) spectra were acquired at 310 K on a Bruker 600 MHz Avance Neo spectrometer equipped with a TXI 600S3 probe head using the jresgpprqf pulse sequence (16 scans, size of fid 16,384 (direct dimension F2)/256 (indirect dimension F1), 10,000.00/78.042 Hz spectral width in F2 (chemical shift axis)/F1 (spin\u0026ndash;spin coupling axis), recycle delay 2 s) with presaturation during the relaxation delay to obtain virtually decoupled spectra. Data were processed in Bruker Topspin version 4.3 using the SINE and QSINE window functions (SSB\u0026thinsp;=\u0026thinsp;0) in F2/F1. Fourier transform was performed with 16,384/256 F2/F1 points of the fid. 2D J-resolved experiments were processed using back prediction implemented in the Bruker au program proc_jres.be [32] The JRES spectra were then projected along F2 and exported as 1D NMR spectra. Quantification of arginine and ADMA was carried out by integration of characteristic peaks as described elsewhere \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eProtein extraction\u003c/h3\u003e\n\u003cp\u003eFor yeast, total proteins were extracted from 15 OD\u003csub\u003e600\u003c/sub\u003e units per sample. Per condition, 4 biological replicates were analyzed. For extraction, pellets were resuspended in 200 \u0026micro;L 1.85 M NaOH with 7.5% (v/v) β-mercaptoethanol and incubated for 10 min on ice. 200 \u0026micro;L 50% TCA were added and samples incubated for further 10 min on ice. Samples were then centrifuged for 20 min at 14000 rpm and 4\u0026deg; C, supernatant removed and samples centrifuged again for 2 min. Pellets were washed twice, centrifuged for 5 min at 14000 rpm and 4\u0026deg; C and supernatant removed. Pellets were then resuspended in 100 \u0026micro;L sample buffer (0.156 M Tris-HCl, 5% SDS, 20% glycerol (87%), 0.01% bromophenol blue, 300 \u0026micro;M DTT) and samples were stored at -20\u0026deg; C.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eDrosophila\u003c/em\u003e, total proteins were extracted from 5 migratory L3 larvae per sample. Per condition, 4 biological replicates were analyzed. Per sample, 100 \u0026micro;L extraction buffer (9.75 mL PBS (pH 7.4)\u0026thinsp;+\u0026thinsp;50 \u0026micro;L Triton X-100\u0026thinsp;+\u0026thinsp;200 \u0026micro;L 0.1 M (in acetone) PMSF) and one 5 mm steel ball were added, and the sample homogenized in a Retsch MM40 homogenizer at 30 Hz and 4\u0026deg; C for 2 min. Samples were incubated for additional 10 min at 4\u0026deg; C on overhead rotator SB3 (Stuart). Afterwards, homogenate was transferred to a new vial and centrifuged for 10 min at 6500 g and 4\u0026deg; C in a 5430 R centrifuge (Eppendorf). The supernatant was removed and the pellet was resuspended in 100 \u0026micro;L 0.4 M HCl followed by 5 min incubation on ice. Samples were then centrifuged for 10 min at 6500 g and 4\u0026deg; C. Supernatant was transferred to a new vial and pH was set to 7 with 1 M NaOH. Sample was mixed with equal volume of sample buffer (0.3125 M Tris-HCl, 10% SDS, 40% glycerol, 0.02% bromophenol blue, 600 \u0026micro;M DTT) and stored at -20\u0026deg; C.\u003c/p\u003e\n\u003ch3\u003eRT-qPCR\u003c/h3\u003e\n\u003cp\u003eSAHH primers were selected from Fly RNAi Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.flyrnai.org/flyprimerbank\u003c/span\u003e\u003cspan address=\"https://www.flyrnai.org/flyprimerbank\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), GAPDH primers were selected according to Beaucher \u003cem\u003eet al\u003c/em\u003e 2007 \u003csup\u003e46\u003c/sup\u003e and are listed in \u003cb\u003eTable\u0026nbsp;3\u003c/b\u003e. RNA from migratory L3 larvae ubiquitously expressing either mCherry RNAi or SAHH RNAi was extracted using RNeasy Minikit according to the manufacturer\u0026rsquo;s instructions for animal tissues. RNA concentration was determined by Nanodrop ND-1000 (Peqlab). 1 \u0026micro;g RNA was treated with DNAse I and reverse transcribed using the Invitrogen superscript III One-Step RT-PCR System according to the manufacturer\u0026rsquo;s instructions. For qPCR 8 ng cDNA, 0.1 nmol forward- and reverse primers (\u003cb\u003eTable\u0026nbsp;3\u003c/b\u003e), 10 \u0026micro;L Taq Universal SYRB Green Supermix and water to reach a final reaction volume of 20 \u0026micro;L were used and analyzed on StepOne Plus RT-PCR system (Applied Biosystems). GAPDH was used as a housekeeping gene (see \u003cb\u003eTable\u0026nbsp;3\u003c/b\u003e) for normalization. Results were calculated in Excel (Office Professional Plus 2021; Microsoft) according to Schmittgen \u003cem\u003eet al\u003c/em\u003e 2008 \u003csup\u003e47\u003c/sup\u003eand depicted as 2\u003csup\u003e\u0026minus;ΔCt\u003c/sup\u003e values. All samples were measured in two biological replicates, each biological replicate in two technical replicates.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003ePolyacrylamide gel electrophoresis (PAGE) was performed using Invitrogen NuPAGE (12% BT 1.0) gels. Per well, 5 \u0026micro;L of sample were applied and consecutively adjusted to equalize signals of normalization antibody. After PAGE, proteins were blotted onto polyvinylidene fluoride (PVDF) Immobilon-P Transfer Membrane (0.45 \u0026micro;m). Western blot analysis was performed using the following antibodies with given dilutions for yeast and \u003cem\u003eDrosophila\u003c/em\u003e samples respectively: GAPDH antibody (1:14000 or 1:5000), MMA antibody (1:1000 or 1:200), ADMA antibody (1:1000 or 1:200), H3 antibody (1:5000 or 1:1000), H3K4Me3 antibody (1:1000 or 1:200), H3K36Me2 antibody (1:1000 or 1:200), H3K36Me3 antibody (1:1000 or 1:200), H3K79Me1 antibody (1:1000 or 1:200), H3K79Me2 antibody (1:1000 or 1:200), H3K79Me3 (1:500 or 1:100) and secondary α-rabbit horseradish peroxidase-conjugated antibody (1:15000).\u003c/p\u003e \u003cp\u003eFor immunostaining membranes were blocked in 2% (w/v) skim milk in TST buffer (0.05 M Tris-HCl, 0.15 M NaCl, 0.1% (v/v) Tween-20, pH 7.4) for 1 h at RT. Primary antibody was diluted in 1% (w/v) skim milk in TST buffer and membranes were incubated in the primary antibodies for 1 h at RT. Membranes were then washed 3 times for 5 min in TST buffer at RT. Secondary antibody was diluted in 1% (w/v) skim milk in TST buffer and membranes were incubated in secondary antibody for 1 h at RT. Membranes were then washed 3 times for 5 min in TST buffer at RT. Protein signals were detected using Clarity Western ECL Substrate Kit and captured with a ChemiDoc Touch Imaging System (Bio-Rad). Quantification was done in ImageLab Software Version 6.0.1 (Bio-Rad). Between different immunostainings primary/secondary antibodies were stripped off the membranes by incubation in stripping buffer (2% (v/v) SDS, 0.064 M Tris-HCl, 0.7% (v/v) β-mercaptoethanol) for 20 min at 60\u0026deg; C before the next immunostaining. Experiment was independently repeated once (Supplemental Data 4). Full blots are shown in Supplemental Data 5.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhospholipid methylation\u003c/h3\u003e\n\u003cp\u003eLipids were extracted from either five \u003cem\u003eDrosophila\u003c/em\u003e L3 larvae or 20 OD\u003csub\u003e600\u003c/sub\u003e units in yeast per sample according to protocol described in Matyash et al. 2008 \u003csup\u003e48\u003c/sup\u003e. Briefly, 700 \u0026micro;L MTBE:MeOH (10:3, v/v) and 50 \u0026micro;L internal standard mix (yeast: PC 38:0, PE 34:0, LPC 17:0 and LPE 17:1; \u003cem\u003eDrosophila\u003c/em\u003e: PC 38:0, LPC 17:0 and LPE 17:10.2 mg/mL in MTBE:MeOH (10:3; v/v)) were added to larvae or yeast cell pellets in addition to either a 5 mm steel ball to \u003cem\u003eDrosophila\u003c/em\u003e larvae or 250 \u0026micro;L glass beads to yeast pellets in 2 mL safe-seal tubes. Samples were homogenized in Homogenizer MM40 (Retsch) at 30 Hz and 4\u0026deg; C for 5 min. 200 \u0026micro;L water (MS-grade) were added and mixed in Homogenizer MM40 (Retsch) at 30 Hz and 4\u0026deg; C for further 5 min. Upper phase was collected and second extraction was performed by adding 700 \u0026micro;L MTBE:MeOH (10:3, v/v), mixing in Homogenizer MM40 (Retsch) at 30 Hz and 4\u0026deg; C for another 5 min and upper phase was pooled with the first extraction. Solvent was removed under nitrogen stream at 38\u0026deg; C. For measurement, samples were resuspended in 1 mL isopropanol with 10 mM ammonium acetate, 0.1% formic acid and 8 \u0026micro;M phosphoric acid.\u003c/p\u003e \u003cp\u003ePhospholipids were analyzed by 1290 Infinity UHPLC coupled to a 6470 Triple-Quadrupole mass spectrometer (Agilent) using a BEH C18 column (3.0 mm\u0026times;150 mm; 1.7 \u0026micro;m) with 50\u0026deg; C column temperature, 5 \u0026micro;L injection volume and a constant flow rate of 200 \u0026micro;L/min. H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;10 mM ammonium acetate\u0026thinsp;+\u0026thinsp;0.1% formic acid\u0026thinsp;+\u0026thinsp;8 \u0026micro;M phosphoric acid (A) and isopropanol\u0026thinsp;+\u0026thinsp;10 mM ammonium acetate\u0026thinsp;+\u0026thinsp;0.1% formic acid\u0026thinsp;+\u0026thinsp;8 \u0026micro;M phosphoric acid (B) were used as solvents. 