Regulatory impact of activating and inactivating histone modifications for epigenetic control of Tet expression during murine brain development and aging

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J. Kraus, Hans A. Kretzschmar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4111305/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 Background: Recent studies demonstrated that 5-hydroxymethylcytosine (5hmC) is particularly enriched in the brain. It is the oxidative product of 5-methylcyotsine (5mC), mediated by the ten-eleven translocation (Tet) genes. Being the intermediate in DNA demethylation, 5hmC itself may also be regulated by certain chromatin structures. As there is an unknown biological mechanism in murine brain and other tissues, there is a need to investigate the biological mechanisms linking 5hmC formation and histone modifications. The aim of this study was to check up the regulation levels of quantified expression levels of genes that are associated with DNA demethylation in murine brain during aging at regions with the histone modifications H3K4me3 (a euchromatin mark) and H3K27me3 (a transcription repressive mark). Results: We show significant reduction of expression levels of the Tet genes compatible for regulation levels at H3K27me3. H3K4me3 displays relatively strong correlations with still known quantified 5hmC amounts in cortex and in cerebellum. Conclusions: Our results demonstrate a new insight into 5hmC availability depending on transcriptional activity and into Tet gene expressions depending on transcriptional repression. DNA modification Histone modification Epigenetics of aging Figures Figure 1 Figure 2 Figure 3 Background The major mechanisms of epigenetic regulation are DNA methylations and histone modifications [1, 2]. Methylation of DNA is a process by which at the 5-position (5mC) a methyl group is added to Cytosine. It is essential for several cellular processes [3, 4] and it is modulated by ten-eleven translocation (Tet) proteins by oxidation into 5-hydroxymethylcytosine (5hmC), further into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) [5-9]. 5hmC, widely accepted as the sixth base in mammalian genome, is known for its region and age dependent distribution in brain [8, 10]. Interestingly, Tet proteins are responsible for decreased expressions in frontal and cerebellar cortex during aging [11], collectively suggesting the initiation of active or passive DNA demethylation [12, 13]. The most extensively studied aforementioned histone modification is methylation, especially of histone H3 lysine 4 (H3K4) and H3K27 [14]. While H3K4 trimethylation (H3K4me3) is an active mark for transcription, H3K27 trimethylation (H3K27me3) is generally associated with transcriptional repression [15, 16]. Key developmental genes in embryonic stem cells are characterized by the co-existence of both modifications establishing the „bivalent domain“ [17, 18]. It is believed that they balance genes for activation or repression [17]. Whole-genome analysis could detect H3K27me3 throughout the whole genome [19-21], and its methyltransferase the Polycomb repressive complex 2 (PRC2) to unmethylated CGIs [16, 19-22]. H3K4me3 is also localized to unmethylated CGIs [23]. It is believed that histone modifications have been shown to regulate DNA methylation. For example DNMT3L which is a enzymatically inactive complex that induces DNA de novo methylation can be blocked by H3K4me3 ensuring the initiation of active gene transcription [8]. Recent studies have demonstrated that TET proteins are regulating the crosstalk between DNA methylation and histone methylation. TET2 has been found to catalyze 5mC oxidation by reactivating genes at repressive chromatin state [24]. In synopsis of these findings another issue is arising like whether there is an interplay between 5hmC, Tet expressions and histone modifications. In this study, we investigated the dynamics of H3K4me3 and H3K27me3 marks in regulating the Tet proteins and the still known quantitative 5hmC levels by focusing in distinct developmental status (embryonic day 16, day of birth, day 7, 15, 30 and 120 after birth) in the frontal cortex for histone modification analysis as well as in frontal and cerebellar cortex of murine brain for gene expression analysis. We uncovered regulatory patterns of histone methylation (H3K4me3 and H3K27me3) on Tet expression and DNA hydroxymethylation. Results Expression differences of Tet genes generating hydroxymethylation during brain aging beginning at embryonic day 16 In order to generate a complete picture of regulation and expression levels of enzymes that are responsible for demethylation during brain development and aging we integrated our previously published Tet expression data [11], attached starting with embryonic day 16 (E16). Investigating the expression levels of the Tet genes in murine frontal cortex and cerebellum from embryonic day 16 (E16) till the age of 120 days (P120) we found distinct differences of gene expression (Fig. 1a-1c, 2d-2f). In frontal cortex the expression levels of Tet 2 significantly decreased starting with E16 and ending with P120 (p<0.05, p<0.01, p<0.001, p<0.0001, ANOVA with Newman-Keuls post hoc) (Fig. 1b). The expression levels of Tet1 in frontal cortex (Fig. 1a) at E16 have significant lower Tet 1 expression levels than at P0 (p<0.001), P7 (p<0.01) and P15 (p<0.001). Comparing P30 and P120 with P0 (p<0.0001), P7 (p<0.001) and P15 (p<0.0001) shows significant decreased expression (ANOVA with Newman-Keuls post hoc). The same applies for Tet3 expression (ANOVA with Newman-Keuls post hoc) in frontal cortex (Fig. 1c) despite of differences in significance. The significantly increased expression levels of Tet 2 in cerebellum (Fig. 2e) are starting with E16 and are lasting till P0. At both days the Tet 2 expression in cerebellum are significantly higher compared with P7, P15, P30 and P120 (p varies between <0,05 and <0,001, ANOVA with Newman-Keuls post hoc). Comparing adult cerebellum of mice at the age of day 30 (p<0.01, ANOVA