ATRX safeguards cellular identity during C. elegans development

ATRX safeguards cellular identity during C. elegans development | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results ATRX safeguards cellular identity during C. elegans development Janie Olver , Mariya Shtumpf , Karim Hussain , Stephen Methot , View ORCID Profile Peter Sarkies , View ORCID Profile Helder Ferreira doi: https://doi.org/10.1101/2025.03.11.641662 Janie Olver 1 School of Biology, University of St Andrews , St Andrews, UK 2 Currently at Rare and Imported Pathogens Laboratory, UK Health Security Agency , Porton Down, Salisbury, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mariya Shtumpf 1 School of Biology, University of St Andrews , St Andrews, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Karim Hussain 1 School of Biology, University of St Andrews , St Andrews, UK 3 Currently at Ingenza Ltd , Midlothian, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stephen Methot 4 Friedrich Miescher Institute for Biomedical Research , Basel, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Peter Sarkies 5 Department of Biochemistry, University of Oxford , Oxford, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Peter Sarkies Helder Ferreira 1 School of Biology, University of St Andrews , St Andrews, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Helder Ferreira For correspondence: hcf2{at}st-andrews.ac.uk Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract ATRX is a member of the SWI/SNF family of ATP-dependent chromatin remodellers. In humans, loss of ATRX function leads to ATRX syndrome, a neurodevelopmental disorder. ATRX mutation in human cell lines is associated with multiple phenotypes including activation of the alternative lengthening of telomere (ALT) pathway, upregulation of retrotransposons and increased sensitivity to replication stress. However, the principal role of ATRX and the reason why its mutation causes such diverse phenotypes is currently unclear. To address this, we studied the role of ATRX in the model organism Caenorhabditis elegans . We find that loss of XNP-1, the C. elegans homologue of ATRX, recapitulates many human phenotypes. Loss of XNP-1 causes ectopic activation of germline genes in somatic cells, indicating a loss of cellular identity control. Strikingly, mutation of the germline transcription factor gsox-1 suppresses both this misexpression and multiple xnp- 1 phenotypes, including developmental delay and telomeric defects. These findings suggest that ectopic germline gene expression underlies the majority of XNP-1-dependent phenotypes, consistent with a role for XNP-1 in maintaining cellular identity, offering insights into the functions of ATRX in humans. Introduction Chromatin structure impacts all processes that require access to DNA. Thus, proteins that alter chromatin structure, particularly ATP-dependent chromatin remodelling enzymes, are important mediators of DNA transcription, replication and repair. Indeed, many disorders are known to be caused by mutation of human ATP-dependent chromatin remodelling genes ( 1 ). One of the best described is the neurodevelopmental disorder ATR-X syndrome, which gave its name to the causal gene, the chromatin remodelling factor, ATRX ( 2 ). Fitting with the ubiquitous roles that chromatin remodelling enzymes play in DNA metabolism, ATRX loss is also strongly associated with cancer, specifically those cancers that use the alternative lengthening of telomere (ALT) pathway ( 3 ). ALT uses break-induced replication to maintain telomere length ( 4 , 5 ) and ATRX has been shown to act as a tumour suppressor by repressing the ALT pathway ( 6 ). Precisely how ATRX influences both ALT and normal development is unclear. This is compounded by the fact that ATRX has been shown to impact many molecular processes. ATRX interacts with DAXX to deposit the replication-independent histone H3.3 at heterochromatin ( 7 , 8 ) and euchromatin ( 9 ). Loss of ATRX leads to increased retrotransposon expression ( 10 – 12 ), increased levels of G-quadruplexes ( 13 ), reduced sister chromatid cohesion ( 14 , 15 ) and increased sensitivity to replication stress ( 16 , 17 ). Unpicking the relative importance of these molecular functions within a developmental context has been made harder by the fact that complete loss of ATRX is embryonic lethal in standard model organisms such as fruit flies, mice and zebrafish ( 18 – 20 ). Conditional mouse knockouts have allowed the study of ATRX in specific tissues, particularly neuronal lineages ( 19 , 21 ). However, the role of ATRX in early embryogenesis is particularly poorly understood. To address this, we used the nematode C. elegans as a model system, because the loss of its ATRX homolog, XNP-1, does not result in embryonic lethality. C. elegans has a particularly well described and stereotypical order of embryonic development where the fate of every cell is known. These decisions are largely governed by the restricted expression of master transcription factors ( 22 , 23 ). However, chromatin structure also plays a complementary role in controlling tissue-specific expression and therefore cellular identity. This is particularly relevant in the very first cell fate decision in C. elegans embryogenesis in which germline versus somatic cell fate is specified ( 24 ). In this manuscript, we show that xnp-1 mutants exhibit ectopic expression of germline genes in somatic cells. We identify the germline transcription factor F33H1.4 (renamed gsox-1 ) as a suppressor of xnp-1 sterility at 25°C. Strikingly, gsox-1 mutation suppresses not only germline misexpression but also seemingly unrelated xnp-1 phenotypes including developmental delay and telomeric defects. Together, our work identifies a novel role for ATRX homologs in maintaining cellular identity by repressing inappropriate germline gene activation. Materials and Methods Brood Size Brood size at 20°C was carried out by singling L4 worms onto OP50 NGM plates and then singling for 3-4 days before counting the total number of progeny. For brood size on plates containing 10 mM hydroxyurea, worms were added to these plates as L1s and then brood size was carried out as above. The worms were maintained on plates containing 10 mM hydroxyurea throughout. Brood size at 25°C was carried out by shifting L4 worms that were previously grown at 20°C to 25°C. The brood size of their progeny (the F1 or the F2 generation) was recorded in the same way as above. C-Circle Assay Animals were synchronised to L1s and approximately 1000 L1s were seeded onto 5-6 OP50 plates (9 cm). Plates were maintained at 20°C or 25°C for 72 hours until animals reached gravid adult stage. The C-circle assay was performed as described ( 25 ) with the difference that the genomic DNA was extracted by bead-beating. In brief, 300 μl of buffer (100 μg/ml RNase A, 10 mM EDTA, 300 mM NaCl) and 400 μl of 0.5 mm glass beads were added to worm pellets and disrupted by mechanical lysis using a cell homogeniser for 3x 20 s at 6 m/s with cooling on ice in between each step. Proteins were denatured by adding 1% SDS and heating the extracts at 65°C for 10 minutes after which the SDS was precipitated with potassium acetate. The supernatant was collected after spinning and DNA purified using phenol:chloroform:isoamyl alcohol (15:24:1, pH 6.7) extraction and a chloroform back-extraction. DNA was ethanol precipitated and eluted in Tris-EDTA buffer (10 mM Tris-HCl, 100 μM EDTA, pH 7.5). Phi29 polymerase (NEB) was added to 1 μg of genomic DNA in a total volume of 20 µl and incubated at 30°C for 8 hours. This was spotted onto a neutral Hybond-N membrane, 1200J/m2 UV cross-linked, and hybridized with a DIG-labelled (GCCTAA) 4 probe at 37°C using DIG Easy Hyb (Roche) according to the manufacturer’s instructions. Telomere length by terminal restriction fragment (TRF) analysis Genomic DNA was extracted 1x NTE buffer (100 mM NaCl, 50 mM Tris pH 7.4, 20 mM EDTA), 1% SDS and 500 μg/ml Proteinase K overnight at 65°C. DNA was purified by two consecutive phenol-chloroform washes (15:24:1, pH 6.7), followed by a chloroform back-extraction and ethanol precipitation. DNA was dissolved in Tris-EDTA pH 8 and incubated with 50 μg/ml RNase A for 30 minutes at 37°C followed by a final round of phenol-chloroform extraction and ethanol precipitation. 