50% solvent A was held for 0.5 min, followed by a change to 80% solvent B over the next 8.5 min and a change to 100% solvent B over next 13 min. 100% solvent B was held for 2.5 min. Re-equilibration was carried out by changing to 50% solvent A within 0.5 min, which was held for 5 min. Total run time was 30 min. All analytes were measured in dynamic MRM mode with optimized individual retention times, retention window of 4 min and cell acceleration voltage of 5 V for all transitions. Fragmentor voltage and collision energy was individually optimized for each lipid class individually. A full list with all analyzed transitions, including individual retention times, fragmentor voltage and collision energy is shown in Supplemental Data 2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure preparations \u0026amp; Statistics\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll diagrams (bar charts, xy-charts and box plots) were prepared in Prism 8 (GraphPad). All collages (yeast- and \u003cem\u003eDrosophila\u003c/em\u003e photographs, western blots and figure blocks) were compiled in PowerPoint (Office Professional Plus 2021; Microsoft). All statistics were calculated in SPSS 27.0 (SPSS Inc.). Data sets were checked for normal distribution by Shapiro Wilk test. If not normally distributed (non-parametric), significances were calculated by Mann-Whitney-U test for single comparisons and Kruskal-Wallis test with Bonferroni correction for multiple testing. If normally distributed (parametric), homogeneity of variance was checked by Levene test. In case of homogenous variances, significances were calculated via Students \u003cem\u003et\u003c/em\u003e-tests for single comparisons and one-way ANOVA with Bonferroni correction for multiple testing or Games-Howell correction for multiple testing for results with non-homogenous variances. All reported p-values are two-sided with an α-level of 0.05. If applicable, parametric significances are marked by asterisks (*) and non-parametric significances are marked by hashtags (#). All results of statistical analyses are shown in Supplemental Data 3.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cp\u003e \u003cb\u003e1. Differential response of SAH and SAM to Hcy supplementation or genetic SAH hydrolase inhibition in yeast and\u003c/b\u003e \u003cb\u003eDrosophila\u003c/b\u003e\u003c/p\u003e \u003c/p\u003e \u003cp\u003eWe have shown previously that Hcy supplementation leads to SAH accumulation in wild type yeast cells (called the yeast Hcy supplementation model in the following). The yeast mutant carrying a deletion of the gene encoding SAHH (∆\u003cem\u003esah1\u003c/em\u003e mutant) accumulates SAH independently of Hcy supplementation (termed the yeast SAHH genetic model in the following) \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Consistent with these published results, SAH accumulates in both the yeast Hcy supplementation and the SAHH genetic model compared to non-supplemented wild type yeast (\u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e). While SAM levels were not increased in Hcy-supplemented wild type yeast, they were significantly elevated in the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant in comparison to non-supplemented wild type (\u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e). This resulted in significantly decreased SAM/SAH ratio in Hcy-supplemented wild type yeast, while the SAM/SAH ratio remained largely unaltered in the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant (\u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e). In line with the yeast results, \u003cem\u003eDrosophila\u003c/em\u003e wild type larvae (Canton-S) grown on food containing 20 mM Hcy (called the fly Hcy supplementation model in the following) showed over 25-fold increase in SAH levels compared to larvae grown on non-supplemented food (\u003cb\u003eFig.\u0026nbsp;1B).\u003c/b\u003e To confirm SAH accumulation to be an universal response of \u003cem\u003eDrosophila\u003c/em\u003e larvae to dietary Hcy supplementation we subjected a genetically unrelated \u003cem\u003ew[1118]\u003c/em\u003e strain to the same food regimen to find similar 25-fold increase in SAH content compared to the same genotype on regular food \u003cb\u003e(Fig. S2\u003c/b\u003e). Moreover, to rule out that SAH accumulation is an indirect effect of dietary supplementation due to an orexigenic effect of Hcy, we performed larval food intake measurements to find no significant difference between larvae on food\u0026thinsp;\u0026plusmn;\u0026thinsp;Hcy (\u003cb\u003eFig. S3\u003c/b\u003e). This SAH accumulation in the fly Hcy supplementation model was similar to the SAH level increase in \u003cem\u003eDrosophila\u003c/em\u003e larvae subject to ubiquitous SAHH gene knockdown mediated by an \u003cem\u003ein vivo\u003c/em\u003e RNAi construct (called the fly SAHH genetic model in the following) compared to control larvae expressing an unrelated RNAi construct (mCherry RNAi) (\u003cb\u003eFig.\u0026nbsp;1B\u003c/b\u003e). The SAHH gene knockdown efficiency is over 70% in this model (\u003cb\u003eFig. S1).\u003c/b\u003e Consistent with the corresponding yeast model, also in the fly Hcy supplementation model, SAM levels were unaffected by the significant increase in SAH (\u003cb\u003eFig.\u0026nbsp;1B\u003c/b\u003e). In contrast to the fly Hcy supplementation model - but in line with the yeast SAHH genetic model \u0026ndash; SAM levels were moderately but significantly increased compared to controls in the fly SAH genetic model (\u003cb\u003eFig.\u0026nbsp;1B\u003c/b\u003e). As a result, the SAM/SAH ratio decreased in both dietary and genetic fly models, compared to the respective controls \u003cb\u003e(Fig.\u0026nbsp;1B\u003c/b\u003e). In addition, while feeding Hcy-containing food to Canton-S larvae did not significantly affect SAM levels, feeding Hcy-containing food to \u003cem\u003ew[1118]\u003c/em\u003e resulted in significant decrease of SAM levels and altogether not as strongly decreased SAM/SAH ratio in response to Hcy supplementation in \u003cem\u003ew[1118]\u003c/em\u003e as compared to Canton-S (\u003cb\u003eFig. S2\u003c/b\u003e and \u003cb\u003e1B\u003c/b\u003e). Collectively, the SAH accumulation response to Hcy supplementation or SAHH inhibition is very similar in yeast and \u003cem\u003eDrosophila\u003c/em\u003e. Differential responses, such as the moderate SAM increase in the fly compared to the yeast genetic model cause different SAH/SAM ratios. These differences can be exploited to correlate phenotypic differences between the organismal models with changes in particular metabolites. Given the role of SAH as competitive inhibitor of SAM-dependent methyltransferases we next compared developmental phenotypes of the fly and yeast models.\u003c/p\u003e \u003cp\u003e \u003cp\u003e \u003cb\u003e2. Growth defects and developmental delay in yeast and\u003c/b\u003e \u003cb\u003eDrosophila\u003c/b\u003e \u003cb\u003emodels of SAH accumulation\u003c/b\u003e\u003c/p\u003e \u003c/p\u003e \u003cp\u003eTight regulation of SAH levels is essential as witnessed by the embryonic lethality of homozygous SAH hydrolase mutant mice \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Similarly, the yeast SAHH genetic model (Δ\u003cem\u003esah1\u003c/em\u003e) exhibits a massive growth defect \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e (and \u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e), which cannot be rescued by methionine supplementation \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. This suggests that SAH accumulation is causal for the growth defect triggered by the block in SAH hydrolysis. We hypothesized that SAH accumulation driven by Hcy supplementation would similarly affect growth in yeast. Accordingly, we measured growth of the Hcy supplementation yeast model in the absence (0 mM) or in the presence of increasing Hcy concentrations (1 mM, 2 mM or 5 mM) in the medium. Indeed, we observed dose-dependent growth inhibition of the yeast Hcy supplementation model. This growth defect is similar to the severe growth defect of the yeast SAHH genetic model grown without Hcy (\u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e). However, while cultures of the SAHH genetic model never reach stationary phase density, the Hcy supplementation model does under all tested Hcy concentrations (\u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e). In accordance, microscopic analysis showed no apparent morphological difference between Hcy-supplemented and non-supplemented yeast cells in stationary phase. In contrast, yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant cells are characterized by multi-budding, altered morphology and aggregation in the same growth phase (\u003cb\u003eFig.\u0026nbsp;2B\u003c/b\u003e), in accordance with previous observations \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo compare the developmental progress and success of the fly Hcy supplementation model to controls on regular food, we assessed the following parameters: the wet weight of individuals at migratory L3 larval stage and the time of/survival rate at two developmental transition states: pupariation (larval to pupal) and hatching (pupal to adult). Migratory L3 larvae grown on Hcy-containing food showed significantly lower body weight compared to larvae grown on food without Hcy supplementation (\u003cb\u003eFig.