with Newman-Keuls post hoc) and 120 (p<0,05, ANOVA with Newman-Keuls post hoc) with embryonic day 16 it confirms a decreased expression level of Tet1 (Fig. 2d). Tet 1 expression at P0 (p<0,05) is significantly higher than at P30 (ANOVA with Newman-Keuls post hoc) (Fig. 2d). At day 7 of adult cerebellum there is a significant increased expression level of Tet 1 compared with the other expressions of adult age (p varies between <0,05 and <0,001, ANOVA with Newman-Keuls post hoc) (Fig. 2d). Tet 3 expression in cerebellar tissue at day 30 is lower than adult day 7 and embryonic day 16 (each p<0,01, ANOVA with Newman-Keuls post hoc) (Fig. 2f). Tet1 regulation at H3K4me3 and H3K27me3 In order to determine the regulation level of Tet genes at transcriptional activated and inactivated chromatin status in adult murine cortex ChIP-qPCR was carried out on H3K4me3 and H3K27me3 marks. ChIP-qPCR data reveal a significant gain of Tet levels at both marks. Analysis confirm in murine cerebrum of fetal (E16), adolescent, adult and elderly (P0, P7, P15, P30) as well as of aged brains (P120) a correlation between H3K4me3 or H3K27me3 and the activation or inactivation status of Tet1. As a mark of transcriptional inactivation H3K27me3 correlates with inactivation status of Tet1. There is increasing inactivation after E16, P7 and P30. In status of transcriptional activation (H3K4me3) there is nearly steady level of Tet 1 activation regardless of the age. These results do not show any significant changes (Fig. 3g). Tet2 regulation at H3K4me3 and H3K27me3 A further evidence for the role of H3K4me3 as a transcriptional activator is shown by the result of the regulation status of Tet2. Assessments of the activation and inactivation levels reveal only a slight change in the age groups. But concurrently, a significant increase of inactivation status in Tet2 in murine brain of P120 is detected (p between <0,01 and <0,001, ANOVA with Newman-Keuls post hoc) (Fig. 3h). In status of transcriptional activation (H3K4me3) there is nearly steady level of Tet 2 activation regardless of the age and without any significant changes (Fig. 3h). Tet3 regulation at H3K4me3 and H3K27me3 Next, we examine the correlation between aging and activation of Tet3. H3K4me3 leads to no significant levels over the life span (Fig. 3i). In contrast, an inverse alteration is observed at H3K27me3 mark: Tet3 in brain of embryonic day E16 and of P120 old mice has a significant higher inactivation compared with the remaining age groups (Fig. 3i). A significant increase of inactivation status in Tet3 is detected in murine brain as well of E16 compared with P0 (p<0,01), P7 (p<0,01), P15 (p<0,05) and P30 (p<0,01) as of P120 compared with P0 (p<0,05), P7 (p<0,05) and P30 (p<0,05) (ANOVA with Newman-Keuls post hoc) (Fig. 3i). Discussion The bivalent chromatin structure is created by H3K4me3 and H3K27me3 modifications. They maintain an appropriate balance in gene regulation of pluripotent stem cells and also in differentiated cells [17, 25, 26]. Our results shed light on the functional role of H3K4me3 and H3K27me3 in healthy adult murine brain. Both modifications have many notable features: Every Tet gene is regulated by H3K4me3 (Fig. 3g-3i). It has been reported before that inactive genes of ES cells with H3K4me3 might poise these genes for activation [17, 27]. Our data suggest that if chromatin signatures are existing in adult neuronal cells because of establishment during genome activation, they are suggested to increase expression of each Tet gene during aging process [11] . As previously reported the region and age dependent amount of 5hmC in brain is at lowest in early developmental stages but is getting more during brain development [8]. To ascertain the possibility that 5hmC is in crosstalk with the „bivalent domain“, we analyzed the ChIP-qPCR results of each Tet proteins at H3K4me3. The data reveal for each no significant effect (Fig. 3g-3i). Given the increase of 5hmC during aging, we conclude that this is due to the transcriptional activation levels shown by H3K4me3. Further we suggest that dosage compensation of H3K27me3 is the likely mechanism which may explain the variable inactivated levels of Tet proteins during the life span. The Polycomb repressive complex PRC2, associated with transcriptionally silent chromatin, has histone methyltransferase activity and trimethylates histone H3 on lysine 27. Loss of PRC2 has been postulated to lose the role of gene inactivation [22]. The inactivation profile of Tet1 at H3K27me3 in our project provides the evidence that the higher or lower the induced chromatin density in aging process by H3K27me3 marks, the lower or higher the gene expression-especially at postnatal day 120 (P120) (Fig. 1a, 1b). Regarding the results of Tet2 gene, we observe clear evidence for increase of Tet2 inactivation at H3K27me3 (Fig. 3h). Our analysis of published datasets suggests that this might be the reason for reduction of Tet2 gene expression in cortex and cerebellum [11] (Fig. 1b) and for an age dependent increase of 5hmC values [8]. This finding could also reveal a regulation of Tet2 by chromatin remodelling. A fundamental issue that remains is to unterstand the result of regulation of Tet3 at H3K27me3. Bivalent domains were found in ES cells poising genes for activation (H3K4me3) while keeping them repressed (H3K27me3) [17]. H3K4me3 and H3K27me3 might co-occupy the same promoters with equal affinity. This occupation would establish the same results of H3K4me3 and H3K27me3 regulation. But indeed, our results in adult neuronal cells indicate that both modifications have different regulation profiles (Fig. 1c, 3i). Basic cytology subdevides heterochromatin into constitutive and facultative heterochromatin, which becomes less-condensed euchromatin during development [28]. If H3K27me3 activates transcription in euchromatic chromosome regions this might be the cause for Tet3 expression in cerebellum [11]. A comparison of the H3K27me3 modification and Tet3 gene demonstrates the association with lineage