5 μg purified genomic DNA was digested overnight with HinfI and HaeIII (NEB) at 37°C and resolved on 1% agarose gel in 1x TAE. Following a 20-minute depurination in 250 mM HCl, the gel was washed 2x in denaturing buffer (1.5 M NaCl, 0.5 M NaOH) and 2x in neutralising buffer (1.5 M NaCl, 0.5 M Tris-HCl, pH 8) at room temperature. DNA was transferred onto neutral nylon membrane (Hybond-NX, GE Healthcare) by capillary transfer in 10x SSC buffer (1.5M NaCl, 150 mM sodium citrate, pH 7). After briefly rinsing in 2x SSC buffer (300 mM NaCl, 30 mM sodium citrate, pH 7) DNA was UV crosslinked at 1200 J/m 2 and hybridised with a digoxygenin-labelled telomere probe (GCCTAA) (1 μg/ml in DIG Easy Hyb ™ hybridisation buffer, Roche) for 2 hours at 37°C. Membranes were washed 2x in MS buffer (100 mM maleic acid, 150 mM NCl, pH 7.5), 0.3% Tween-20, blocked in MS buffer, 1% BSA, 1% milk, 0.1% Tween-20, probed with AP-conjugated anti-digoxigenin Fab fragments (Roche) at 1:20 000 dilution in the same buffer (1:20000), followed by 3 washes in MS buffer, 0.3% Tween-20. Membranes were equilibrated in AP buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl) followed by chemiluminescent detection with CDP-Star ® substrate (Roche). Quantifying C. elegans growth rates Post-embryonic growth rates were measured using a luciferase-based assay performed as described previously ( 26 ), based on ( 27 ). Briefly, wells in luminometer compatible 384- well plates (Berthold Technologies, 32505) were filled with 90 μ S- Basal medium containing E. coli OP50 (OD600 = 1) and 100 μM Firefly D- Luciferin (p.j.k., #102111). Single eggs, from animals expressing luciferase under a constitutive and ubiquitous promoter (xeSi312[eft-3p::luc::gfp::unc-54 3′UTR, unc-119(+)] IV) ( 28 ), were then loaded into individual wells and plates were sealed with Breathe Easier sealing membrane (Diversified Biotech, BERM-2000). Development was monitored over 96 hrs by measuring luminescence in every well (every 10 mins for 0.5 secs) using a luminometer (Berthold Technologies, Centro XS3 LB 960) placed in a temperature- controlled incubator set to 18.5°C. Luminescence data were analyzed similarly to before ( 26 ), but using the Python-based ‘PyLuc’ tool developed by L. Morales Moya ( 28 ). The identification of molts (the final phase of each larval stage) was based on a drop in luminescence signal, which results from animals not feeding during this period. For each animal, larval stage durations were then calculated as the time from hatching to the end of the first molt (L1), or from the end of one molt to the end of the subsequent molt (L2 to L4). RNA Experiments RNA extraction was performed as described in ( 29 ). Approximately 800 ng of RNA was used as input to each reverse transcription reaction (GoScript Reverse Transcriptase, Promega). The qPCR reaction was carried out on a QuantStudioTM Real-Time PCR system machine (Thermo Scientific) with the following reaction steps: 95°C for 60” ➔ 40x(95°C for 15” ➔ 59°C for 30”). The qPCR primers used are given in Supplementary Table 2. RNA sequencing of embryos and L1 was carried out at the Oxford Genomics Centre and for the L4 samples was carried out at Novogene, Cambridge. All genotypes were sequenced in biological triplicate. Briefly, reads were trimmed to remove Illumina adaptors using Trim_galore, a wrapper script from Cutadapt (version 0.6.7) ( 30 ) using default settings. Trimmed reads were mapped to the reference genome (release WS280, BioProject PRJNA13758) using STAR (version 2.7.10a) ( 31 ), without limits on the mapped length and with maximum number of mismatches per pair set to 2. The reads were re-sorted by read name using Samtools (version 1.6) ( 32 ). FeatureCounts (Subread, version 2.0.1) ( 33 ) was used to generate a count matrix at gene-level. Differential gene expression was conducted in edgeR (version 3.40.0) ( 34 ) on a local machine using R (version 4.2.2) applying a minimum gene count of four per replicate and 20 counts across all three replicates per genotype. All scripts can be found in our Github repository ( https://gitlab.com/mariya_s/draft_scripts_olver_et_al_2025/-/tree/4aab25604a4a4c716fb24962c6437338bb094249/ ). SimpleMine web tool from WormBase was used to convert WormBase gene IDs to gene names. Gene set enrichment analysis was also ran on the WormBase web server and the results were plotted using the bubble plot function provided by the SRplot web server ( https://www.bioinformatics.com.cn/srplot ) ( 35 ). Small non-coding RNA sequencing and analysis was performed as described ( 29 ). Briefly, 1ug total RNA was treated with RppH (NEB) for 1 hour at 37C to remove 5’ triphosphates. RNA was then extracted by phenol chloroform and precipitated with 3M sodium acetate and ethanol. The resulting RNA was subjected to small RNA sequencing by Oxford Genomics. Fastq files were processed to remove adaptors using Fastx Toolkit. To obtain genome-wide alignments, all reads were aligned to the reference genome (WS280 as above) using Bowtie with the following parameters: -k 1 -v 0 --best to produce alignment sam files, which were further converted to bam files. Bedtools ( 36 ) intersect was used to assign reads to different genomic features. 22G-RNAs aligning to transposable elements were specifically interrogated by first selecting reads that were 22 nucleotides long and with a G as the 5’ nucleotide using a custom perl script. These reads were then aligned to the consensus sequences for transposable elements in C. elegans downloaded from RepBase ( https://www.girinst.org/repbase/ ) using Bowtie with parameters as above. Antisense reads corresponding to each transposable element were counted using Bedtools genomecov and quantified and visualised using R (version 4.2.2). Forward genetic screen and DNA sequencing Mutagenesis was carried out as described ( 37 ). Briefly, synchronized L4 xnp-1(tm678) worms were exposed to 50mM ethyl methanesulfonate (EMS) for 4 hours at room temperature, before being washed and placed on OP50 at 20°C until the F1 generation was gravid. At this point ∼250 worms were singled onto new OP50 plates and kept at 25°C. Three fertile lines were isolated and one of these, xnp-1(hcf3) was pursued further. xnp-1(hcf3) was backcrossed three times to xnp-1(tm678) before genomic DNA was extracted (as described in TRF analysis) and sent for sequencing at Novogene (Cambridge Science Park). Potential mutations were mapped, and homozygous mutations identified using the MiModD (version 0.1.9) package developed in the Baumeister lab ( https://mimodd.readthedocs.io/en/latest/index.html ). The reference genome used was release WS283 (BioProject PRJNA13758). Strains All strains used and their availability are listed in Supplementary Table 1. Results XNP-1 loss phenocopies several aspects of ATRX loss in human cells Chromatin remodellers are defined by their ATPase activity and domain swap experiments show that this region dictates remodelling outcomes ( 38 ). C. elegans XNP-1 and human ATRX share 70% sequence identity within this catalytic domain ( Figure 1A ), suggesting that despite being half the size of ATRX, XNP-1 likely performs similar chromatin remodelling reactions ( 39 , 40 ). Download figure Open in new tab Figure One Loss of C. elegans XNP-1 mimics several phenotypes of human ATRX mutation A) XNP-1 is the C. elegans homologue of human ATRX. The ADD (ATRX- DNMT3-DNMT3L) domain is not found in ATRX homologues of lower eukaryotes, but the catalytic ATPase domain is highly conserved between humans and worms. (B) xnp-1 mutant worms display elevated C-circle levels at 25°C. Representative blot from a C-circle assay both at 20°C or 25°C with and without the polymerase Phi29. Quantification of data from five independent