\u0026nbsp;2C\u003c/b\u003e). Moreover, pupariation and hatching of the fly Hcy supplementation model was substantially delayed compared to controls. The first pupae on Hcy food emerged after 232 h after egg laying (AEL) compared to 136 h on standard food (\u003cb\u003eFig.\u0026nbsp;2D\u003c/b\u003e). Additionally, the last larvae pupariated after 401.5 h AEL on Hcy-containing food compared to 232 h on standard food (\u003cb\u003eFig.\u0026nbsp;2D\u003c/b\u003e). This extended pupariation phase in the population might indicate a Hcy-dependent desynchronization of developmental time by individually different growth retardation. Both aspects, developmental delay and extended developmental phase equally manifest when comparing Hcy-fed flies to controls at pupal hatching to adults (\u003cb\u003eFig.\u0026nbsp;2D\u003c/b\u003e). This indicates that metamorphosis is unaffected by Hcy exposure during larval feeding. Of note, despite the developmental delay, Hcy supplementation did not decrease overall survival rates at pupariation or hatching (\u003cb\u003eFig.\u0026nbsp;2D\u003c/b\u003e). Reduced larval body weight and developmental delay combined with unaffected pupariation and hatching rates appears to be a universal signature of Hcy-fed flies. In support of this, the genotypically unrelated \u003cem\u003ew[1118]\u003c/em\u003e stock display the same set of phenotypes as Canton-S wild type strain in response to Hcy feeding (compare \u003cb\u003eFig. S4\u003c/b\u003e to \u003cb\u003eFig.\u0026nbsp;2C-D\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn accordance with SAH accumulation playing a central role in growth and development, larvae of the fly SAHH genetic model are severely retarded at pupariation and hatching (\u003cb\u003eFig.\u0026nbsp;2E\u003c/b\u003e). The first pupae of the genetic SAHH model only emerged 192 h AEL compared to 144 h in the case of control larvae with normal SAHH activity (\u003cb\u003eFig.\u0026nbsp;2E\u003c/b\u003e). While the onset of the developmental delay is similar in the fly Hcy supplementation and SAHH genetic models, the pupariation phase at the population level is much more extended in the genetic model (compare \u003cb\u003eFig.\u0026nbsp;2D to 2E\u003c/b\u003e). Consistently, not only the growth of larvae subjected to ubiquitous SAHH gene knockdown is retarded but size heterogeneity of larvae of the same chronological age (\u003cb\u003eFig.\u0026nbsp;2F\u003c/b\u003e) also indicated developmental desynchronization. In line with the severe growth phenotype, but unlike the Hcy supplementation model, drastically reduced survival rates by 70% and 85% at pupariation and hatching, respectively, characterize the SAHH genetic model (\u003cb\u003eFig.\u0026nbsp;2E\u003c/b\u003e). These data suggest the growth retardation to result in developmental arrest at larval stages for the majority of individuals in response to global reduction in SAHH activity.\u003c/p\u003e \u003cp\u003eWe asked next, in which organs SAHH activity is particularly relevant for proper development. To this aim, we targeted the SAHH gene knockdown selectively to different organs and scored for the hatching rate of the respective flies compared to controls subject to organ-specific expression of an mCherry RNAi construct (\u003cb\u003eFig.\u0026nbsp;2G\u003c/b\u003e). SAHH gene knockdown in the tracheal system (insect respiratory system) and in muscle significantly reduced the hatching rate as does the ubiquitous knockdown of the gene as shown above (\u003cb\u003eFig.\u0026nbsp;2G\u003c/b\u003e). In contrast, targeted SAHH gene knockdown in the central nervous system (CNS) neurons, the endocrine cells of the gut or the Malpighian tubules (insect kidneys) did not impact hatching (\u003cb\u003eFig.\u0026nbsp;2G\u003c/b\u003e). Of note, organ-specificity of the SAHH knockdown was confirmed by GFP-reporter gene control crosses (\u003cb\u003eFig. S6\u003c/b\u003e). Interestingly, targeted SAHH impairment in the fat body (insect liver and adipose tissue equivalent), does not significantly affect developmental success, but causes developmental delay and developmental phase extension at the population level (\u003cb\u003eFig.\u0026nbsp;2H\u003c/b\u003e) reminiscent to the fly Hcy supplementation model.\u003c/p\u003e \u003cp\u003eCollectively, the fly Hcy supplementation and SAHH genetic models demonstrate that SAH accumulation severely affects \u003cem\u003eDrosophila\u003c/em\u003e growth and development. We also present first evidence for organ-selective roles of SAHH in these processes. Characterization of the lethal phase and the disclosure of the underlying mechanisms of developmental delay and arrest deserve future research attention. It is noteworthy that presumable melanotic tumors were frequently observed in larvae subject to ubiquitous SAHH gene knockdown (\u003cb\u003eFig. S5\u003c/b\u003e), which might contribute to death during development. Given the central role of SAH as inhibitor of SAM-dependent methyltransferases, we asked next whether the methylation profiles of proteins and phospholipids were selectively altered in the yeast and fly models.\u003c/p\u003e \u003cp\u003e \u003cp\u003e \u003cb\u003e3. Hcy supplementation similarly to genetic SAH hydrolase inhibition leads to impaired protein methylation in yeast and\u003c/b\u003e \u003cb\u003eDrosophila\u003c/b\u003e\u003c/p\u003e \u003c/p\u003e \u003cp\u003eBoth elevated SAH and Hcy levels are known to inhibit methylation \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. To analyze whether Hcy supplementation, which leads to SAH accumulation, results in inhibition of protein methylation, we first analyzed total protein asymmetrically dimethylated arginine versus total arginine (ADMA/Arg) ratio in wild type yeast grown in the presence of Hcy and in the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant. Indeed, wild type yeast cultivated in the presence of Hcy, similarly to the Δ\u003cem\u003esah1\u003c/em\u003e mutant, exhibits a decreased ratio of ADMA/Arg in proteins (\u003cb\u003eFig.\u0026nbsp;3A\u003c/b\u003e). Cultivation of wild type yeast in the presence of Hcy also resulted in altered intensities of distinct protein bands detected by an anti-monomethylarginine (MMA)-specific antibody (\u003cb\u003eFig.\u0026nbsp;3B\u003c/b\u003e). While the signals of two protein bands detected by the MMA antibody were increased, two others were decreased (\u003cb\u003eFig.\u0026nbsp;3B\u003c/b\u003e). In contrast, the total protein ADMA/Arg ratio in the fly Hcy supplementation model was not significantly changed in comparison to non-supplemented larvae (\u003cb\u003eFig.\u0026nbsp;3C\u003c/b\u003e). In the fly Hcy supplementation and SAHH genetic models the MMA antibody detected differentially monomethylated proteins, similarly to observations made in yeast (compare \u003cb\u003eFig.\u0026nbsp;3B to 3D\u003c/b\u003e). These observations suggest that the inhibition of protein methylation by SAH may vary depending on specific proteins or residues involved. To directly assess whether different methylation sites are differentially affected, we next used site-specific antibodies to detect distinct methylated residues.\u003c/p\u003e \u003cp\u003eFirst, we analyzed whether Hcy supplementation, which leads to SAH accumulation, leads to inhibition of lysine methylation of histone 3. Indeed, wild type yeast grown in the presence of Hcy exhibited significantly decreased levels of all analyzed tri-methylated sites, H3K4Me3, H3K36Me3, and H3K79Me3, with H3K79Me3 being the most affected one in response to Hcy supplementation (\u003cb\u003eFig.\u0026nbsp;4A\u003c/b\u003e). Similarly, the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant shows significant decrease in tri-methylated H3K36Me3 and H3K4Me3, however not in H3K79Me3 (\u003cb\u003eFig.\u0026nbsp;4A\u003c/b\u003e). Methylation of H3K79Me3 in the Δ\u003cem\u003esah1\u003c/em\u003e mutant is also decreased, however, it is higher than in the Hcy-supplemented wild type yeast (\u003cb\u003eFig.\u0026nbsp;4A\u003c/b\u003e). Hcy supplementation has no effect on methylation of H3K79Me2 and a weak effect on methylation of H3K36Me2 (\u003cb\u003eFig.\u0026nbsp;4A\u003c/b\u003e). In accordance, methylation of H3K79Me2 in the Δ\u003cem\u003esah1\u003c/em\u003e mutant is unaltered, however methylation of H3K36Me2 is significantly decreased (\u003cb\u003eFig.\u0026nbsp;4A\u003c/b\u003e). Noteworthy, Hcy supplementation also led to significantly increased levels of H3K79Me1 as compared to non-supplemented wild type yeast (\u003cb\u003eFig.\u0026nbsp;4A\u003c/b\u003e). In accordance, a non-significant trend to elevated H3K79Me1 levels was also observed in the Δ\u003cem\u003esah1\u003c/em\u003e mutant (\u003cb\u003eFig.\u0026nbsp;4A\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eHistone 3 methylation levels in the \u003cem\u003eDrosophila\u003c/em\u003e Hcy supplementation model were not affected compared to controls in any of the tested histone methylation sites (\u003cb\u003eFig.\u0026nbsp;4B\u003c/b\u003e). In contrast, histone 3 methylation in larvae of the SAHH genetic model was decreased at all the tested sites, with H3K4Me3, H3K36Me2, and H3K79Me1 being significantly decreased, and H3K36Me3 and H3K79Me2 being non-significantly decreased with exception of H3K79Me1, which was unaffected (\u003cb\u003eFig.