differentiation. H3K27me3 has a role in X chromosome inactivation. Gene expression of Tet3 has been observed in oocytes and cygotes [9]. Our experiments suggest that inactivation of one of the X chromosomes in female cells is the mechanism of the inactivation profile of Tet3 at H3K27me3. For validation future experiments directly seperating into female and male brain tissues will be required. An inheritance model implies patterning of histone modifications during early development. While chromatin signatures in human and mouse spermatozoa and also before zygotic genome activation support this view, data in Xenopus and zebrafish embryos from the time of zygotic gene activation onward as well as in adult Drosophila show H3K4me3 or H3K27me3 enrichment [27, 29-33]. In contrast to those recent proposals we observe clear evidence for modified histones (K4 and K27) in adult murine brain. We suggest that H3K27me3 mark may established de novo , and would not require the faithful inheritance of histone modifications. This discrepancy could be due to the fact that gene regulation of H3K27me3 shows lower influence on inactivation resulting in increased Tet3 expression of adult and postnatal day 7 (P7) mouse cerebellum, at the peak of many differentiation events at this time [11, 34] (Fig. 2f, 3i). Prospective functional analysis of non-coding RNAs, of histone modifications and of sequence-specific transcription factors are essential. Tumor diseases are supporting our suggestion of de novo establishment of H3K27me3. Previous studies about myeloid malignancies showed loss 5hmC caused by TET gene mutations [35, 36]. In contrast, there was no association between loss of 5hmC and TET2 and TET3 gene alterations in human gliomas [37]. In summary, our data demonstrate that two modified histones (H3K4me3 and H3K27me3) in healthy brain tissue are influencing Tet enzyme activity. Further research will be necessary to understand the functional role of the bivalent domain in tumors of same dignity. Methods All experimental protocols were approved by Center of Neuropathology and Prion Research LMU München. All methods were carried out in accordance with relevant internal guidelines and regulations and were reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Brain samples For this study, we selected brain tissue samples of 30 mice (strain C57Bl6) per Tet gene analysis. Both female and male mice were used. Samples were assigned to six age groups with five cases per group. Each five mice were sacrificed at embryonic day 16 of development and day 0, 7, 15, 30, 120 after birth. Till the age of three weeks mice were housed together with the mother. Afterwards, they were removed from the mother and each five were housed in standard cages (30 x 15 x 120 cm). To identify the expression and regulation levels of genes each five mice were sacrificed at each time point. The brains were removed then stored at -80°C. Frontal cortex and cerebellum were selected as target regions. Extraction of RNA and reverse transcription reaction RNA extraction and reverse transcription were performed as previously described [11]. Quantification of gene expression using real-time PCR The expression levels of Tet genes at certain regions and age groups were analyzed as previously described [11]. Chromatin immunoprecipitation assay Frozen brain tissue was performed on ice beginning by homogenizing in 250 μl PBS. Cross-linking was done by incubation in 1% formaldehyde at 37% for 10 minutes, followed by adding glycine to a final concentration of 0.125 M. Genomic DNA was sheared to 200-500 bp using EpiSheary™ Probe Sonicator (Active Motif, California, USA: Catalog No. 53051) at optimal shearing conditions. The remaining steps based on the protocol MAGnify™ Chromatin Immunoprecipitation System (invitrogen, California, USA: Catalog No. 492024). Diluting sheared chromatin by adding Proteinase K followed by binding to Dynabeads (Dynabeads™ Protein G Immunoprecipitation Kit; invitrogen, California, USA: Catalog No. 10007D). Diluting sheared chromatin to 1:10 by adding Proteinase K. Lysates were then separated to pellet debris by binding to an Antibody-Dynabead complex which includes the primary antibody (anti-H3K4me3, anti-H3K27me3) (Active Motif, California, USA: Catalog No. 39060, 39055) or the rabbit IgG antibody (control) coupled to Dynabeads. Immunoprecipitation was carried out on rotator at 4°C overnight. Then the bound chromatin was washed three times with 100 µl IP Buffer 1 and two times with IP Buffer 2. Cross-linking was reverted by adding Proteinase K and incubating by heating at 55°C for 15 minutes, followed by a further boiling at 65°C for 15 minutes. Finally, un-crosslinked DNA was purified using magnetic beads and isolated by incubation for 20 minutes at 55°C. The resulting immunoprecipitated DNA was quantified with the Qubit fluorometer by using the Qubit™ dsDNA HS Assay Kit (invitrogen, California, USA: Catalog No. Q32851) and stored at -20°C until subjecting quantitative real-time PCR. All primers were synthetized by Eurofins MWG Operon despite Gapdh-2 and were performed specific for 17-28 bp. Gapdh-2 amplification was selected for data normalization. qRT-PCR analysis The regulation of all Tet genes at genomic DNA with the modifications H3K4me3 and H3K27me3 was investigated using the quantitative real-time PCR (qRT-PCR) technique. qRT-PCR was performed on the LightCycler 480 II (Roche, Germany) using SYBR™ Gren (Applied Biosystems, USA: Catalog No. A25741). Selected reference gene was Gapdh-2 (Active Motif, California, USA: Catalog No. 71018). The primer sequences for the Tet1 gene: forward 5'-CCAGCTCACCCTAAACTGC-3', reverse 5'-CTGGAAAGTTTGTCCAAGGATTG-3'; for Tet2 gene: forward 5'-CCGTCAAGAGCGAGGAAAG-3', reverse 5'-GGTGGACTGCGAGGCTG-3'; for Tet3 gene: forward 5'-GCAAGCCACTTTAGAACTTGC-3', reverse 5'-CATCACAGGTCATTTTGTAAAATAAAAG-3'. The experiment was repeated in triplicates with a final volume of 10 μl. Each sample contained 3 μl of gDNA, 2 μl of primer pair and 5 μl of SYBR Green (Applied Biosystems, Germany) in