C-circle assay experiments using worms synchronised to L1s and then grown for 72 hours at either 20°C or 25°C. Normalised values are presented as percent of pot-2(tm1400) signal and the mean across different experiments is shown. Error bars represent the standard deviation from the mean, ns = not significant, * = p < 5 x 10 -2 (two-tailed Mann-Whitney U test). (C) xnp-1 mutant worms have lower brood sizes than wildtype and are sensitive to hydroxyurea (HU). Synchronised L1 wildtype or xnp-1(tm678) larvae were placed on either OP50 plates or OP50 plates supplemented with 10 mM HU and maintained at 20°C. The brood sizes of 12 - 20 adults per genotype are displayed as box and whisker plots, ** = p < 1 x 10 -3 , *** = p < 1 x 10 -5 (Welch’s test). (D) xnp-1 mutant worms develop slower than wildtype. Synchronised L1 wildtype or xnp-1(tm678) larvae were singled into individual wells and imaged over two days to monitor growth. Box and whisker plots of data from at least 70 worms per genotype indicating the total time to complete each larval stage, *** = p < 1 x 10 -6 (two tailed T-test). (E) The LTR retrotransposon Cer10 is de-repressed most strongly in larval stages of xnp-1 animals that contain a germline. RT-qPCR of Cer10 with tba-1 used as the reference in wildtype and xnp-1(tm678) animals with Cer10 expression normalised to wildtype. The mean and standard deviation is shown for three biological replicates. ns = not significant, * = p ≤ 5 x 10 -2 , ** = p ≤ 10 x 10 -3 (two tailed T-test). To study the effects of XNP-1 loss, we used a null mutant, xnp-1(tm678) . This allele contains a 673bp deletion in the xnp-1 gene and additionally introduces a premature stop codon before the ATPase domain ( 39 , 40 ). Loss of ATRX in human cells has been linked to several phenotypes such as ALT activation, sensitivity to replication inhibitors, development delay and de-repression of retrotransposons ( 3 , 12 , 16 ). We therefore tested whether loss of XNP-1 induced similar phenotypes in C. elegans . Human ALT positive tumours are defined clinically by the presence of C-circles: partially single-stranded circles of extrachromosomal telomeric DNA ( 25 ). The connection between ALT and C-circles extends beyond humans as C. elegans ALT-like strains also show increased C-circle levels ( 41 – 43 ). When we examined synchronized populations of wildtype and xnp-1 gravid adults grown at 20°C, we detected no significant differences in C-circle levels. However, when grown at 25°C, a known stress condition, xnp-1 showed significantly elevated C-circle levels compared to wildtype ( Figure 1B ). These levels were comparable to those in pot-2(tm1400) mutants, which form long-term ALT survivors in telomerase-null backgrounds ( 42 , 43 ). This stress-dependent C-circle induction parallels the human situation, where additional factors such as telomeric DNA breaks ( 44 ) or protein-DNA adducts ( 45 ) are required for robust C-circle formation in ATRX mutant cells. ATRX mutant cells show signs of replication stress and are sensitive to hydroxyurea (HU) ( 16 ). This drug induces replication stress by reducing the levels of deoxyribonucleotides in S phase, thereby uncoupling the movement of DNA polymerases from the replicative CMG helicase. We exposed worms to 10mM hydroxyurea from the L1 stage onwards and quantified its effect by measuring brood size of gravid adults two days later. Even without HU treatment, xnp-1 worms had significantly smaller brood sizes than wildtype ( Figure 1C ). However, xnp-1 mutants showed markedly increased HU sensitivity compared to wildtype. While 10mM HU caused an approximately two-fold decrease in wildtype brood size, it resulted in a more than nine-fold drop in xnp-1 , rendering them nearly sterile ( Figure 1C ). ATRX syndrome causes neurodevelopmental delay in humans ( 46 ). As this is difficult to assay in worms, we instead assayed whether XNP-1 affects the rate of post-embryonic development. We observed that xnp-1 mutants took significantly longer than wildtype to develop at every larval stage ( Figure 1D ). Moreover, the normally tight synchrony of worm development was weakened in xnp-1 (Supplementary Figure S1). Loss of ATRX leads to increased expression of endogenous retroviruses (ERVs) in mammalian stem cells ( 12 ). To test whether XNP-1 similarly regulates transposable elements, we examined both individual retrotransposons and genome-wide transposon expression. We focused on Cer10, an LTR retrotransposon for which validated qPCR primers are available ( 47 ), as LTR retrotransposons are the elements in worms most similar to mammalian ERVs ( 48 ). When we examined Cer10 expression across developmental stages, levels were similar between xnp-1 and wildtype in embryos and L1 larvae but were significantly elevated in xnp-1 L4s and adults ( Figure 1E ). RNA-seq analysis revealed that retrotransposon upregulation was not restricted to later stages. Multiple transposon families were significantly upregulated in xnp-1 mutant embryos and L1 larvae, including other LTR retrotransposons and PIF-Harbinger DNA transposons (Supplementary Figure S2A). Small RNA sequencing revealed no differences in 22G-siRNA levels targeting these transposons, indicating that XNP-1 regulates retrotransposons independently of the siRNA pathway (Supplementary Figure S2B). To determine whether XNP-1 silences retrotransposons through H3K9me3-dependent mechanisms, we examined the role of SET-25, the only C. elegans H3K9 trimethylase ( 49 ). Consistent with previous data ( 56 ), set-25 single mutants showed increased Cer10 retrotransposon expression. However, xnp-1; set-25 double mutants had significantly higher Cer10 levels than set-25 single mutants (Supplementary Figure S3), indicating that XNP-1 and SET-25 function in independent pathways. Overall, our data demonstrates that loss of XNP-1 in C. elegans recapitulates multiple key phenotypes seen in ATRX mutant human cells, supporting functional conservation despite structural differences between organisms. XNP-1 represses germline genes distinct from SynMuvB Analysis of polyA-enriched RNA sequencing data revealed relatively few differentially expressed genes in either xnp-1 embryos or L1 larvae when grown at 20°C ( Figure 2A and Supplementary Table 3). Downregulated genes between xnp-1 embryo and L1 larvae were largely distinct ( Figure 2B ) but tissue enrichment analysis displayed an association with the intestine (Supplementary figure S4A). In contrast, there was a large degree of overlap between genes upregulated in embryos and L1s ( Figure 2B ). Tissue enrichment analysis revealed that these genes were very strongly associated with the germline ( Figure 2C ). This was surprising as neither embryos nor starved L1 larvae have a germline per se . Indeed, the only two germline progenitor cells (Z2 and Z3) in both these developmental stages are maintained in a transcriptionally quiescent state ( 50 ). Therefore, the increased level of germline expression we see in xnp-1 embryos and L1 larvae is most likely to be due to ectopic expression of germline genes from somatic cells. The ectopic activation of germline genes in somatic cells indicates a loss of cellular identity control. This suggests XNP-1 normally functions to maintain the transcriptional boundary between germline and somatic cell fates by repressing germline-specific genes in somatic cells. This resembles the phenotype caused by loss of SynMuvB genes ( 51 ), sometimes referred to as the DREAM complex in other species ( 52 ). A hallmark of synMuvB mutation in worms is increased expression of PGL-1, a P granule component found in all germ cells ( 51 ). We monitored pgl-1 expression in xnp-1 embryos or L1 larvae at 25°C, a condition in which the increase in PGL-1 in SynMuvB mutants is particularly pronounced ( 51 ). However, we found no significant increase in pgl-1 mRNA levels in xnp-1 compared to wildtype control ( Figure 2D ), suggesting that xnp-1 may not be a SynMuvB gene. Indeed, when we looked beyond pgl-1 to include all genes that become