\u0026nbsp;4B\u003c/b\u003e). We conclude that while SAH inhibits histone 3 lysine methylation both in yeast and \u003cem\u003eDrosophila\u003c/em\u003e, the extent of inhibition varies across different methylation sites. This further supports the hypothesis that SAH accumulation selectively inhibits distinct protein methylation processes.\u003c/p\u003e \u003cp\u003e \u003cp\u003e \u003cb\u003e4. Hcy supplementation similarly to genetic SAH hydrolase inhibition leads to impaired phospholipid methylation in yeast and\u003c/b\u003e \u003cb\u003eDrosophila\u003c/b\u003e\u003c/p\u003e \u003c/p\u003e \u003cp\u003ePhospholipid (PL) methylation is a major consumer of SAM both in yeast and mammals, and requires three-step methylation via monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE) for the synthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Alternatively, PC can be synthesized both in yeast and mammals by the salvage Kennedy pathway using choline \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We have shown previously that PL methylation is sensitive to SAH accumulation as well as Hcy supplementation in choline free-medium \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. However, the medium we used for the cultivation of yeast cells in the current study contained choline. Thus, next we analyzed whether Hcy supplementation impairs PC synthesis by the methylation pathway also in the presence of choline, and whether it leads to a drop in PC levels and interferes with PL metabolism. For this we analyzed the levels of PE, MMPE, DMPE and PC in wild type yeast grown in the presence or absence of Hcy in the medium and in the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant, as well as in larvae of the corresponding \u003cem\u003eDrosophila\u003c/em\u003e Hcy supplementation and SAHH genetic models.\u003c/p\u003e \u003cp\u003eIn yeast, both Hcy-supplemented wild type and the Δ\u003cem\u003esah1\u003c/em\u003e mutant exhibited decreased total PC levels compared to non-supplemented wild type yeast, although only Hcy-supplemented wild type yeast showed significantly decreased PC (\u003cb\u003eFig.\u0026nbsp;5A\u003c/b\u003e). In accordance, total PE levels were significantly increased only in Hcy-supplemented wild type, but not in the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant, in which they were even significantly decreased (\u003cb\u003eFig.\u0026nbsp;5A\u003c/b\u003e). However, both Hcy-supplemented wild type and the Δ\u003cem\u003esah1\u003c/em\u003e mutant displayed significantly increased relative PE levels as well as decreased relative PC levels (\u003cb\u003eFig. S8A\u003c/b\u003e). In accordance, PC/PE ratios were significantly decreased in both Hcy-supplemented wild type as well as in the Δ\u003cem\u003esah1\u003c/em\u003e mutant compared to non-supplemented wildtype, but the Δ\u003cem\u003esah1\u003c/em\u003e mutant showed significantly higher PC/PE ratios compared to Hcy-supplemented wild type (\u003cb\u003eFig.\u0026nbsp;5C\u003c/b\u003e). Both Hcy-supplemented wild type and the Δ\u003cem\u003esah1\u003c/em\u003e mutant displayed significantly decreased total levels of the intermediates of PL methylation, MMPE and DMPE (\u003cb\u003eFig.\u0026nbsp;5A\u003c/b\u003e). It has to be noted, however, that the levels of MMPE and DMPE were stronger decreased in Hcy-supplemented wild type than in the Δ\u003cem\u003esah1\u003c/em\u003e mutant in line with significantly higher PC/PE ratios in the Δ\u003cem\u003esah1\u003c/em\u003e mutant compared to Hcy-supplemented wild type yeast (\u003cb\u003eFig.\u0026nbsp;5A\u003c/b\u003e and \u003cb\u003eC\u003c/b\u003e). Similarly, larvae of the \u003cem\u003eDrosophila\u003c/em\u003e SAHH genetic model exhibited significantly decreased PC, DMPE and MMPE levels compared to controls (\u003cb\u003eFig.\u0026nbsp;5B\u003c/b\u003e). Though PE levels were unaltered, the PC/PE ratio was significantly decreased in this model (\u003cb\u003eFig.\u0026nbsp;5B\u003c/b\u003e and \u003cb\u003eD\u003c/b\u003e). Surprisingly, larvae of the fly Hcy supplementation model contained significantly more PC as well as markedly, but non-significantly elevated PE compared to larvae grown in the absence of Hcy (\u003cb\u003eFig.\u0026nbsp;5B\u003c/b\u003e). Even so, larvae grown on Hcy containing food showed a non-significant trend of decreased PC/PE ratios compared to non-supplemented larvae (\u003cb\u003eFig.\u0026nbsp;5D\u003c/b\u003e). Interestingly, while levels of MMPE were significantly increased, the levels of DMPE were significantly decreased in larvae of the Hcy supplementation model compared to control food (\u003cb\u003eFig.\u0026nbsp;5B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eInhibition of PL methylation alters PC to PE levels and consequently the content of nonbilayer-forming PLs in the membranes which might lead to up-regulation of PL remodeling in response to Hcy/SAH accumulation. Therefore, we analyzed the levels of the lysophospholipids, LPE, LMMPE, LDMPE and LPC, which are central to PL remodeling. Analysis of lysophospholipids in yeast showed that LPE, LMMPE, LDMPE and LPC levels were deregulated following exactly the same pattern as PE, MMPE, DMPE and PC both in Hcy-supplemented wild type and the Δ\u003cem\u003esah1\u003c/em\u003e mutant (\u003cb\u003eFig. S7A\u003c/b\u003e). In particular, LMMPE, LDMPE and LPC were significantly decreased in Hcy-supplemented wild type similarly as MMPE, DMPE and PC (\u003cb\u003eFig. S7A\u003c/b\u003e). In contrast, in the Δ\u003cem\u003esah1\u003c/em\u003e mutant only LPC levels unlike PC levels were significantly decreased, while LPE, LMMPE and LDMPE levels differently to PE, MMPE and DMPE levels were only insignificantly decreased compared to non-supplemented wild type (\u003cb\u003eFig. S7A\u003c/b\u003e). LysoPL molecular species analysis of the \u003cem\u003eDrosophila\u003c/em\u003e genetic model showed the same pattern as the analyzed PLs except for LPC (\u003cb\u003eFig. S7B\u003c/b\u003e). While PC levels are significantly decreased in \u003cem\u003eDrosophila\u003c/em\u003e larvae carrying SAHH RNAi, LPC levels are only insignificantly decreased in response to reduced SAH hydrolase activity (\u003cb\u003eFig. S7B\u003c/b\u003e). Furthermore, in \u003cem\u003eDrosophila\u003c/em\u003e dietary model the levels of analyzed lysoPLs molecular species did not change compared to controls, in contrast to significant elevation of MMPE and PC and significant decrease of DMPE in larvae fed Hcy-containing food compared to larvae on control diet (\u003cb\u003eFig. S7B\u003c/b\u003e). It has to be noted that the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant exhibited a significant decrease of relative PC levels and \u003cem\u003eDrosophila\u003c/em\u003e larvae carrying SAHH RNAi showed significant increase of relative PE and a decrease of relative LPE levels (\u003cb\u003eFig. S8A-D).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe conclude that Hcy even in the presence of choline leads to inhibition of PL methylation, altered PC/PE ratio and PL remodeling in wild type yeast similarly to the yeast and \u003cem\u003eDrosophila\u003c/em\u003e SAHH genetic models., however, the extent of perturbations varies depending on the secondary effects. This further supports the hypothesis that inhibition of PL methylation by SAH accumulation along with inhibition of protein methylation is one of the central mechanisms how SAH leads to pathological consequences. Altogether, developed yeast and \u003cem\u003eDrosophila\u003c/em\u003e dietary and genetic models of HHcy-associated SAH accumulation show that methylation inhibition is an important mechanism how elevated Hcy can lead to pathological consequences. Functional characterization of evolutionary conserved Hcy/SAH-dependent methylation targets in \u003cem\u003eDrosophila\u003c/em\u003e and yeast will reveal mechanisms of SAH toxicity which may be operative in HHcy-associated human pathologies.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCVD, the leading cause of death worldwide, can only be to 50% explained by established risk factors including cholesterol \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Hcy, a sulfur containing amino acid involved in methylation metabolism, is an independent risk factor for the development of atherosclerosis, increases CVD risk in combination with hypercholesterolemia \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, is linked to cardiac pathologies \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and further human diseases including neurological disorders, fatty liver disease, insulin resistance, and cancer as well as to CVD and all-cause mortality \u003csup\u003e11\u0026ndash;14,16\u0026minus;18\u003c/sup\u003e. In accordance with the central role of Hcy, CKD, which is associated with highly elevated Hcy, is also linked to dramatically increased CVD as well as increased all-cause mortality \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Elevated Hcy is found in 5\u0026ndash;10% of the general population, in up to 30% of the elderly and in as many as 70% of men over 80 years of age \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In most cases it is associated with deficiency of vitamins required for Hcy degradation \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and is also linked to physical inactivity \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, low skeletal mass \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, high fat diet \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and obesity \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Attempts to decrease CVD and neurological outcomes associated with elevated Hcy by lowering plasma Hcy levels were disappointing \u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e indicating necessity to understand detailed mechanisms downstream Hcy leading to pathological consequences.