PCR 96-Well-Plate (Catalog No. 712282) (Biozym Scientific GmbH, Germany). The PCR program was carried out under thermal cycle conditions beginning with an initial preliminary denaturation at 50°C for 2 min, further 95°C for 10 min for denaturation, followed by 40 cycles of 94 °C for 15 sec, 53°C for 20 sec for primer annealing and elongation at 60°C for 1 min. Additionally, all samples were analyzed by a melting temperature. In cases of forming primer dimers the reaction was repeated. A standard curve was generated by a cDNA serial dilution and amplification efficiencies were calculated to correct gene regulation. Regulation levels of each gene were calculated relative to the regulation level of the reference gene Gapdh-2 by using the comparative Ct-method (∆∆Ct-method). Data analysis All data were statistically analyzed with Prism 8 (GraphPad) software. Post-hoc comparison was applied by one-way ANOVA using Neuman-Keuls Multiple Comparison Test. Samples per age group were considered an n=5 and data are shown as the mean ± standard error of mean (SEM). Statistical significance was assumed for p values <0.05. Abbreviations 5caC: 5-carboxylcytosine; 5fC: 5-formylcytosine; 5hmC: 5-hydroxymethylcytosine; 5mC: 5-methylcyotsine; bp: base pair; cDNA: complementary deoxyribonucleic acid; CGIs: CpG islands; ChIP: Chromatin Immunoprecipitation; ∆∆Ct: delta-delta-threshold cycle; DNMT3L: DNA methyltransferase 3 like; dsDNA: double-stranded deoxyribonucleic acid; E16: embryonic day 16; ES cells: Embryonic stem cells; Gapdh-2: Glyceraldehyde 3-phosphate dehydrogenase 2; gDNA: genomic deoxyribonucleic acid; H3K4me3: trimethylation at histone H3 on lysine 4; H3K27me3: trimethylation at histone H3 on lysine 27; P0: postnatal day 0; PBS: Phosphate buffered saline; PCR: Polymerase chain reaction; PRC2: Polycomb repressive complex 2; qRT-PCR: quantitative real-time PCR; RNA: ribonucleic acid; Tet: ten-eleven translocation; TET: ten-eleven translocation. Declarations Author's contribution All the experiments were performed by SK. SK and TK designed the experiments. SK wrote the manuscript. TK participated in advision and active discussion. Both authors read and approved the final manuscript. HAK participated in supervision. Acknowledgments We thank the veterinary physicians of CNP (Center for Neuropathology and Prion Research) for providing brain speciments. Competing interests The authors declare that they have no competing interests. Data availability All data generated or analysed during this study are included in this published article [and its supplementary file]. Further data that support the findings of this study are available from the corresponding author on reasonable request. Consent for publication Not applicable. Ethics approval All procedures performed in this study involving murine participants were in accordance with the ethical standards of the institute at which the experiments were conducted. Funding This work was supported by the Förderprogramm für Forschung und Lehre (FöFoLe) of the Ludwig-Maximilians-University Munich. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4111305","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":289561923,"identity":"499da4b8-f814-45b0-a531-0cd34f48b81c","order_by":0,"name":"Selma Kilinc","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYFAC5gYGhgMSMmwMDIyPIQJAER68WhjBWniAWpiNGRgMgBQjUVrAatikwVoYCGiRb29s3fDjjAUPn3Tzs+qCij/R/O1ALW8qcGsxOHOw7WbPDaDDZI6Z3Z5xxiB3xmHGBsY5Z/BokUhsu8HzAahFIoftNm+bQW4DUAszbxseh81/2HbzD1RLMUjLfLCWf3g8c4Ox7TbPDYgWZpCWDWAtDfj8kth2W+YMSEuasTTPGePcjUAtB+ccw+Ow9sPHbr45VicnPyP54WeeCrnceecPH3zwpgaPw7CCA6RqGAWjYBSMglGACgAOX1HdnmAS/gAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Selma","middleName":"","lastName":"Kilinc","suffix":""},{"id":289561925,"identity":"fedc56ce-1190-44cf-93b3-d5577fce743f","order_by":1,"name":"Theo F. J. Kraus","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Theo","middleName":"F. J.","lastName":"Kraus","suffix":""},{"id":289561926,"identity":"82f2d14f-bf99-41b5-a76c-f8139823d725","order_by":2,"name":"Hans A. Kretzschmar","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hans","middleName":"A.","lastName":"Kretzschmar","suffix":""}],"badges":[],"createdAt":"2024-03-16 04:59:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4111305/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4111305/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54499501,"identity":"b6518f7f-0542-4e4d-90f6-5b047aed7eb1","added_by":"auto","created_at":"2024-04-11 12:35:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":389639,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4111305/v1/2a86551332ae1a40f4f79236.png"},{"id":54499212,"identity":"06ab73cf-75bb-493c-811a-6acbbb431153","added_by":"auto","created_at":"2024-04-11 12:27:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":379942,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4111305/v1/29f894ae50d69420a7e7f558.png"},{"id":54499214,"identity":"a8da8a43-961a-4b82-8c87-3b596d54b1c8","added_by":"auto","created_at":"2024-04-11 12:27:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":451729,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4111305/v1/92860f87b683c56d40283406.png"},{"id":56509906,"identity":"b2d1696a-e9d4-4c7b-b7f8-1211a361b295","added_by":"auto","created_at":"2024-05-15 06:20:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1755356,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4111305/v1/629baa63-b16c-4e1d-98d3-cd59ce39baf0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regulatory impact of activating and inactivating histone modifications for epigenetic control of Tet expression during murine brain development and aging","fulltext":[{"header":"Background","content":"\u003cp\u003eThe major mechanisms of epigenetic regulation are DNA methylations and histone modifications\u0026nbsp;[1, 2]. Methylation of DNA is a process by which at the 5-position (5mC) a methyl group is added to Cytosine. It is essential for several cellular processes\u0026nbsp;[3, 4]\u0026nbsp;and it is modulated by ten-eleven translocation (Tet) proteins by oxidation into 5-hydroxymethylcytosine (5hmC), further into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) \u0026nbsp;[5-9]. 