upregulated in lin-35, a key member of the SynMuvB family ( 51 ), we saw very little overlap with genes that become upregulated in xnp-1 (Supplementary Figure S4B). Another hallmark of SynMuvB mutants is that they display larval arrest at elevated temperatures, which is suppressed by RNAi of the H3K36 methyltransferase mes-4 ( 51 ). However, loss of XNP-1 did not show a larval arrest phenotype at 25°C ( Figure 2E ) and mes-4(RNAi) did not rescue the reduced brood size xnp-1 at 25°C ( Figure 2F and Supplementary Figure S4C). Altogether, these data show that XNP-1 and SynMuvB repress germline genes through distinct pathways. Alongside the upregulation of germline genes, we observed downregulation of somatic genes in xnp-1 embryos and L1 larvae. Download figure Open in new tab Figure Two XNP-1 prevents ectopic germline expression in a different manner to the SynMuvB pathway (A) Loss of XNP-1 leads to changes to a modest number of differentially expressed genes (DEGs) in both embryos and L1s grown at 20°C. Genes whose expression is significantly altered in xnp-1 are marked in red within the MA plot, false discovery rate (FDR) < 0.05. (B) Upregulated genes in xnp-1 are more likely to behave similarly in both embryo and L1 samples compared to downregulated genes. (C) Analysis of genes that are significantly upregulated in xnp-1 samples (from part A) indicates that they are normally expressed within the germline and typically have functions in DNA repair and meiosis. (D) pgl-1 is not upregulated in xnp-1 mutants. RT-qPCR of pgl-1 normalised to tba-1 in wildtype and xnp-1(tm678) embryos and L1 larvae grown at 25°C. The mean and standard deviation is shown for three biological replicates. ns = p > 5 x 10 -2 (two tailed T-test). (E) xnp-1 animals (in contrast to lin-35 ) do not show L1 larval arrest at 25°C. Synchronised L4s were placed at 25°C and left to produce offspring. The number of offspring scored for an arrest phenotype and the percentage of L1 larval arrest is indicated. (F) The low brood size of xnp-1 animals at 25°C is not rescued by mes-4(RNAi). Synchronised xnp-1 L1s were placed at 25°C onto control or mes-4(RNAi) plates. Once they developed to gravid adults, they were singled and the brood size of those adults (F1) scored. Box and whisker plots from 15 - 20 adults are displayed, ns = not significant. Identification of gsox-1 as a suppressor of xnp-1 sterility at 25°C To identify genes that function with XNP-1, we turned to genetic suppressor analysis. Forward genetic screens reveal functionally relevant genes in living animals, where transcription, development, and stress responses are tightly integrated. C. elegans lacking XNP-1 become completely sterile within two generations when grown at 25°C ( Figure 3A ), providing a robust phenotype for screening. We used ethyl methanesulfonate (EMS) to mutagenise xnp-1 mutants and screen for suppressor mutants that restored fertility at 25°C ( Figure 3B ). One suppressor, xnp-1(hcf3) , could be maintained indefinitely at 25°C with significantly larger brood sizes than the original xnp-1(tm678) strain ( Figure 3C ). Genome sequencing of this strain identified a locus on chromosome II containing G/C to A/T mutations characteristic of EMS mutagenesis. Within this locus were four genes ( toe-1 , mog-5 , hgap-2 and F33H1.4 ) with single point mutations within their exons that resulted in protein-coding changes. To definitively link the suppressor phenotype to a single mutation, we independently introduced each of these mutations using CRISPR into a wildtype background and then crossed them into unmutagenised xnp-1(tm678). Only the F33H1.4 mutation suppressed xnp-1 sterility at 25°C ( Figure 3D ). This gene encodes a nematode-specific transcription factor expressed in the germline that binds to germline gene promoters ( 53 , 54 ). Based on this, we re-named F33H1.4 as gsox-1 (germline suppressor of xnp-1 ). Download figure Open in new tab Figure Three Identification of gsox-1 as a suppressor of xnp-1 sterility at 25°C (A) Loss of XNP-1 leads to rapid sterility at 25°C. Synchronised xnp-1 L1s were placed at 25°C onto OP50 plates. Once they developed to gravid adults, they were singled and the brood size of those adults (F1) scored. The subsequent generation at 25°C (F2) were sterile. In contrast, xnp-1 worms can be maintained indefinitely at 20°C. Box and whisker plots from 15 - 20 adults are displayed, ** = p ≤ 10 x 10 -4 (two tailed T-test). (B) Schematic of forward genetic screen to isolate xnp-1 suppressors. (C) Identification of xnp-1(hcf3) as a weak suppressor of temperature induced sterility. Box and whisker plot of the number of F2 progeny of wildtype, the original xnp-1(tm678) strain and the suppressor line xnp-1(hcf3) , * = p 19 for all genotypes. (D) gsox-1 is the causal gene behind the fertility of xnp-1(hcf3) at 25°C. Box and whisker plot of the number of F2 progeny of wildtype, xnp-1(tm678) , toe-1(syb6057) , mog-5(syb5976) , hgap-2(syb7188) , gsox-1(syb7245) and double mutants of these with xnp-1(tm678) , *** = p 10 for all genotypes. Mutation of gsox-1 suppresses the majority of misregulated germline expression in xnp-1 The connection of gsox-1 to germline gene expression was intriguing given the ectopic germline gene expression seen in xnp-1 mutants at 20°C ( Figure 2A-C ). We therefore examined transcriptional changes in xnp-1 animals at 25°C, the condition causing rapid sterility. Wildtype and xnp-1 mutant embryos were hatched at 20°C and then grown from L1 to L4 larvae at 25°C ( Figure 4A ). Transcriptomic analysis revealed that >6000 genes were significantly altered (FDR <0.05) compared to wildtype ( Figure 4A ), far more than observed at 20°C ( Figure 2A ). Download figure Open in new tab Figure Four Mutation of gsox-1 suppresses the majority of transcriptional changes in xnp-1 (A) Schematic of growth conditions of wildtype and xnp-1 worms prior to processing for polyA-RNA sequencing. MA plot illustrating that a very large number of transcripts are misrergulated in xnp-1 relative to wildtype. Differentially expressed genes (DEGs) were defined using a false discovery rate (FDR) of less than 0.05. (B) Upregulated and downregulated DEGs are associated with distinct tissues and molecular functions. Upregulated DEGs in xnp-1 are typically expressed in the germline (similar to what is seen in Figure 2C ). In contrast, downregulated DEGs are typically expressed in males and the hypodermis. Enriched gene ontology terms for up- and downregulated DEGs are ordered by enrichment over the background set (all genes detected) and colour coded by their FDR. Upregulated DEGs in xnp-1 are typically associated with functions in DNA repair and meiotic cell cycle (again similar to what is seen in Figure 2C ). In contrast, downregulated DEGs have functions in cuticle formation and moulting. (C) Mutation of gsox-1 by itself does not cause significant transcriptional disruption. (D) Mutation of gsox-1 reduces the number of DEGs in an xnp-1 background. MA plot, as described in part A, showing that the number of DEGs in xnp-1; gsox-1 is more than 25-fold lower than in xnp-1 . (E) GSOX-1 shows robust binding at promoters of genes upregulated in xnp-1 but not at downregulated genes. ChIP-seq data from ( 53 ) analysed using deepTools. Top panel: Average GSOX-1 signal across all genes in each category, cantered on the TSS +/- 3kb. Bottom panel: Heatmap showing GSOX-1 binding at individual genes, with each row representing one gene. More importantly, when we looked at the identity of upregulated genes, we saw that they were again very strongly associated with the germline and the meiotic cell cycle ( Figure 4B ), similar to what was seen at 20°C ( Figure 2C ). This meant that the same types of genes were affected in xnp-1 worms at both 20°C (relatively normal physiology) and 25°C (profound developmental defects), but that the scale of the transcriptional changes was much larger at 25°C. Altogether, this suggested a link between germline upregulation and sterility in xnp-1 . Down-regulated genes in