\u003c/p\u003e \u003cp\u003eMethylation of different macromolecules is an important regulatory mechanism involved in a wide variety of cellular processes \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and its dysregulation is increasingly recognized as a risk factor for CVD development\u003csup\u003e\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. CVD has been linked to increased levels of SAH, a universal strong product inhibitor of SAM-dependent methylation \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Furthermore, both elevated SAH and Hcy levels are known to inhibit methylation, however, detailed mechanistic understanding of their downstream signaling cascades leading to CVD is largely missing \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Further, lowering of elevated plasma Hcy levels in addition to the inability to reduce associated clinical outcomes \u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e does not decrease SAH levels, indicating a central role of SAH in Hcy-associated pathology \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our recent publication we showed that Hcy independently of cholesterol alters aortic wall structure and functionality as well as leads to deregulation of lipoprotein metabolism and interferes with organization of elastin, which was reported to be linked to progressive aortic stiffening and all-cause mortality in CKD patients \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Furthermore, elevation of Hcy levels in rabbits fed diet blocking Hcy degradation was associated with a drop of total protein methylated arginine as well as decreased levels of creatine, which requires methylation for its synthesis \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, suggesting involvement of deficient methylation in pathological consequences associated with elevated Hcy.\u003c/p\u003e \u003cp\u003eInhibition of methylation is an overlooked mechanism how elevated Hcy can lead to pathological consequences. More than 200 SAM-dependent methyltransferases in humans that transfer the methyl group of SAM to numerous cellular acceptors including nucleic acids, proteins (including histones) as well as low molecular weight compounds that require SAM for their synthesis, e.g. phospholipids and creatine are centrally involved in many processes in the organism \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. An accumulation of SAH in response to elevation of Hcy levels may inhibit respective enzymes and interfere with numerous crucial methylation-dependent processes including epigenetic regulation of gene expression, signaling, lipid and energy metabolism. Understanding sensitivity of methylation-dependent processes to Hcy and SAH accumulation will help to understand pathological consequences associated with elevated Hcy.\u003c/p\u003e \u003cp\u003eSAH is degraded to Hcy and adenosine in a reversible reaction catalyzed by SAH hydrolase \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. SAH hydrolase offers the sole possibility of SAH degradation in mammals and is very highly evolutionary conserved exhibiting over 70% of identity between yeast and human orthologs \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. High evolutionary conservation of SAH hydrolase as well as of methylation metabolism \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e suggest their central role in the regulation of cellular function.\u003c/p\u003e \u003cp\u003eDeletion of SAH hydrolase in yeast is lethal unless a yeast-specific sulfur assimilation pathway is active \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Similarly, interference with SAH hydrolase locus in mice is embryonically lethal \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Here, we developed a dietary \u003cem\u003eDrosophila\u003c/em\u003e model of HHcy and compared it with a genetic \u003cem\u003eDrosophila\u003c/em\u003e model of SAH-mediated methylation inhibition and corresponding yeast models to reveal evolutionarily conserved Hcy/SAH-sensitive methylation patterns. Feeding \u003cem\u003eDrosophila\u003c/em\u003e larvae a Hcy-containing diet or growing yeast on Hcy-supplemented medium similarly to blocking SAH degradation leads to SAH accumulation, developmental delay and growth defects. Surprisingly, while Hcy-supplementation of wild type yeast resulted only in SAH accumulation, in the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant both SAH and SAM accumulated leading to virtually unaltered SAM/SAH ratio. Similarly, in \u003cem\u003eDrosophila\u003c/em\u003e carrying SAHH RNAi but not in \u003cem\u003eDrosophila\u003c/em\u003e fed Hcy-containing food SAM levels were significantly increased leading altogether to a much smaller drop of SAM/SAH ratio in genetic compared to dietary \u003cem\u003eDrosophila\u003c/em\u003e model.\u003c/p\u003e \u003cp\u003eBlock of cystathionine \u0026szlig;-synthase in CBS mice leads to a massive decrease in glutathione levels \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e suggesting that also deletion of SAH hydrolase in the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant or its downregulation in \u003cem\u003eDrosophila\u003c/em\u003e larvae carrying SAHH RNAi, which is likely to be associated with a decrease in Hcy levels, may lead to glutathione depletion. CBS is allosterically activated by SAM \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Moreover, it was shown that during switch from methionine to Hcy-containing medium, CBS protein levels are drastically decreased and binding of SAM stabilizes CBS against degradation \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Thus, it appears likely that both the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant and \u003cem\u003eDrosophila\u003c/em\u003e larvae carrying SAHH RNAi require (elevated) SAM to maintain redox capacity.\u003c/p\u003e \u003cp\u003eAs observed previously, interference with SAH hydrolase in yeast leads to altered morphology and impaired growth \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Similarly, we observed a massive growth defect of the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant as well as its altered morphology. Hcy-supplementation resulted in the gradual inhibition of yeast growth, in accordance with a detrimental role of SAH. Aggregation of the Δ\u003cem\u003esah1\u003c/em\u003e mutant at the end of cultivation in contrast to Hcy-supplemented wild type yeast cells will be studied in the future.\u003c/p\u003e \u003cp\u003eIn accordance with the growth defect of wild type yeast cultivated in the presence of Hcy, \u003cem\u003eDrosophila\u003c/em\u003e larvae fed Hcy-containing food exhibited developmental delay as shown by delayed pupariation and hatching rates. Moreover, larvae fed Hcy-containing food exhibited significantly lower weight, in accordance with the detrimental impact of SAH on cellular functionality. Noteworthy, despite developmental delay survival of larvae during pupariation as well as survival of pupae during hatching was unaffected similarly to unaffected survival of yeast cells despite gradual inhibition of growth in Hcy-containing medium.\u003c/p\u003e \u003cp\u003eIn contrast, \u003cem\u003eDrosophila\u003c/em\u003e carrying SAHH RNAi exhibited massively reduced survival both at pupariation as well as at hatching in addition to drastically delayed pupariation and hatching rates. Additionally, the size of \u003cem\u003eDrosophila\u003c/em\u003e carrying SAHH RNAi was markedly decreased and these larvae in addition to delayed development, poor viability and reduced size exhibited black spots reminiscent of melanotic tumors. Formation of melanized bodies can occur for instance through dysregulated expression of Hox genes \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Moreover, it has been reported that misexpression of various Hox genes leads not only to formation of melanized (pseudo-) tumors, but also to pupal lethality in \u003cem\u003eDrosophila\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, which is in line with our observation of strongly decreased survival rates, especially at pupal stage, in \u003cem\u003eDrosophila\u003c/em\u003e larvae expressing SAHH RNAi. Noteworthy, expression of Hox genes is regulated via H3 lysine methylation, which is highly conserved across animal species \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Furthermore, H3K79 methylation is critical for mammalian HOX gene expression \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlso, other nuclear co-regulators were shown to be linked to deregulated methylation. Monomethylated Yap by Set7 methyltransferase at lysine 494 controls an evolutionarily conserved Hippo signaling pathway that regulates organ size