5hmC, widely accepted as the sixth base in mammalian genome, is known for its region and age dependent distribution in brain\u0026nbsp;[8, 10]. Interestingly, Tet proteins are responsible for decreased expressions in frontal and cerebellar cortex during aging\u0026nbsp;[11], collectively suggesting the initiation of active or passive DNA demethylation\u0026nbsp;[12, 13].\u003c/p\u003e\n\u003cp\u003eThe most extensively studied aforementioned histone modification is methylation, especially of histone H3 lysine 4 (H3K4) and H3K27\u0026nbsp;[14]. While H3K4 trimethylation (H3K4me3) is an active mark for transcription, H3K27 trimethylation (H3K27me3) is generally associated with transcriptional repression\u0026nbsp;[15, 16]. Key developmental genes in embryonic stem cells are characterized by the co-existence of both modifications establishing the \u0026bdquo;bivalent domain\u0026ldquo;\u0026nbsp;[17, 18]. It is believed that they balance genes for activation or repression\u0026nbsp;[17].\u003c/p\u003e\n\u003cp\u003eWhole-genome analysis could detect H3K27me3 throughout the whole genome\u0026nbsp;[19-21], and its methyltransferase the Polycomb repressive complex 2 (PRC2) to unmethylated CGIs\u0026nbsp;[16, 19-22]. H3K4me3 is also localized to unmethylated CGIs\u0026nbsp;[23].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is believed that histone modifications have been shown to regulate DNA methylation. For example DNMT3L which is a enzymatically inactive complex that induces DNA \u003cem\u003ede novo\u003c/em\u003e methylation can be blocked by H3K4me3 ensuring the initiation of active gene transcription\u0026nbsp;[8].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRecent studies have demonstrated that TET proteins are regulating the crosstalk between DNA methylation and histone methylation. TET2 has been found to catalyze 5mC oxidation by reactivating genes at repressive chromatin state\u0026nbsp;[24].\u003c/p\u003e\n\u003cp\u003eIn synopsis of these findings another issue is arising like whether there is an interplay between 5hmC, Tet expressions and histone modifications. In this study, we investigated the dynamics of H3K4me3 and H3K27me3 marks in regulating the Tet proteins and the still known quantitative 5hmC levels by focusing in distinct developmental status (embryonic day 16, day of birth, day 7, 15, 30 and 120 after birth) in the frontal cortex for histone modification analysis as well as in frontal and cerebellar cortex of murine brain for gene expression analysis. We uncovered regulatory patterns of histone methylation (H3K4me3 and H3K27me3) on Tet expression and DNA hydroxymethylation.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eExpression differences of Tet genes generating hydroxymethylation during brain aging beginning at embryonic day 16\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to generate a complete picture of regulation and expression levels of enzymes that are responsible for demethylation during brain development and aging we integrated our previously published Tet expression data\u0026nbsp;[11], attached starting with embryonic day 16 (E16). Investigating the expression levels of the Tet genes in murine frontal cortex and cerebellum from embryonic day 16 (E16) till the age of 120 days (P120) we found distinct differences of gene expression (Fig. 1a-1c, 2d-2f). In frontal cortex the expression levels of Tet 2 significantly decreased starting with E16 and ending with P120 (p\u0026lt;0.05, p\u0026lt;0.01, p\u0026lt;0.001, p\u0026lt;0.0001, ANOVA with Newman-Keuls post hoc) (Fig. 1b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe expression levels of Tet1 in frontal cortex (Fig. 1a) at E16 have significant lower Tet 1 expression levels than at P0 (p\u0026lt;0.001), P7 (p\u0026lt;0.01) and P15 (p\u0026lt;0.001). Comparing P30 and P120 with P0 (p\u0026lt;0.0001), P7 (p\u0026lt;0.001) and P15 (p\u0026lt;0.0001) shows significant decreased expression (ANOVA with Newman-Keuls post hoc). The same applies for Tet3 expression (ANOVA with Newman-Keuls post hoc) in frontal cortex (Fig. 1c) despite of differences in significance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe significantly increased expression levels of Tet 2 in cerebellum (Fig. 2e) are starting with E16 and are lasting till P0. At both days the Tet 2 expression in cerebellum are significantly higher compared with P7, P15, P30 and P120 (p varies between \u0026lt;0,05 and \u0026lt;0,001, ANOVA with Newman-Keuls post hoc).\u003c/p\u003e\n\u003cp\u003eComparing adult cerebellum of mice at the age of day 30 (p\u0026lt;0.01, ANOVA with Newman-Keuls post hoc) and 120 (p\u0026lt;0,05, ANOVA with Newman-Keuls post hoc) with embryonic day 16 it confirms a decreased expression level of Tet1 (Fig. 2d). Tet 1 expression at P0 (p\u0026lt;0,05) is significantly higher than at P30 (ANOVA with Newman-Keuls post hoc) (Fig. 2d). At day 7 of adult cerebellum there is a significant increased expression level of Tet 1 compared with the other expressions of adult age (p varies between \u0026lt;0,05 and \u0026lt;0,001, ANOVA with Newman-Keuls post hoc) (Fig. 2d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTet 3 expression in cerebellar tissue at day 30 is lower than adult day 7 and embryonic day 16 (each p\u0026lt;0,01, ANOVA with Newman-Keuls post hoc) (Fig. 2f). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTet1 regulation at H3K4me3 and H3K27me3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to determine the regulation level of Tet genes at transcriptional activated and inactivated chromatin status in adult murine cortex ChIP-qPCR was carried out on H3K4me3 and H3K27me3 marks. ChIP-qPCR data reveal a significant gain of Tet levels at both marks. Analysis confirm in murine cerebrum of fetal (E16), adolescent, adult and elderly (P0, P7, P15, P30) as well as of aged brains (P120) a correlation between H3K4me3 or H3K27me3 and the activation or inactivation status of Tet1. As a mark of transcriptional inactivation H3K27me3 correlates with inactivation status of Tet1. There is increasing inactivation after E16, P7 and P30. In status of transcriptional activation (H3K4me3) there is nearly steady level of Tet 1 activation regardless of the age. These results do not show any significant changes (Fig. 3g).