xnp-1 were associated with skin (hypodermis), the cuticle and moulting cycle ( Figure 4B ). Cuticle formation and the moulting cycle drive the growth of larval stages in C. elegans . It is possible that the disruption of these pathways contributes to the slow post-embryonic development of xnp-1 animals ( Figure 1D ). We observed very few significant transcriptional changes in our single gsox-1 mutant compared to wildtype ( Figure 4C ). This is consistent with the gsox-1 allele (R1192Q) being a hypomorph since RNAi knockdown of gsox- 1 is embryonic lethal ( 55 ). Despite having a minimal effect in wildtype, gsox-1 mutation had a marked effect in an xnp-1 background, supressing the vast majority of transcriptional misregulation. The double xnp-1; gsox-1 mutant had only 234 differentially expressed genes (DEGs) relative to wildtype ( Figure 4D ), which is much less than the 6781 DEGs between xnp-1 and wildtype ( Figure 4A ). To distinguish between direct and indirect effects, we leveraged existing ChIP-seq data for GSOX-1 in wildtype young adults ( 53 ). GSOX-1 displayed robust binding at the promoters of upregulated genes but essentially no binding at xnp-1 downregulated genes ( Figure 4E ). This was specific to GSOX-1 as we did not see the same binding pattern in the unrelated transcription factor NHR-23 (Supplementary figure S5). This enrichment is consistent with GSOX-1’s known role as a germline transcription factor ( 54 ) and suggest a hierarchy of effects: gsox-1 directly suppresses xnp-1 upregulated (germline) genes, whereas its effects on downregulated genes are indirect. This argues that ectopic germline gene expression is the primary defect in xnp-1 , with disruption of somatic programs as downstream consequences and is consistent with the pattern of misregulation seen in embryos and L1 larvae at 20°C. Accordingly, these data support a direct link between fertility defects in xnp-1 and germline gene misregulation. Altered cellular identity likely drives the developmental and telomeric phenotypes of xnp-1 We wondered whether gsox-1 could also suppress other xnp-1 phenotypes. Indeed, we found that gsox-1 significantly suppressed the slow growth of xnp-1 at 20°C. The xnp-1; gsox-1 double mutant developed significantly faster than xnp-1 at every larval stage ( Figure 5A ). This included the first larval stage, L1, which has no significant germline and only two germline progenitor cells per animal. Therefore, gsox-1 can suppress xnp-1 developmental phenotypes that are not directly linked to the germline. This interpretation was strengthened when we looked at telomeric phenotypes. We observed that gsox-1 partially suppressed the long telomere phenotype of xnp-1 animals ( Figure 5B ) and almost completely supressed the increased C-circle levels at 25°C ( Figure 5C ). Similarly, gsox-1 largely suppressed the upregulation of the Cer10 retrotransposon in xnp-1 L4 larvae at 25°C ( Figure 5D ). Our data show that these seemingly unconnected xnp-1 mutant phenotypes are all rescued by mutation of a germline transcription factor. This raises the possibility that these xnp-1 phenotypes are driven by altered cellular identity, specifically an increase in a germline-like cell fate. Download figure Open in new tab Figure Five The developmental and telomeric phenotypes of xnp-1 are linked and likely due to altered cellular identity (A) gsox-1 partially recues the slow development phenotype of xnp-1 at 20°C. Synchronised L1 larvae were singled into individual wells and imaged over two days to monitor growth. Data for wildtype and xnp-1 as in Figure 1D . Box and whisker plots of data from at least 70 worms per genotype indicating the total time to complete each larval stage, *** = p < 1 x 10 -6 (two tailed T-test). (B) xnp-1 worms have longer telomeres than wildtype which is partially suppressed by gsox-1 . Southern blot, probed with a telomere-specific sequence, of an asynchronous population of the indicated strains grown at 20°C. (C) gsox-1 recues the elevated C-circle levels of xnp-1 at 25°C. Quantification of data from five independent C-circle assay experiments using worms synchronised to L1s and then grown for 72 hours at either 20°C or 25°C. Data for wildtype and xnp-1 as in Figure 1B , * = p < 5 x 10 -2 (two-tailed Mann-Whitney U test) (D) gsox-1 partially recues the increased Cer10 expression observed in xnp-1. RT-qPCR of Cer10 levels normalised to tba-1 in L4 stage animals of the indicated genotypes grown at 25°C from L1 onwards. The level of wildtype Cer10 expression is set to 1. The graph displays the mean and standard deviation from three biological replicates. * = p ≤ 5 x 10 -2 , ** = p ≤ 5 x 10 -3 (two tailed T-test). (E) gsox-1 does not supress the HU sensitivity of xnp-1. Synchronised L1 larvae of the indicated genotypes were placed on either OP50 plates or OP50 plates supplemented with 10 mM HU and maintained at 20°C. Data for wildtype and xnp-1 as in Figure 1C . The brood sizes of 12 - 20 adults per genotype are displayed as box and whisker plots, ns = not significant. (F) Model for how XNP-1 works in C. elegans . XNP-1 represses the ectopic expression of germline genes in a manner distinct form the SynMuvB pathway. The inappropriate activation of a germline-like fate is the likely cause of the developmental, telomeric and retrotransposon repression defects seen in in xnp-1 as all these phenotypes are suppressed by gsox-1 . The one xnp- 1 phenotype which gsox-1 could not suppress was sensitivity to HU ( Figure 5E ). We observed that the brood size of xnp-1; gsox-1 double mutants on 10mM HU was no higher than xnp-1 . Instead, gsox-1 mutants were themselves mildly sensitive to HU. Thus, XNP-1’s role in replisome stability operates independently of GSOX-1 and is unlikely to be linked to germline identity, unlike its other functions described here ( Figure 5F ). Discussion The ATRX gene has long been linked to human disease; it is the causal mutation behind ATRX syndrome ( 2 ) and is mutated in 90% of ALT positive cancers ( 3 ). In this study, we find that loss of the ATRX homologue, XNP-1 in C. elegans , leads to many of the same phenotypes in nematodes as in human cells, including: de-repression of the ALT pathway, increased sensitivity to replication stress, de-repressed retrotransposon expression and disrupted development. Given that nematodes and humans diverged over a billion years ago ( 56 ), this suggests that ATRX syndrome and upregulation of ALT are caused by disruption of an evolutionarily ancient function of the ATRX gene. One of the striking aspects of ATRX has been the multiple, seemingly disparate, phenotypes observed when it is mutated ( 57 ). It has not been clear how these various ATRX mutant phenotypes are linked, or whether they instead represent separate, independent functions. The fact that we see conservation of so many ATRX phenotypes in C. elegans , hints at a common underlying mechanism. This is strengthened by the fact that a single suppressor mutant of xnp-1 sterility ( gsox-1) also supressed other xnp-1 phenotypes that it was not selected for. The simplest explanation for this observation is that these different xnp-1 phenotypes are all distinct downstream consequences of a common upstream problem. Given that GSOX-1 is a germline transcription factor ( 53 , 54 ), we propose that the initial trigger in xnp-1 mutants is misregulated cellular identity, more specifically due to cells taking on a more germline-like fate. Although GSOX-1 functions primarily in the germline, it is also expressed in some somatic tissues, such as glia ( 58 ). Thus, reducing GSOX-1 levels likely suppresses inappropriate germline programs in both tissues. GSOX-1 binds robustly at the promoters of genes that are upregulated in xnp-1 mutants (germline genes) but shows no binding at downregulated genes (somatic genes) in wildtype. This indicates that GSOX-1 directly activates these germline genes under normal conditions. Consequently, when gsox-1 is mutated in an xnp-1 background, the suppression of these upregulated genes