and function in mice \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Of note, the Hippo pathway has been also linked to vascular smooth muscle cell proliferation during vascular remodeling in CVD \u003csup\u003e\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Similarly, arginine and lysine methylation of transcriptional coactivator BRD4 that has a central role in regulating transcription and genome stability was shown to be linked to transcription deregulation and DNA repair \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e as well as negative regulation of genes that are involved in translation and total mRNA translation inhibition in mammalian cells \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, respectively. Furthermore, arginine methylation of mammalian DEAD-box family RNA helicase, DDX5, was shown to regulate resolution of aberrant transcription-associated RNA:DNA hybrid (R-loop) formation, which often causes catastrophic conflicts during replication, resulting in DNA double-strand breaks and genomic instability \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Moreover, SAH hydrolase was shown to be essential for cyclic H3K4 trimethylation, genome-wide recruitment of BMAL1 to chromatin and subsequent circadian transcription promoting rhythmic H3K4 trimethylation and cyclic BMAL1 recruitment to target genes in mice \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo understand how Hcy/SAH affect methylation of different molecules we next analyzed the total protein ADMA/Arg ratios by NMR as well as MMA methylation by western blotting. Indeed, both Hcy-supplementation of wild type yeast as well as the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant exhibited significantly decreased ADMA/Arg ratios. These findings suggest that SAH inhibits protein methylation. However, our data suggest that not all protein methylations are affected to the same extent: the signals of two specific yeast protein bands detected by an MMA-specific antibody were increased; one can speculate that the methylation reactions mediating monomethylation of the corresponding proteins are not (or less) inhibited by SAH or alternatively decreased levels of dimethylated arginines leads to relative increase of MMRs. Similarly, comparing the two \u003cem\u003eDrosophila\u003c/em\u003e models we also observed decreased as well as increased signals of specific (but unidentified) fly proteins detected by an MMA-specific antibody. However, in contrast to yeast, \u003cem\u003eDrosophila\u003c/em\u003e fed Hcy-containing food exhibited unaltered ADMA/Arg ratio. Of note, only one of nine protein arginine methyltransferases identified in \u003cem\u003eDrosophila\u003c/em\u003e is abundantly expressed in larvae with next two being moderately expressed in this developmental stage \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMoreover, we observed different extents of inhibition of different types of lysine methylations both in yeast and \u003cem\u003eDrosophila\u003c/em\u003e. In particular, in yeast histone 3 (H3) K36 trimethylation was more inhibited in response to Hcy than dimethylation at the same site. Similarly, H3K79 mono-, di- and trimethylation were differently altered in response to Hcy and SAH both in yeast and \u003cem\u003eDrosophila\u003c/em\u003e. This suggests that SAH has different inhibitory capacity for different protein methylation reactions. In line with this observation is a previous report that SAH inhibits protein methylation at much lower concentrations than DNA methylation\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHistone methylation is a major consumer of methyl groups particularly in the absence of PL methylation \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e, affects metabolism independently of transcriptional regulation \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e and together with histone acetylation influences cellular metabolism \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. H3K4 and H3K36 methylations are catalyzed by SET domain lysine methyltransferases, however H3K79 methylation is catalyzed by a lysine methyltransferase without a SET domain (Dot1 in yeast or Grappa in \u003cem\u003eDrosophila\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. A distributive mechanism and highly diverged catalytic properties reported for Dot1 methyltransferases may explain the different inhibitory potential of Hcy and SAH in our models toward H3K79 compared to histone lysine methylation sites methylated by SET domain containing methyltransferases \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. Interestingly, H3K36 methylation is regulated by demethylation of PP2A, which activates demethylation of H3K36 through hyperphosphorylation of H3K36 demethylase Rph1 in yeast \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Moreover, in accordance with observed elevated SAM levels in the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant (as well as in \u003cem\u003eDrosophila\u003c/em\u003e SAHH RNAi) yeast PP2A mutants as well as yeast mutant lacking H3K36 demethylase Rph1 spares SAM by limiting histone methylation and exhibit elevated SAM levels \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSynthesis of PC via three step methylation of PE is another major consumer of SAM \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e,\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. In contrast to mammals the first methylation from PE to MMPE in yeast is catalyzed by Cho2 and further methylations to DMPE and PC are catalyzed by Opi3 \u003csup\u003e70\u003c/sup\u003e. Deficiencies of either of these enzymes were reported to lead to accumulation of SAM and increased SAM to SAH ratios in yeast \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. In accordance with inhibition of Cho2 and Opi3 we observed drastically decreased MMPE, DMPE and PC levels, while PE accumulated, resulting in drastically decreased PC/PE ratios in wild type yeast supplemented with Hcy. Similar effects can be seen in respective lysoPLs, with a significant elevation of LPE and significantly decreased LMMPE, LDMPE and LPC in response to Hcy supplementation in wild type yeast.\u003c/p\u003e \u003cp\u003eIn contrast, despite highly increased SAH levels, block of SAH hydrolase in the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant did not lead to decreased overall SAM to SAH ratio and resulted in less drastic decrease in PE, MMPE, DMPE and PC levels as compared to wild type yeast supplemented with Hcy. This is mirrored in PC/PE ratio, which is lower compared to untreated wild type, but higher compared to wild type yeast supplemented with Hcy. Furthermore, no elevation of LPE and even an increase in LMMPE were observed in wild type yeast supplemented with Hcy, while LDMPE and LPC were decreased, however to a lower degree. This suggests further mechanisms are likely to be operative in the yeast Δ\u003cem\u003esah1\u003c/em\u003e mutant compared to wild type yeast supplemented with Hcy.\u003c/p\u003e \u003cp\u003eIn contrast to yeast, in mammals PL methylation is catalyzed by a single enzyme, phosphatidylethanolamine methyltransferase (PEMT), catalyzing all three methylation steps \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eDrosophila\u003c/em\u003e exact mechanisms of PL methylation are still unknown, but PL methyltransferase activity has been detected in \u003cem\u003eDrosophila\u003c/em\u003e in the past \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Increased PE and decreased DMPE levels as well as decreased PC/PE ratio in \u003cem\u003eDrosophila\u003c/em\u003e larvae fed Hcy-supplemented food further suggest PL methylation in \u003cem\u003eDrosophila.\u003c/em\u003e Similarly, \u003cem\u003eDrosophila\u003c/em\u003e carrying SAHH RNAi exhibited drastically decreased MMPE, DMPE and PC levels and slightly increased PE levels as well as a massively decreased PC/PE ratio. Unexpectedly, we also observed increased MMPE and PC levels in \u003cem\u003eDrosophila\u003c/em\u003e larvae fed Hcy-supplemented food but not in \u003cem\u003eDrosophila\u003c/em\u003e carrying SAHH RNAi. Similar to major changes in PL methylation pathway being observed for \u003cem\u003eDrosophila\u003c/em\u003e carrying SAHH RNAi, we found a decrease in all lysoPLs with strongest decrease in LMMPE and LDMPE levels in the genetic \u003cem\u003eDrosophila\u003c/em\u003e model, while Hcy supplementation of \u003cem\u003eDrosophila\u003c/em\u003e larvae did not lead to any change in lysoPLs. Interestingly, it was also reported that PEMT-deficiency in mice results in inability to gain weight even on high fat diet, leading to significantly decreased body weight \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, comparison of \u003cem\u003eDrosophila\u003c/em\u003e and yeast models of Hcy-associated SAH-mediated methylation inhibition showed similar as well as different Hcy/SAH-sensitive methylation patterns. Hcy supplementation or blocking of SAH degradation in both model organisms lead to impaired protein and PL methylation. While in yeast Hcy/SAH accumulation leads to decreased total protein ADMA/Arg ratio, impaired MMA, histone and PL methylation, in \u003cem\u003eDrosophila\u003c/em\u003e fed Hcy-containing food total protein ADMA/Arg ratio was unaffected, while MMA, histone and PL methylation, similarly to yeast, was sensitive to Hcy/SAH accumulation in \u003cem\u003eDrosophila\u003c/em\u003e fed Hcy-containing food or carrying SAHH RNAi. Furthermore, both Hcy supplementation as well as genetically triggered SAH accumulation are linked to developmental delay and growth defect both in yeast and \u003cem\u003eDrosophila\u003c/em\u003e models. \u003cem\u003eDrosophila\u003c/em\u003e larvae carrying SAH hydrolase RNAi in addition to development delay, poor viability and reduced size exhibited black spots reminiscent of melanotic tumors. Employing yeast and \u003cem\u003eDrosophila\u003c/em\u003e genetic screens will reveal evolutionary conserved Hcy/SAH-dependent mechanisms with high potential relevance for HHcy-associated human pathologies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank\u0026nbsp;Fred van Leeuwen\u0026nbsp;for providing H3K79 antibodies. The authors thank Raphael K\u0026uuml;hnlein and Lydia Misslinger for excellent technical assistance in the context of organ-specific developmental analysis and fly food preparation, respectively.