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTet2 regulation at H3K4me3 and H3K27me3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA further evidence for the role of H3K4me3 as a transcriptional activator is shown by the result of the regulation status of Tet2. Assessments of the activation and inactivation levels reveal only a slight change in the age groups. But concurrently, a significant increase of inactivation status in Tet2 in murine brain of P120 is detected (p between \u0026lt;0,01 and \u0026lt;0,001, ANOVA with Newman-Keuls post hoc) (Fig. 3h). In status of transcriptional activation (H3K4me3) there is nearly steady level of Tet 2 activation regardless of the age and without any significant changes (Fig. 3h).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTet3 regulation at H3K4me3 and H3K27me3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we examine the correlation between aging and activation of Tet3. H3K4me3 leads to no significant levels over the life span (Fig. 3i). In contrast, an inverse alteration is observed at H3K27me3 mark: Tet3 in brain of embryonic day E16 and of P120 old mice has a significant higher inactivation compared with the remaining age groups (Fig. 3i). \u0026nbsp;A significant increase of inactivation status in Tet3 is detected in murine brain as well of E16 compared with P0 (p\u0026lt;0,01), P7 (p\u0026lt;0,01), P15 (p\u0026lt;0,05) and P30 (p\u0026lt;0,01) as of P120 compared with P0 (p\u0026lt;0,05), P7 (p\u0026lt;0,05) and P30 (p\u0026lt;0,05) (ANOVA with Newman-Keuls post hoc) (Fig. 3i).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe bivalent chromatin structure is created by H3K4me3 and H3K27me3 modifications. They maintain an appropriate balance in gene regulation of pluripotent stem cells and also in differentiated cells\u0026nbsp;[17, 25, 26].\u003c/p\u003e\n\u003cp\u003eOur results shed light on the functional role of H3K4me3 and H3K27me3 in healthy adult murine brain. Both modifications have many notable features: Every Tet gene is regulated by H3K4me3 (Fig. 3g-3i). It has been reported before that inactive genes of ES cells with H3K4me3 might poise these genes for activation\u0026nbsp;[17, 27]. Our data suggest that if chromatin signatures are existing in adult neuronal cells because of establishment during genome activation, they are suggested to increase expression of each Tet gene during aging process\u0026nbsp;[11]\u003cem\u003e.\u003c/em\u003e As previously reported the region and age dependent amount of 5hmC in brain is at lowest in early developmental stages but is getting more during brain development\u0026nbsp;[8]. To ascertain the possibility that 5hmC is in crosstalk with the \u0026bdquo;bivalent domain\u0026ldquo;, we analyzed the ChIP-qPCR results of each Tet proteins at H3K4me3. The data reveal for each no significant effect (Fig. 3g-3i). Given the increase of 5hmC during aging, we conclude that this is due to the transcriptional activation levels shown by H3K4me3. Further we suggest that dosage compensation of H3K27me3 is the likely mechanism which may explain the variable inactivated levels of Tet proteins during the life span.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Polycomb repressive complex PRC2, associated with transcriptionally silent chromatin, has histone methyltransferase activity and trimethylates histone H3 on lysine 27. Loss of PRC2 has been postulated to lose the role of gene inactivation\u0026nbsp;[22]. The inactivation profile of Tet1 at H3K27me3 in our project provides the evidence that the higher or lower the induced chromatin density in aging process by H3K27me3 marks, the lower or higher the gene expression-especially at postnatal day 120 (P120) (Fig. 1a, 1b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding the results of Tet2 gene, we observe clear evidence for increase of Tet2 inactivation at H3K27me3 (Fig. 3h). Our analysis of published datasets suggests that this might be the reason for reduction of Tet2 gene expression in cortex and cerebellum\u0026nbsp;[11]\u0026nbsp;(Fig. 1b) and for an age dependent increase of 5hmC values\u0026nbsp;[8]. This finding could also reveal a regulation of Tet2 by chromatin remodelling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA fundamental issue that remains is to unterstand the result of regulation of Tet3 at H3K27me3. Bivalent domains were found in ES cells poising genes for activation (H3K4me3) while keeping them repressed (H3K27me3)\u0026nbsp;[17]. H3K4me3 and H3K27me3 might co-occupy the same promoters with equal affinity. This occupation would establish the same results of H3K4me3 and H3K27me3 regulation. But indeed, our results in adult neuronal cells indicate that both modifications have different regulation profiles (Fig. 1c, 3i).\u003c/p\u003e\n\u003cp\u003eBasic cytology subdevides heterochromatin into constitutive and facultative heterochromatin, which becomes less-condensed euchromatin during development\u0026nbsp;[28]. If H3K27me3 activates transcription in euchromatic chromosome regions this might be the cause for Tet3 expression in cerebellum\u0026nbsp;[11].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA comparison of the H3K27me3 modification and Tet3 gene demonstrates the association with lineage differentiation. H3K27me3 has a role in X chromosome inactivation. Gene expression of Tet3 has been observed in oocytes and cygotes\u0026nbsp;[9]. Our experiments suggest that inactivation of one of the X chromosomes in female cells is the mechanism of the inactivation profile of Tet3 at H3K27me3. For validation future experiments directly seperating into female and male brain tissues will be required.