restores proper germline identity, with down-regulation of soma-specific genes being indirect secondary consequences. How does loss of cellular identity control explain the diverse xnp-1 phenotypes we observe? Disruption of gene regulation within the germline is likely to impair its development, which can directly reduce fertility. Ectopic expression of germline transcripts in somatic cells could also explain the slower development of xnp-1 animals during developmental stages (L1) where they do not contain a germline per se ( Figure 1D ). Since germline genes are upregulated in xnp-1 ( Figure 2A and 4A ), and retrotransposons normally increase during meiosis ( 59 , 60 ), the Cer10 upregulation we observe is consistent with an ectopic meiotic environment caused by inappropriate germline gene expression. Similarly, amongst the genes whose expression increases significantly in xnp-1 L4 larvae at 25°C are the telomerase catalytic subunit ( trt-1 ), the telomere shelterin components ( pot-1 and pot-2) as well as several DNA repair proteins (Supplementary Table 3). Since ALT is driven by activation of inappropriate DNA repair pathways at dysfunctional telomeres ( 5 ), aberrant germline gene expression could pre-dispose telomeres to inappropriate lengthening and dysfunction. However, not all xnp-1 phenotypes are explained by loss of cellular identity control. We find that gsox-1 does not suppress the HU sensitivity of xnp-1 ( Figure 5E ). This suggests that the fork stability phenotype of xnp-1 represents a functionally distinct role of XNP-1 ( Figure 5F ). Most models of ALT invoke aberrant DNA repair at a damaged replication fork ( 61 ). However, our data indicates that the fork stability phenotype of xnp-1 (HU sensitivity) is not linked to other xnp-1 phenotypes. At first glance this is surprising as expressing meiotic proteins in human cells is sufficient to induce replication stress ( 62 ). It is possible that XNP-1 plays a more direct role at the replication fork. Human ATRX associates physically with the MCM complex ( 63 ), perhaps pointing to a mechanism by which it travels with the fork to promote replisome stability. The precise mechanism by which XNP-1 controls gene expression is not clear. Our data on retrotransposon expression indicated that XNP-1’s effect does not function via the siRNA pathway and is independent of H3K9me3-dependent silencing. As a member of the ATP-dependent family of chromatin remodeling enzymes, ATRX may play a role in regulating the chromatin template. However, interestingly, ATRX is also involved in functions beyond chromatin formation ( 64 ) and a recent study found that most genes whose expression increased following ATRX mutation did not show a corresponding increase in promoter chromatin accessibility ( 65 ). This suggests that ATRX may not regulate transcription merely via heterochromatin formation. Our findings in C. elegans are consistent with observations in mammalian systems. ATRX is involved in regulating gene expression and maintaining cellular identity in mammalian cells. Loss of ATRX leads to altered differentiation of mouse oligodendrocyte and human mesenchymal progenitor cells ( 65 , 66 ). Moreover, a mouse model of ATRX syndrome shows signs of early developmental defects ( 67 ). Thus, loss of ATRX leads to changes in stem cell identity and a reduction in their ability to resist differentiation into inappropriate cell fates. This connection might also be relevant to ATRX’s role in supressing ALT. A recent study shows that induction of differentiation is a key trigger for ALT in ATRX-deficient stem cells ( 68 ). ALT is more frequent in childhood cancers ( 69 ). In these cancers, cells have less time to accumulate additional cancer-driving mutations, suggesting that changes in their initial cellular identity may play a more significant role in their eventual malignant transformation. Mutation of xnp-1 manifests as a large increase in germline gene expression ( Figure 3C , 5B ). However, this may be because germline is hypothesized to be the default cell fate in C. elegans ( 70 ). Thus, XNP-1 may be required to restrict cell fate specification more broadly rather than specifically repress germline genes per se . In C. elegans , GSOX-1 serves as the key downstream effector of this identity control, yet this transcription factor is not conserved beyond nematodes, whereas XNP-1/ATRX is. This suggests that while the specific transcriptional effectors may differ across species, preventing inappropriate cell fate specification is a fundamental and conserved role of ATRX. Acknowledgements Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). We thank Prof. Julie Ahringer and Dr Chantal Wicky for helpful discussions. The authors acknowledge Research Computing at the James Hutton Institute for providing computational resources and technical support for the UK’s Crop Diversity Bioinformatics HPC (BBSRC grants BB/S019669/1 and BB/X019683/1), use of which has contributed to the results reported within this paper. MS was supported by an EASTBIO studentship, (BBSRC) [grant number BB/M010996/1]. We thank Peter Thorpe and Marco Ferreira Fernandes for help with bioinformatics. Footnotes New analysis of GSOX-1 ChIP added and figures have been re-organised to streamline the manuscript References 1. ↵ Amberger , J.S. , Bocchini , C.A. , Schiettecatte , F. , Scott , A.F. and Hamosh , A. ( 2015 ) OMIM.org: Online Mendelian Inheritance in Man (OMIM(R)), an online catalog of human genes and genetic disorders . Nucleic Acids Res , 43 , D789 – 798 . OpenUrl CrossRef PubMed 2. ↵ Gibbons , R.J. , Picketts , D.J. , Villard , L. and Higgs , D.R. ( 1995 ) Mutations in a putative global transcriptional regulator cause X-linked mental retardation with alpha-thalassemia (ATR-X syndrome) . Cell , 80 , 837 – 845 . OpenUrl CrossRef PubMed Web of Science 3. ↵ Lovejoy , C.A. , Li , W. , Reisenweber , S. , Thongthip , S. , Bruno , J. , de Lange , T. , De , S. , Petrini , J.H. , Sung , P.A. , Jasin , M. , et al. ( 2012 ) Loss of ATRX, Genome Instability, and an Altered DNA Damage Response Are Hallmarks of the Alternative Lengthening of Telomeres Pathway . PLoS genetics , 8 , e1002772 . OpenUrl 4. ↵ Zhang , J.M. , Yadav , T. , Ouyang , J. , Lan , L. and Zou , L. ( 2019 ) Alternative Lengthening of Telomeres through Two Distinct Break-Induced Replication Pathways . Cell reports , 26 , 955 – 968 e953. OpenUrl PubMed 5. ↵ Dilley , R.L. , Verma , P. , Cho , N.W. , Winters , H.D. , Wondisford , A.R. and Greenberg , R.A. ( 2016 ) Break-induced telomere synthesis underlies alternative telomere maintenance . Nature , 539 , 54 – 58 . OpenUrl CrossRef PubMed 6. ↵ Clynes , D. , Jelinska , C. , Xella , B. , Ayyub , H. , Scott , C. , Mitson , M. , Taylor , S. , Higgs , D.R. and Gibbons , R.J. ( 2015 ) Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX . Nature communications , 6 , 7538 . OpenUrl PubMed 7. ↵ Lewis , P.W. , Elsaesser , S.J. , Noh , K.M. , Stadler , S.C. and Allis , C.D. ( 2010 ) Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres . Proceedings of the National Academy of Sciences of the United States of America , 107 , 14075 – 14080 . OpenUrl Abstract / FREE Full Text 8. ↵ Goldberg , A.D. , Banaszynski , L.A. , Noh , K.M. , Lewis , P.W. , Elsaesser , S.J. , Stadler , S. , Dewell , S. , Law , M. , Guo , X. , Li , X. et al. ( 2010 ) Distinct factors control histone variant H3.3 localization at specific genomic regions . Cell , 140 , 678 – 691 . OpenUrl CrossRef PubMed Web of Science 9. ↵ Truch , J. , Downes , D.J. , Scott , C. , Gur , E.R. , Telenius , J.M. , Repapi , E. , Schwessinger , R. , Gosden , M. , Brown , J.M. , Taylor , S. et al. ( 2022 ) The chromatin remodeller ATRX facilitates diverse nuclear processes, in a stochastic manner, in both heterochromatin and euchromatin . Nature communications , 13 , 3485 . OpenUrl PubMed 10. ↵ Sadic , D. , Schmidt , K. , Groh , S. , Kondofersky , I. , Ellwart , J. , Fuchs , C. , Theis , F.J. and Schotta , G. ( 2015 ) Atrx promotes heterochromatin formation at retrotransposons . EMBO Rep , 16 , 836 – 850 . OpenUrl Abstract / FREE Full Text 11. Elsasser , S.J. , Noh , K.M. , Diaz , N. , Allis , C.D. and Banaszynski , L.A. ( 2015 ) Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells . Nature , 522 , 240 – 244 . OpenUrl CrossRef PubMed 12. ↵ Robbez-Masson , L. , Tie , C.H.C. , Conde , L. , Tunbak , H. , Husovsky , C. , Tchasovnikarova , I.A. , Timms , R.T. , Herrero , J. , Lehner , P.J. and Rowe , H.M. ( 2018 ) The HUSH complex cooperates with TRIM28 to repress young retrotransposons and new genes . Genome Res , 28 , 836 – 845 . OpenUrl Abstract / FREE Full Text 13. ↵ Law , M.J. , Lower , K.M. , Voon , H.P. , Hughes , J.R. , Garrick , D. , Viprakasit , V. , Mitson , M. , De Gobbi , M. , Marra , M. , Morris , A. , et al. ( 2010 ) ATR-X syndrome protein targets tandem repeats and influences allele-specific expression in a size-dependent manner . Cell , 143 , 367 – 378 . OpenUrl CrossRef PubMed Web of Science 14. ↵ Ramamoorthy , M. and Smith , S. ( 2015 ) Loss of ATRX Suppresses Resolution of Telomere Cohesion to Control Recombination in ALT Cancer Cells . Cancer Cell , 28 , 357 – 369 . OpenUrl CrossRef PubMed 15. ↵ Ritchie , K. , Seah , C. , Moulin , J. , Isaac , C. , Dick , F. and Berube , N.G. ( 2008 ) Loss of ATRX leads to chromosome cohesion and congression defects . J Cell Biol , 180 , 315 – 324 . OpenUrl Abstract / FREE Full Text 16. ↵ Clynes , D. , Jelinska , C. , Xella , B. , Ayyub , H. , Taylor , S. , Mitson , M. , Bachrati , C.Z. , Higgs , D.R. and Gibbons , R.J. ( 2014 ) ATRX dysfunction induces replication defects in primary mouse cells . PLoS One , 9 , e92915 . OpenUrl CrossRef PubMed 17. ↵ Leung , J.W. , Ghosal , G. , Wang , W. , Shen , X. , Wang , J. , Li , L. and Chen , J. ( 2013 ) Alpha thalassemia/mental retardation syndrome X-linked gene product ATRX is required for proper replication restart and cellular resistance to replication stress . J Biol Chem , 288 , 6342 – 6350 . OpenUrl Abstract / FREE Full Text 18. ↵ Oppel , F. , Tao , T. , Shi , H. , Ross , K.N. , Zimmerman , M.W. , He , S. , Tong , G. , Aster , J.C. and Look , A.T. ( 2019 ) Loss of atrx cooperates with p53-deficiency to promote the development of sarcomas and other malignancies . PLoS Genet , 15 , e1008039 . OpenUrl CrossRef PubMed 19. ↵ Berube , N.G. , Mangelsdorf , M. , Jagla , M. , Vanderluit , J. , Garrick , D. , Gibbons , R.J. , Higgs , D.R. , Slack , R.S. and Picketts , D.J. ( 2005 ) The chromatin-remodeling protein ATRX is critical for neuronal survival during corticogenesis . J Clin Invest , 115 , 258 – 267 . OpenUrl CrossRef PubMed Web of Science 20. ↵ Bassett , A.R. , Cooper , S.E. , Ragab , A. and Travers , A.A. ( 2008 ) The chromatin remodelling factor dATRX is involved in heterochromatin formation . PLoS One , 3 , e2099 . OpenUrl CrossRef PubMed 21. ↵ Medina , C.F. , Mazerolle , C. , Wang , Y. , Berube , N.G. , Coupland , S. , Gibbons , R.J. , Wallace , V.A. and Picketts , D.J. ( 2009 ) Altered visual function and interneuron survival in Atrx knockout mice: inference for the human syndrome . Hum Mol Genet , 18 , 966 – 977 . OpenUrl CrossRef PubMed 22. ↵ Horner , M.A. , Quintin , S. , Domeier , M.E. , Kimble , J. , Labouesse , M. and Mango , S.E. ( 1998 ) pha-4, an HNF-3 homolog, specifies pharyngeal organ identity in Caenorhabditis elegans . Genes Dev , 12 , 1947 – 1952 . OpenUrl Abstract / FREE Full Text 23. ↵ McGhee , J.D. , Fukushige , T. , Krause , M.W. , Minnema , S.E. , Goszczynski , B. , Gaudet , J. , Kohara , Y. , Bossinger , O. , Zhao , Y. , Khattra , J. et al. ( 2009 ) ELT-2 is the predominant transcription factor controlling differentiation and function of the C. elegans intestine, from embryo to adult . Dev Biol , 327 , 551 – 565 . OpenUrl CrossRef PubMed 24. ↵ Wang , J.T. and Seydoux , G. ( 2013 ) Germ cell specification . Adv Exp Med Biol , 757 , 17 – 39 . OpenUrl CrossRef PubMed 25. ↵ Henson , J.D. , Cao , Y. , Huschtscha , L.I. , Chang , A.C. , Au , A.Y. , Pickett , H.A. and Reddel , R.R. ( 2009 ) DNA C-circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity . Nat Biotechnol , 27 , 1181 – 1185 . OpenUrl CrossRef PubMed Web of Science 26. ↵ Meeuse , M.W. , Hauser , Y.P. , Morales Moya , L.J. , Hendriks , G.J. , Eglinger , J. , Bogaarts , G. , Tsiairis , C. and Grosshans , H. ( 2020 ) Developmental function and state transitions of a gene expression oscillator in Caenorhabditis elegans . Mol Syst Biol , 16 , e9498 . OpenUrl CrossRef PubMed 27. ↵ Olmedo , M. , Geibel , M. , Artal-Sanz , M. and Merrow , M. ( 2015 ) A High-Throughput Method for the Analysis of Larval Developmental Phenotypes in Caenorhabditis elegans . Genetics , 201 , 443 – 448 . OpenUrl Abstract / FREE Full Text 28. ↵ Nahar , S. , Morales Moya , L.J. , Brunner , J. , Hendriks , G.J. , Towbin , B. , Hauser , Y.P. , Brancati , G. , Gaidatzis , D. and Grosshans , H. ( 2024 ) Dynamics of miRNA accumulation during C. elegans larval development . Nucleic Acids Res , 52 , 5336 – 5355 . OpenUrl CrossRef PubMed 29. ↵ Beltran , T. , Pahita , E. , Ghosh , S. , Lenhard , B. and Sarkies , P. ( 2021 ) Integrator is recruited to promoter-proximally paused RNA Pol II to generate Caenorhabditis elegans piRNA precursors . EMBO J , 40 , e105564 . OpenUrl CrossRef PubMed 30. ↵ Martin , M. ( 2011 ) Cutadapt removes adapter sequences from high-throughput sequencing reads . EMBnet.journal 17 , 10 – 12 . OpenUrl 31. ↵ Dobin , A. , Davis , C.A. , Schlesinger , F. , Drenkow , J. , Zaleski , C. , Jha , S. , Batut , P. , Chaisson , M. and Gingeras , T.R. ( 2013 ) STAR: ultrafast universal RNA-seq aligner . Bioinformatics , 29 , 15 – 21 . OpenUrl CrossRef PubMed Web of Science 32. ↵ Danecek , P. , Bonfield , J.K. , Liddle , J. , Marshall , J. , Ohan , V. , Pollard , M.O. , Whitwham , A. , Keane , T. , McCarthy , S.A. , Davies , R.M. et al. ( 2021 ) Twelve years of SAMtools and BCFtools . Gigascience , 10 . 33. ↵ Liao , Y. , Smyth , G.K. and Shi , W. ( 2014 ) featureCounts: an efficient general purpose program for assigning sequence reads to genomic features . Bioinformatics , 30 , 923 – 930 . OpenUrl CrossRef PubMed Web of Science 34. ↵ Robinson , M.D. , McCarthy , D.J. and Smyth , G.K. ( 2010 ) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data . Bioinformatics , 26 , 139 – 140 . OpenUrl CrossRef PubMed Web of Science 35. ↵ Tang , D. , Chen , M. , Huang , X. , Zhang , G. , Zeng , L. , Zhang , G. , Wu , S. and Wang , Y. ( 2023 ) SRplot: A free online platform for data visualization and graphing . PLoS One , 18 , e0294236 . OpenUrl CrossRef PubMed 36. ↵ Quinlan , A.R. and Hall , I.M. ( 2010 ) BEDTools: a flexible suite of utilities for comparing genomic features . Bioinformatics , 26 , 841 – 842 . OpenUrl CrossRef PubMed Web of Science 37. ↵ Brenner , S. ( 1974 ) The genetics of Caenorhabditis elegans . Genetics , 77 , 71 – 94 . OpenUrl Abstract / FREE Full Text 38. ↵ Fan , H.Y. , Trotter , K.W. , Archer , T.K. and Kingston , R.E. ( 2005 ) Swapping function of two chromatin remodeling complexes . Mol Cell , 17 , 805 – 815 . OpenUrl CrossRef PubMed Web of Science 39. ↵ Cardoso , C. , Couillault , C. , Mignon-Ravix , C. , Millet , A. , Ewbank , J.J. , Fontes , M. and Pujol , N. ( 2005 ) XNP-1/ATR-X acts with RB, HP1 and the NuRD complex during larval development in C. elegans . Developmental biology , 278 , 49 – 59 . OpenUrl CrossRef PubMed 40. ↵ Bender , A.M. , Wells , O. and Fay , D.S. ( 2004 ) lin-35/Rb and xnp-1/ATR-X function redundantly to control somatic gonad development in C. elegans . Developmental biology , 273 , 335 – 349 . OpenUrl CrossRef PubMed Web of Science 41. ↵ Lackner , D.H. , Raices , M. , Maruyama , H. , Haggblom , C. and Karlseder , J. ( 2012 ) Organismal propagation in the absence of a functional telomerase pathway in Caenorhabditis elegans . EMBO J ., 31 , 2024 – 2033 . OpenUrl Abstract / FREE Full Text 42. ↵ Cheng , C. , Shtessel , L. , Brady , M.M. and Ahmed , S. ( 2012 ) Caenorhabditis elegans POT-2 telomere protein represses a mode of alternative lengthening of telomeres with normal telomere lengths . Proceedings of