\u0026nbsp;Stocks obtained from the Bloomington \u003cem\u003eDrosophila\u003c/em\u003e Stock Center (NIH P40OD018537), from the Vienna \u003cem\u003eDrosophila\u0026nbsp;\u003c/em\u003eResource Center (VDRC,\u0026nbsp;\u003ca href=\"http://www.vdrc.at\"\u003ewww.vdrc.at\u003c/a\u003e) and\u0026nbsp;from the KYOTO \u003cem\u003eDrosophila\u003c/em\u003e Stock Center (Kyoto Institute of Technology)\u0026nbsp;were used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSources of funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded in whole, or in part, by the Austrian Science Fund (FWF) [Grant-DOIs: 10.55776/P31105 and 10.55776/P33672 to OT and grant DOIs 10.55776/P27996 and 10.55776/DOC50 to BP]. Further, this work was supported by the Doctoral Academy of the University of Graz (Consortium Molecular Metabolism MOBILES), the Integrative Metabolism Research Center Graz, Austrian Infrastructure Program 2016/2017, the BioTechMed-Graz Flagship project DYNIMO, and the Province of Styria (Zukunftsfonds, doc.fund)\u0026nbsp;and the City of Graz.\u0026nbsp;The authors acknowledge the financial support by the University of Graz.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMSB\u0026nbsp;\u0026ndash; Investigation, Data curation, Methodology, Formal analysis, Validation, Visualization, Software, Resources (\u003cem\u003eDrosophila\u003c/em\u003e \u0026amp; yeast experiments, analytics), Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing; HH \u0026amp; TM\u0026nbsp;\u0026ndash; Investigation, Data curation, Methodology, Formal analysis, Validation, Visualization, Software, Resources (NMR of global ArgMet),\u0026nbsp;Writing \u0026ndash; review and editing; JH \u0026amp; MM\u0026nbsp;\u0026ndash; Methodology, Formal analysis (yeast experiments),\u0026nbsp;Writing \u0026ndash; review and editing; ZN \u0026ndash;\u0026nbsp;Investigation, Data curation, Methodology, Formal analysis, Validation, Visualization, Software, Resources (establishment of\u003cem\u003e\u0026nbsp;Drosophila\u0026nbsp;\u003c/em\u003edietary model), Writing \u0026ndash; review and editing; HW\u0026nbsp;\u0026ndash; Methodology, Formal analysis, Visualization, Software, Resources (microscopy),\u0026nbsp;Writing \u0026ndash; review and editing; GNR\u0026nbsp;\u0026ndash; Data curation, Methodology, Software, Resources (analytic),\u0026nbsp;Writing \u0026ndash; review and editing;\u0026nbsp;BR\u003csup\u003e\u0026nbsp;\u0026nbsp;\u003c/sup\u003e\u0026ndash; Conceptualization, Investigation, Methodology, Supervision (yeast subproject: western blots),\u0026nbsp;Writing \u0026ndash; review and editing;\u0026nbsp;RPK\u0026nbsp;\u0026ndash; Conceptualization, Investigation, Methodology, Supervision, Funding acquisition, Project administration (\u003cem\u003eDrosophila\u003c/em\u003e subproject),\u0026nbsp;Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing; OT\u003csup\u003e\u0026nbsp; \u0026nbsp;\u003c/sup\u003e\u0026ndash;\u0026nbsp;\u003csup\u003e\u0026nbsp;\u003c/sup\u003eConceptualization, Investigation, Methodology, Supervision, Funding acquisition, Project administration (yeast subproject and Hcy-associated models of SAH accumulation),\u0026nbsp;Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors of the manuscript declare no conflicts of interests.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHcy \u0026ndash; homocysteine, SAH \u0026ndash; \u003cem\u003eS\u003c/em\u003e-adenosyl-\u003cem\u003eL\u003c/em\u003e-homocysteine, SAM \u0026ndash; \u003cem\u003eS\u003c/em\u003e-adenosyl-\u003cem\u003eL\u003c/em\u003e-methionine, SAHH \u0026ndash; SAH hydrolase, HHcy \u0026ndash; hyperhomocysteinemia, CVD \u0026ndash; cardiovascular disease, CKD \u0026ndash; chronic kidney disease, ADMA/Arg \u0026ndash; ratio of total protein asymmetrically dimethylated arginines versus arginines, MMA \u003cem\u003e\u0026ndash;\u003c/em\u003e monomethylarginine, H3 \u0026ndash; histone 3, PL \u0026ndash; phospholipid, PE \u0026ndash; phosphatidylethanolamine, PC \u0026ndash; phosphatidylcholine, MMPE \u0026ndash; monomethylphosphatidylethanolamine, DMPE \u0026ndash;dimethylphosphatidylethanolamine, LPE \u0026ndash; lysophosphatidylethanolamine, LPC \u0026ndash; lysophosphatidylcholine, LMMPE \u0026ndash; lysomonomethylphosphatidylethanolamine, LDMPE \u0026ndash; lysodimethylphosphatidylethanolamine, AEL \u0026ndash; after egg laying\u003c/p\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLefkowitz RJ, Willerson JT (2001) Prospects for cardiovascular research. 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J Biol Chem 285:22403\u0026ndash;22413. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1074/jbc.M110.108514\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M110.108514\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eTable 1: Yeast strains used in this study\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"714\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3697%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.9552%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenotype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.6751%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3697%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.9552%;\"\u003e\n \u003cp\u003e\u003cem\u003eMAT a his3\u003c/em\u003e\u0026Delta;\u003cem\u003e1 leu2\u003c/em\u003e\u0026Delta;\u003cem\u003e0 ura3\u003c/em\u003e\u0026Delta;\u003cem\u003e0\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.6751%;\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5900771/\"\u003eVisram et al. (2018)\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3697%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026Delta;\u003cem\u003esah1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.9552%;\"\u003e\n \u003cp\u003e\u003cem\u003eMAT a his3\u003c/em\u003e\u0026Delta;\u003cem\u003e1 leu2\u003c/em\u003e\u0026Delta;\u003cem\u003e0 ura3\u003c/em\u003e\u0026Delta;\u003cem\u003e0 sah1::kanMX4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.6751%;\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5900771/\"\u003eVisram et al. (2018)\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eTable 2: Drosophila fly lines used in this study\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"714\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTrivial Name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFunction\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenotype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInternal Stock No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource Stock No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCanton-S\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003ewild type control stock\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003eRKF 1679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003eBDSC* 64349\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ew[1118]\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003ewhite mutant control stock\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cem\u003ew\u003csup\u003e1118\u003c/sup\u003e; +/+; +/+;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003eRKF 1084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003eVDRC** 6000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUbiquitous\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003eUbiquitous driver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cem\u003ew*; +/+; P{GAL4-da.G32\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003eRKF 908\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003e\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/7606787/\"\u003eWodarz et al. (1995)\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRNAi ctrl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003emCherry RNAi effector\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cem\u003ey[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=VALIUM20-mCherry}attP2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003eRKF 1645\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003eBDSC 35785\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSAHH RNAi\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003eSAH hydrolase RNAi effector\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cem\u003ew[1118] y[1] fl. Sc[*] fl. V[1] sev[21] fl. ; P{y[+t7.7] v[+t1.8]=TriP.HMS05799}attP40;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003eRKF 2416\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003eThis work based on BDSC\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003ebased on BDSC 67848\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGFP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003eGFP reporter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cem\u003e+; P{w[+mC]=UAS-Stinger}2; + / +\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003eRKF 1171\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003e\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/11056799/\"\u003eBarolo et al. (2000)\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCNS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003eCentral nervous system-specific driver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cem\u003ew*; +/+; P{w[+mW.hs]=GAL4-Nrv2-3} P{w[+m*]UAS-GFP}\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003eRKF 211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003e\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/10468627/\"\u003eSun et al. (1999)\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGut\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003eGut enterocyte-specific driver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cem\u003ew[1118]; P{w[+mC]=mex1-GAL4.2.1}10-8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003eCHF 2396\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003eBDSC 91368\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTracheal System\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003eTracheal system-specific driver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cem\u003ew*; P{UAS-GFP::nLacZ} / CyO float; P{btl-GAL4} / TM3 Sb* Ser*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003eRKF 1912\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003eKSC*** 109132\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMuscle\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003eMuscle-specific driver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cem\u003emef2-Gal4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003eIPF 2174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003eBDSC 27390\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMalpighian Tubules\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003eMalpighian tubules-specific driver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cem\u003e+/+; UO-Gal4/CyO float; +/+\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003eJRF 1254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003e\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/20060894/\"\u003eTerhzaz et al. (2010)\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFat body\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003eFat body-specific driver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 0%;\"\u003e\n \u003cp\u003e\u003cem\u003ew*; +/+; P{Lpp-GAL4.B}/TM3, P{w[+mC]=ActGFP}JMR2, Ser[1]\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28.6713%;\"\u003e\n \u003cp\u003eRKF 1582\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.7622%;\"\u003e\n \u003cp\u003eThis work is based on \u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/20685986/\"\u003eBrankatschk and Eaton (2010)\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.5664%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e* = Bloomington Drosophila Stock Centre, ** = Vienna Drosophila Resource Center, *** = KYOTO Stock Center\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eTable 3: \u003cem\u003eDrosophila\u003c/em\u003e RT-qPCR primers\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"714\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 20.5882%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTarget\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.1092%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eForward primer sequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3697%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReverse primer sequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.9328%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 20.5882%;\"\u003e\n \u003cp\u003eSAHH (exon spanning)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.1092%;\"\u003e\n \u003cp\u003e5\u0026rsquo;- AGT\u0026nbsp;ACG GCC CAT CTA AGC C - 3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3697%;\"\u003e\n \u003cp\u003e5\u0026apos; - CGG CAG CAT TAT CCT GGG T - 3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.9328%;\"\u003e\n \u003cp\u003eFly RNAi Database, identifier PP25254\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 20.5882%;\"\u003e\n \u003cp\u003eSAHH (both isoforms)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.1092%;\"\u003e\n \u003cp\u003e5\u0026apos; - AGC CCC TGA ACA TGA TCC TG - 3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3697%;\"\u003e\n \u003cp\u003e5\u0026apos; - CGA CCC TCC TTG AAC ATC TTG T - 3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.9328%;\"\u003e\n \u003cp\u003eFly RNAi Database, identifier PP36991\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 20.5882%;\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.1092%;\"\u003e\n \u003cp\u003e5\u0026apos; - GTC GGG CTT GTA GGC ATC C - 3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.3697%;\"\u003e\n \u003cp\u003e5\u0026apos; - AGG CAT CCA CTC ACT TGA AGG - 3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.9328%;\"\u003e\n \u003cp\u003e\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/17239363/\"\u003eBeaucher et al. (2007)\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Graz","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"S-adenosyl-L-homocysteine, homocysteine, protein and phospholipid methylation, yeast, Drosophila","lastPublishedDoi":"10.21203/rs.3.rs-5144569/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5144569/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eS\u003c/em\u003e-adenosyl-\u003cem\u003eL\u003c/em\u003e-homocysteine (SAH), the product inhibitor of \u003cem\u003eS\u003c/em\u003e-adenosyl-\u003cem\u003eL\u003c/em\u003e-methionine (SAM)-dependent methyltransferases, and its degradation product homocysteine (Hcy) are evolutionarily conserved master regulators of methylation metabolism. They can affect more than 200 methyltransferases in humans interfering with numerous methylation-dependent processes. Hyperhomocysteinemia (HHcy), characterized by elevated Hcy levels in the blood, is an independent risk factor for atherosclerosis and a strong predictor of cardiovascular mortality, yet, the mechanisms by which elevated Hcy contributes to pathological consequences remain poorly understood.\u003c/p\u003e \u003cp\u003eHere we developed a \u003cem\u003eDrosophila\u003c/em\u003e dietary HHcy model, as well as a genetic SAH hydrolase (SAHH) \u003cem\u003ein vivo\u003c/em\u003e knockdown model and compared them to corresponding yeast models to reveal evolutionarily conserved developmental effects and methylation pattern changes. Feeding \u003cem\u003eDrosophila\u003c/em\u003e a Hcy-containing diet or growing yeast on Hcy-supplemented medium similarly to genetically blocking SAH degradation leads to SAH accumulation, developmental delay and growth defects. Furthermore, Hcy supplementation or genetically induced SAH accumulation leads to impaired protein and phospholipid (PL) methylation in both model organisms. While total protein arginine methylation is significantly decreased in wild type yeast grown in presence of Hcy or in the ∆\u003cem\u003esah1\u003c/em\u003e yeast mutant, it is unaffected in \u003cem\u003eDrosophila\u003c/em\u003e larvae raised on Hcy-supplemented diet. In contrast, histone methylation is affected in \u003cem\u003eDrosophila\u003c/em\u003e and yeast, but exhibited differences in responses of particular histone methylation sites. Similarly, PL methylation was reduced in both organisms and resulted in deregulation of lysoPL metabolism suggesting PL remodeling. Functional characterization of evolutionary conserved Hcy/SAH-dependent methylation targets in \u003cem\u003eDrosophila\u003c/em\u003e and yeast will reveal mechanisms of SAH toxicity which may be operative in HHcy-associated human pathologies.\u003c/p\u003e","manuscriptTitle":"Accumulation of S-adenosyl-L-homocysteine impairs methylation and development in Saccharomyces cerevisiae and Drosophila melanogaster","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-24 15:51:59","doi":"10.21203/rs.3.rs-5144569/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"759c9f94-64f1-4b08-ab7e-b06913fdf820","owner":[],"postedDate":"September 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38133219,"name":"General Biochemistry"}],"tags":[],"updatedAt":"2024-09-24T15:51:59+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-24 15:51:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5144569","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5144569","identity":"rs-5144569","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}