Demethylation of the protein phosphatase PP2A promotes the demethylation of histones to enable their function as a methyl group sink
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE121635 | GEO | 2019/02/15
REPOSITORIES: GEO
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Project description:S-adenosylmethionine (SAM) is the methyl donor for biological methylation modifications that regulate protein and nucleic acid functions. Here we show that methylation of a phospholipid, phosphatidylethanolamine (PE), is the major consumer of SAM in budding yeast. The induction of phospholipid biosynthetic genes is accompanied by induction of the enzyme that hydrolyzes S-adenosylhomocysteine (SAH), a product and inhibitor of methyltransferases. Beyond its function for the synthesis of phosphatidylcholine (PC), the methylation of PE facilitates the turnover of SAM for the synthesis of cysteine and glutathione. Strikingly, cells that lack PE methylation accumulate SAM, which leads to hypermethylation of histones and the major phosphatase PP2A, dependency on cysteine, and sensitivity to oxidative stress. Without PE methylation, particular sites on histones then become methyl sinks to enable the turnover of SAM. These findings reveal an unforeseen metabolic function for phospholipid and histone methylation intrinsic to the life of a cell.
2017-03-30 | GSE84059 | GEO
Project description:The arginine or lysine methylation status of histones dynamically changes during many essential cellular processes, particularly during embryonic and hematopoietic stem cell development. The enzymes demethylate methyllysine residues have been well defined, but the enzymes demethylate the methylarginine residues during different cellular processes are unknown. In current study, we demonstrate that JMJD1B is a lysine demethylase for H3K9me2 and an arginine demethylase for H4R3me2s. To reveal the biological significance of JMJD1B as an arginine demethylase, we isolate hematopoietic stem/progenitor cells (HSPC) from wild-type and JMJD1B knockout (JKO) bone marrow. We then conduct ChIP-seq on H4R3me2s and H3K9me2 histone markers and perform RNA-seq to determine the global gene expression profiles. We have observed global demethylation of H4R3me2s at the gene body but not the intergenic regions in hematopoietic stem/progenitor cells. H4R3me2s demethylation at the gene body region is directly correlated with gene expression in these cells. Furthermore, knockout of JMJD1B causes defects in removing the H4R3me2s epigenetic marker, leading to down-regulation of genes important for blood cells differentiation and development. Altogether, our current study demonstrates that arginine demethylases exist in cellular systems and that JMJD1B demethylates H4R3me2s for proper epigenetic programming during development.
2018-03-14 | GSE94966 | GEO
Project description:The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes with catalytic, scaffolding, and regulatory subunits. Although there are only ten catalytic PPP subunits encoded in the human genome, the formation of holoenzymes creates hundreds of unique enzymes that oppose kinases and carry out specific dephosphorylation reactions. Thus, to achieve the correct cellular phosphorylation state, the assembly of PPP holoenzymes needs to be tightly controlled. Indeed, changes in the cellular repertoire of PPPs are frequently linked to human disease, including cancer and neurodegeneration. The Protein Phosphatase 2A (PP2A) subfamily of PPPs consists of PP2A, PP4, PP6, and holoenzyme formation is at least in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as methylation) of the catalytic subunits. This reversible modification is catalyzed by a Leucine Carboxyl Methyltransferase-1 (LCMT1) that utilizes S-adenosyl-methionine (SAM) as the methyl donor and removed by Protein Phosphatase Methylesterase 1 (PME1). For PP2A, C-terminal methylation controls regulatory subunit selection. Notably, different types of regulatory subunits display differential methylation sensitivity. For PP4 and PP6, the role of C-terminal methylation is less well defined. Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to comprehensively elucidate the role of C-terminal methylation in PP2A, PP4, and PP6 function in multiple cell lines. Using these approaches, we quantitatively determine the effects of reduced C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly in an unbiased, isoform-specific manner.