the National Academy of Sciences of the United States of America , 109 , 7805 – 7810 . OpenUrl Abstract / FREE Full Text 43. ↵ Shtessel , L. , Lowden , M.R. , Cheng , C. , Simon , M. , Wang , K. and Ahmed , S. ( 2013 ) Caenorhabditis elegans POT-1 and POT-2 repress telomere maintenance pathways . G3 , 3 , 305 – 313 . OpenUrl CrossRef PubMed 44. ↵ Lovejoy , C.A. , Takai , K. , Huh , M.S. , Picketts , D.J. and de Lange , T. ( 2020 ) ATRX affects the repair of telomeric DSBs by promoting cohesion and a DAXX-dependent activity . PLoS Biol , 18 , e3000594 . OpenUrl CrossRef PubMed 45. ↵ Rose , A.M. , Goncalves , T. , Cunniffe , S. , Geiller , H.E.B. , Kent , T. , Shepherd , S. , Ratnaweera , M. , O’Sullivan , R.J. , Gibbons , R.J. and Clynes , D. ( 2023 ) Induction of the alternative lengthening of telomeres pathway by trapping of proteins on DNA . Nucleic Acids Res , 51 , 6509 – 6527 . OpenUrl CrossRef PubMed 46. ↵ Gibbons , R. ( 2006 ) Alpha thalassaemia-mental retardation, X linked . Orphanet J Rare Dis , 1 , 15 . OpenUrl CrossRef PubMed 47. ↵ Zeller , P. , Padeken , J. , van Schendel , R. , Kalck , V. , Tijsterman , M. and Gasser , S.M. ( 2016 ) Histone H3K9 methylation is dispensable for Caenorhabditis elegans development but suppresses RNA:DNA hybrid-associated repeat instability . Nat Genet , 48 , 1385 – 1395 . OpenUrl CrossRef PubMed 48. ↵ Ganko , E.W. , Fielman , K.T. and McDonald , J.F. ( 2001 ) Evolutionary history of Cer elements and their impact on the C. elegans genome . Genome Res , 11 , 2066 – 2074 . OpenUrl Abstract / FREE Full Text 49. ↵ Towbin , B.D. , Gonzalez-Aguilera , C. , Sack , R. , Gaidatzis , D. , Kalck , V. , Meister , P. , Askjaer , P. and Gasser , S.M. ( 2012 ) Step-wise methylation of histone H3K9 positions heterochromatin at the nuclear periphery . Cell , 150 , 934 – 947 . OpenUrl CrossRef PubMed Web of Science 50. ↵ Wong , M.M. , Belew , M.D. , Kwieraga , A. , Nhan , J.D. and Michael , W.M. ( 2018 ) Programmed DNA Breaks Activate the Germline Genome in Caenorhabditis elegans . Dev Cell , 46 , 302 – 315 e305. OpenUrl CrossRef PubMed 51. ↵ Petrella , L.N. , Wang , W. , Spike , C.A. , Rechtsteiner , A. , Reinke , V. and Strome , S. ( 2011 ) synMuv B proteins antagonize germline fate in the intestine and ensure C. elegans survival . Development , 138 , 1069 – 1079 . OpenUrl Abstract / FREE Full Text 52. ↵ Hoareau , M. , Rincheval-Arnold , A. , Gaumer , S. and Guenal , I. ( 2024 ) DREAM a little dREAM of DRM: Model organisms and conservation of DREAM-like complexes: Model organisms uncover the mechanisms of DREAM-mediated transcription regulation . Bioessays , 46 , e2300125 . OpenUrl CrossRef PubMed 53. ↵ Kudron , M. , Gevirtzman , L. , Victorsen , A. , Lear , B.C. , Gao , J. , Xu , J. , Samanta , S. , Frink , E. , Tran-Pearson , A. , Huynh , C. , et al. ( 2024 ) Binding profiles for 954 Drosophila and C. elegans transcription factors reveal tissue specific regulatory relationships . bioRxiv. 54. ↵ Cao , W. , Fan , Q. , Amparado , G. , Begic , D. , Godini , R. , Gopal , S. and Pocock , R. ( 2024 ) A nucleic acid binding protein map of germline regulation in Caenorhabditis elegans . Nature communications , 15 , 6884 . OpenUrl PubMed 55. ↵ Maeda , I. , Kohara , Y. , Yamamoto , M. and Sugimoto , A. ( 2001 ) Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi . Curr Biol , 11 , 171 – 176 . OpenUrl CrossRef PubMed Web of Science 56. ↵ Wang , D.Y. , Kumar , S. and Hedges , S.B. ( 1999 ) Divergence time estimates for the early history of animal phyla and the origin of plants, animals and fungi . Proc Biol Sci , 266 , 163 – 171 . OpenUrl CrossRef GeoRef PubMed 57. ↵ Aguilera , P. and Lopez-Contreras , A.J. ( 2023 ) ATRX, a guardian of chromatin . Trends Genet , 39 , 505 – 519 . OpenUrl CrossRef PubMed 58. ↵ Ghaddar , A. , Armingol , E. , Huynh , C. , Gevirtzman , L. , Lewis , N.E. , Waterston , R. and O’Rourke , E.J. ( 2023 ) Whole-body gene expression atlas of an adult metazoan . Sci Adv , 9 , eadg0506 . OpenUrl CrossRef PubMed 59. ↵ van der Heijden , G.W. and Bortvin , A. ( 2009 ) Transient relaxation of transposon silencing at the onset of mammalian meiosis . Epigenetics , 4 , 76 – 79 . OpenUrl CrossRef PubMed Web of Science 60. ↵ Dennis , S. , Sheth , U. , Feldman , J.L. , English , K.A. and Priess , J.R. ( 2012 ) C. elegans germ cells show temperature and age-dependent expression of Cer1, a Gypsy/Ty3-related retrotransposon . PLoS Pathog , 8 , e1002591 . OpenUrl CrossRef PubMed 61. ↵ Lu , R. and Pickett , H.A. ( 2022 ) Telomeric replication stress: the beginning and the end for alternative lengthening of telomeres cancers . Open Biol , 12 , 220011 . OpenUrl CrossRef PubMed 62. ↵ Lingg , L. , Rottenberg , S. and Francica , P. ( 2022 ) Meiotic Genes and DNA Double Strand Break Repair in Cancer . Front Genet , 13 , 831620 . OpenUrl CrossRef PubMed 63. ↵ Teng , Y.C. , Sundaresan , A. , O’Hara , R. , Gant , V.U. , Li , M. , Martire , S. , Warshaw , J.N. , Basu , A. and Banaszynski , L.A. ( 2021 ) ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress . Nature communications , 12 , 3887 . OpenUrl PubMed 64. ↵ Scott , W.A. , Dhanji , E.Z. , Dyakov , B.J.A. , Dreseris , E.S. , Asa , J.S. , Grange , L.J. , Mirceta , M. , Pearson , C.E. , Stewart , G.S. , Gingras , A.C. et al. ( 2021 ) ATRX proximal protein associations boast roles beyond histone deposition . PLoS Genet , 17 , e1009909 . OpenUrl CrossRef PubMed 65. ↵ Fang , Y. , Barrows , D. , Dabas , Y. , Carroll , T.S. , Singer , S. , Tap , W.D. and Nacev , B.A. ( 2024 ) ATRX guards against aberrant differentiation in mesenchymal progenitor cells . Nucleic Acids Res , 52 , 4950 – 4968 . OpenUrl CrossRef PubMed 66. ↵ Rowland , M.E. , Jiang , Y. , Shafiq , S. , Ghahramani , A. , Pena-Ortiz , M.A. , Dumeaux , V. and Berube , N.G. ( 2023 ) Systemic and intrinsic functions of ATRX in glial cell fate and CNS myelination in male mice . Nature communications , 14 , 7090 . OpenUrl PubMed 67. ↵ Quesnel , K.M. , Martin-Kenny , N. and Berube , N.G. ( 2023 ) A mouse model of ATRX deficiency with cognitive deficits and autistic traits . J Neurodev Disord , 15 , 39 . OpenUrl CrossRef PubMed 68. ↵ Turkalo , T.K. , Maffia , A. , Schabort , J.J. , Regalado , S.G. , Bhakta , M. , Blanchette , M. , Spierings , D.C.J. , Lansdorp , P.M. and Hockemeyer , D. ( 2023 ) A non-genetic switch triggers alternative telomere lengthening and cellular immortalization in ATRX deficient cells . Nature communications , 14 , 939 . OpenUrl PubMed 69. ↵ Claude , E. and Decottignies , A. ( 2020 ) Telomere maintenance mechanisms in cancer: telomerase, ALT or lack thereof . Curr Opin Genet Dev , 60 , 1 – 8 . OpenUrl CrossRef PubMed 70. ↵ Cockrum , C.S. and Strome , S. ( 2022 ) Maternal H3K36 and H3K27 HMTs protect germline development via regulation of the transcription factor LIN-15B . Elife , 11 . View the discussion thread. Back to top Previous Next Posted January 14, 2026. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Share ATRX safeguards cellular identity during C. elegans development Janie Olver , Mariya Shtumpf , Karim Hussain , Stephen Methot , Peter Sarkies , Helder Ferreira bioRxiv 2025.03.11.641662; doi: https://doi.org/10.1101/2025.03.11.641662 Share This Article: Copy Citation Tools ATRX safeguards cellular identity during C. elegans development Janie Olver , Mariya Shtumpf , Karim Hussain , Stephen Methot , Peter Sarkies , Helder Ferreira bioRxiv 2025.03.11.641662; doi: https://doi.org/10.1101/2025.03.11.641662 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Molecular Biology Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17648) Bioengineering (13870) Bioinformatics (41880) Biophysics (21423) Cancer Biology (18553) Cell Biology (25458) Clinical Trials (138) Developmental Biology (13364) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15589) Genomics (22475) Immunology (17711) Microbiology (40326) Molecular Biology (17145) Neuroscience (88471) Paleontology (666) Pathology (2826) Pharmacology and Toxicology (4815) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)