\u003c/p\u003e\n\u003cp\u003eAn inheritance model implies patterning of histone modifications during early development. While chromatin signatures in human and mouse spermatozoa and also before zygotic genome activation support this view, data in Xenopus and zebrafish embryos from the time of zygotic gene activation onward as well as in adult Drosophila show H3K4me3 or H3K27me3 enrichment\u0026nbsp;[27, 29-33].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast to those recent proposals we observe clear evidence for modified histones (K4 and K27) in adult murine brain. We suggest that H3K27me3 mark may established \u003cem\u003ede novo\u003c/em\u003e, and would not require the faithful inheritance of histone modifications. This discrepancy could be due to the fact that gene regulation of H3K27me3 shows lower influence on inactivation resulting in increased Tet3 expression of adult and postnatal day 7 (P7) mouse cerebellum, at the peak of many differentiation events at this time\u0026nbsp;[11, 34]\u0026nbsp;(Fig. 2f, 3i). Prospective functional analysis of non-coding RNAs, of histone modifications and of sequence-specific transcription factors are essential.\u003c/p\u003e\n\u003cp\u003eTumor diseases are supporting our suggestion of \u003cem\u003ede novo\u0026nbsp;\u003c/em\u003eestablishment of H3K27me3. Previous studies about myeloid malignancies showed loss 5hmC caused by TET gene mutations [35, 36]. In contrast, there was no association between loss of 5hmC and TET2 and TET3 gene alterations in human gliomas [37]. In summary, our data demonstrate that two modified histones (H3K4me3 and H3K27me3) in healthy brain tissue are influencing Tet enzyme activity. Further research will be necessary to understand the functional role of the bivalent domain in tumors of same dignity.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eAll experimental protocols were approved by Center of Neuropathology and Prion Research LMU M\u0026uuml;nchen. All methods were carried out in accordance with relevant internal guidelines and regulations and were reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBrain samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor this study, we selected brain tissue samples of 30 mice (strain C57Bl6) per Tet gene analysis. Both female and male mice were used. Samples were assigned to six age groups with five cases per group. Each five mice were sacrificed at embryonic day 16 of development and day 0, 7, 15, 30, 120 after birth. Till the age of three weeks mice were housed together with the mother. Afterwards, they were removed from the mother and each five were housed in standard cages (30 x 15 x 120 cm). To identify the expression and regulation levels of genes each five mice were sacrificed at each time point. The brains were removed then stored at -80\u0026deg;C. Frontal cortex and cerebellum were selected as target regions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eExtraction of RNA and reverse transcription reaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA extraction and reverse transcription were performed as previously described\u0026nbsp;[11].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of gene expression using real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression levels of Tet genes at certain regions and age groups were analyzed as previously described\u0026nbsp;[11].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eChromatin immunoprecipitation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrozen brain tissue was performed on ice beginning by homogenizing in 250 \u0026mu;l PBS. Cross-linking was done by incubation in 1% formaldehyde at 37% for 10 minutes, followed by adding glycine to a final concentration of 0.125 M. Genomic DNA was sheared to 200-500 bp using EpiSheary\u0026trade; Probe Sonicator (Active Motif, California, USA: Catalog No. 53051) at optimal shearing conditions. The remaining steps based on the protocol MAGnify\u0026trade; Chromatin Immunoprecipitation System (invitrogen, California, USA: Catalog No. 492024). Diluting sheared chromatin by adding Proteinase K followed by binding to Dynabeads (Dynabeads\u0026trade; Protein G Immunoprecipitation Kit; invitrogen, California, USA: Catalog No. 10007D). Diluting sheared chromatin to 1:10 by adding Proteinase K. Lysates were then separated to pellet debris by binding to an Antibody-Dynabead complex which includes the primary antibody (anti-H3K4me3, anti-H3K27me3) (Active Motif, California, USA: Catalog No. 39060, 39055) or the rabbit IgG antibody (control) coupled to Dynabeads. Immunoprecipitation was carried out on rotator at 4\u0026deg;C overnight. Then the bound chromatin was washed three times with 100 \u0026micro;l IP Buffer 1 and two times with IP Buffer 2. Cross-linking was reverted by adding Proteinase K and incubating by heating at 55\u0026deg;C for 15 minutes, followed by a further boiling at 65\u0026deg;C for 15 minutes. Finally, un-crosslinked DNA was purified using magnetic beads and isolated by incubation for 20 minutes at 55\u0026deg;C. The resulting immunoprecipitated DNA was quantified with the Qubit fluorometer by using the Qubit\u0026trade; dsDNA HS Assay Kit (invitrogen, California, USA: Catalog No. Q32851) and stored at -20\u0026deg;C until subjecting quantitative real-time PCR. All primers were synthetized by Eurofins MWG Operon despite Gapdh-2 and were performed specific for 17-28 bp. Gapdh-2 amplification was selected for data normalization.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe regulation of all Tet genes at genomic DNA with the modifications H3K4me3 and H3K27me3 was investigated using the quantitative real-time PCR (qRT-PCR) technique. qRT-PCR was performed on the LightCycler 480 II (Roche, Germany) using SYBR\u0026trade; Gren (Applied Biosystems, USA: Catalog No. A25741). Selected reference gene was Gapdh-2 (Active Motif, California, USA: Catalog No. 71018). The primer sequences for the Tet1 gene: forward 5\u0026apos;-CCAGCTCACCCTAAACTGC-3\u0026apos;, reverse 5\u0026apos;-CTGGAAAGTTTGTCCAAGGATTG-3\u0026apos;; for Tet2 gene: \u0026nbsp; forward 5\u0026apos;-CCGTCAAGAGCGAGGAAAG-3\u0026apos;, reverse 5\u0026apos;-GGTGGACTGCGAGGCTG-3\u0026apos;; for Tet3 gene: \u0026nbsp; forward 5\u0026apos;-GCAAGCCACTTTAGAACTTGC-3\u0026apos;, reverse 5\u0026apos;-CATCACAGGTCATTTTGTAAAATAAAAG-3\u0026apos;. The experiment was repeated in triplicates with a final volume of 10 \u0026mu;l. Each sample contained 3 \u0026mu;l of gDNA, 2 \u0026mu;l of primer pair and 5 \u0026mu;l of SYBR Green (Applied Biosystems, Germany) in PCR 96-Well-Plate (Catalog No. 712282) (Biozym Scientific GmbH, Germany). The PCR program was carried out under thermal cycle conditions beginning with an initial preliminary denaturation at 50\u0026deg;C for 2 min, further 95\u0026deg;C for 10 min for denaturation, followed by 40 cycles of 94 \u0026deg;C for 15 sec, 53\u0026deg;C for 20 sec for primer annealing and elongation at 60\u0026deg;C for 1 min. Additionally, all samples were analyzed by a melting temperature. In cases of forming primer dimers the reaction was repeated. A standard curve was generated by a cDNA serial dilution and amplification efficiencies were calculated to correct gene regulation. Regulation levels of each gene were calculated relative to the regulation level of the reference gene Gapdh-2 by using the comparative Ct-method (∆∆Ct-method).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were statistically analyzed with Prism 8 (GraphPad) software. Post-hoc comparison was applied by one-way ANOVA using Neuman-Keuls Multiple Comparison Test. Samples per age group were considered an n=5 and data are shown as the mean \u0026plusmn; standard error of mean (SEM). Statistical significance was assumed for \u003cem\u003ep\u003c/em\u003e values \u0026lt;0.05.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e5caC: 5-carboxylcytosine; 5fC: 5-formylcytosine; 5hmC: 5-hydroxymethylcytosine; 5mC: 5-methylcyotsine; bp: base pair; cDNA: complementary deoxyribonucleic acid; CGIs: CpG islands; ChIP: Chromatin Immunoprecipitation; ∆∆Ct: delta-delta-threshold cycle; DNMT3L: DNA methyltransferase 3 like; dsDNA: double-stranded deoxyribonucleic acid; E16: embryonic day 16; ES cells: Embryonic stem cells; Gapdh-2: Glyceraldehyde 3-phosphate dehydrogenase 2; gDNA: genomic deoxyribonucleic acid; H3K4me3: trimethylation at histone H3 on lysine 4; H3K27me3: trimethylation at histone H3 on lysine 27; P0: postnatal day 0; PBS: Phosphate buffered saline; PCR: Polymerase chain reaction; PRC2: Polycomb repressive complex 2; qRT-PCR: quantitative real-time PCR; RNA: ribonucleic acid; Tet: ten-eleven translocation; TET: ten-eleven translocation.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor\u0026apos;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the experiments were performed by SK. SK and TK designed the experiments. SK wrote the manuscript. TK participated in advision and active discussion. Both authors read and approved the final manuscript. HAK participated in supervision.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the veterinary physicians of CNP (Center for Neuropathology and Prion Research) for providing brain speciments. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary file]. Further data that support the findings of this study are available from the corresponding author on reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in this study involving murine participants were in accordance with the ethical standards of the institute at which the experiments were conducted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the F\u0026ouml;rderprogramm f\u0026uuml;r Forschung und Lehre (F\u0026ouml;FoLe) of the Ludwig-Maximilians-University Munich. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026lsquo;s information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026sup1;,\u0026sup2;,\u0026sup3; Center for Neuropathology and Prion Research, LMU M\u0026uuml;nchen, Munich, Germany\u003c/p\u003e\n\u003cp\u003e*Corresponding author. 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It is the oxidative product of 5-methylcyotsine (5mC), mediated by the ten-eleven translocation (Tet) genes. Being the intermediate in DNA demethylation, 5hmC itself may also be regulated by certain chromatin structures. As there is an unknown biological mechanism in murine brain and other tissues, there is a need to investigate the biological mechanisms linking 5hmC formation and histone modifications. The aim of this study was to check up the regulation levels of quantified expression levels of genes that are associated with DNA demethylation in murine brain during aging at regions with the histone modifications H3K4me3 (a euchromatin mark) and H3K27me3 (a transcription repressive mark).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e We show significant reduction of expression levels of the Tet genes compatible for regulation levels at H3K27me3. H3K4me3 displays relatively strong correlations with still known quantified 5hmC amounts in cortex and in cerebellum.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Our results demonstrate a new insight into 5hmC availability depending on transcriptional activity and into Tet gene expressions depending on transcriptional repression.\u003c/p\u003e","manuscriptTitle":"Regulatory impact of activating and inactivating histone modifications for epigenetic control of Tet expression during murine brain development and aging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-11 12:27:30","doi":"10.21203/rs.3.rs-4111305/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":"99f6d30a-d3d0-49d1-97d6-a9df86bb6bc4","owner":[],"postedDate":"April 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-15T06:11:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-11 12:27:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4111305","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4111305","identity":"rs-4111305","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}