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Project description:Histone lysine methylation is a key epigenetic modification that regulates eukaryotic transcription. In Saccharomyces cerevisiae, it is controlled by a reduced but evolutionarily conserved suite of methyltransferase (Set1p, Set2p, Dot1p, and Set5p) and demethylase (Jhd1p, Jhd2p, Rph1p, and Gis1p) enzymes. Many of these enzymes are extensively phosphorylated in vivo; however, the functions of specific phosphosites are poorly understood. Here, we comprehensively investigate the phosphoregulation of the yeast histone methylation network by analysing 40 phosphosites on six enzymes through mutagenesis. A total of 82 genomically-edited S. cerevisiae strains were generated and screened for changes in native H3K4, H3K36, and H3K79 methylation levels, and for sensitivity to environmental stress conditions. This demonstrated the functional relevance of phosphosites on methyltransferase Set2p (S6, S8, S10, and T127) and demethylase Jhd1p (S44) in the regulation of H3K36 methylation in vivo, and in the coordination of specific stress response pathways in budding yeast. Proteomic analysis of SET2 mutants revealed that phosphorylation site mutations lead to significant downregulation of membrane-associated proteins and processes, consistent with changes brought about by SET2 deletion. This study represents the first systematic investigation into the phosphoregulation of an entire epigenetic network in any eukaryote, and our findings establish phosphorylation as an important regulator of histone lysine methylation in S. cerevisiae.
2022-03-07 | PXD029088 | Pride
Project description:Demethylation of the protein phosphatase PP2A promotes the demethylation of histones to enable their function as a methyl group sink
| PRJNA497903 | ENA
Project description:Histone H3 lysine 4 tri-methylation (H3K4me3) is a hallmark of transcription initiation, but how H3K4me3 is demethylated during gene repression is poorly understood. Jhd2, a JmjC domain protein, was recently identified as the major H3K4me3 histone demethylase (HDM) in S. cerevisiae. While JHD2 is required for removal of methylation upon gene repression, deletion of JHD2 does not result in increased levels of H3K4me3 in bulk histones, indicating that this HDM is unable to demethylate histones during steady state conditions. In this study, we showed that this was due to the negative regulation of Jhd2 activity by histone H3 lysine 14 acetylation, which co-localizes with H3K4me3 across the yeast genome. We demonstrated that loss of the histone H3-specific acetyltransferases (HATs) resulted in genome-wide-depletion of H3K4me3, and this was not due to a transcription defect. Moreover, H3K4me3 levels were reestablished in HAT mutants following loss of JHD2, which suggested that H3-specific HATs and Jhd2 served opposing functions in regulating H3K4me3 levels. We revealed the molecular basis for this suppression by demonstrating that histone H3K14 acetylation negatively regulated Jhd2 demethylase activity on an acetylated peptide in vitro. These results revealed the existence of a general mechanism for removal of H3K4me3 following gene repression. Examination of H3K4me3 in WT, ada2sas3, ada2sas3jhd2, and jhd2 strains.
2012-11-05 | E-GEOD-41424 | biostudies-arrayexpress
Project description:Cytosine DNA methylation is considered to be a stable epigenetic mark, but active demethylation has been observed in both plants and animals. In Arabidopsis thaliana, DNA glycosylases of the DEMETER (DME) family remove methylcytosines from DNA. Demethylation by DME is necessary for genomic imprinting and demethylation by a related protein, REPRESSOR OF SILENCING1, which prevents gene silencing in a transgenic background. However, the extent and function of demethylation by DEMETER-LIKE (DML) proteins in WT plants is not known. Using genome-tiling microarrays, we mapped DNA methylation in mutant and WT plants and identified 179 loci actively demethylated by DML enzymes. Mutations in DML genes lead to locus-specific DNA hypermethylation. Reintroducing WT DML genes restores most loci to the normal pattern of methylation, although at some loci, hypermethylated epialleles persist. Of loci demethylated by DML enzymes, >80% are near or overlap genes. Genic demethylation by DML enzymes primarily occurs at the 5' and 3' ends, a pattern opposite to the overall distribution of WT DNA methylation. Our results show that demethylation by DML DNA glycosylases edits the patterns of DNA methylation within the Arabidopsis genome to protect genes from potentially deleterious methylation. Keywords: whole genome profiling, DNA methylation, demethylase
2007-04-02 | GSE7336 | GEO
Project description:N6-methyladenosine (m6A) is the most abundant internal modification in the messenger RNA (mRNA) of all higher eukaryotes. This modification has been shown to be reversible in mammals; it is installed by a methyltransferase heterodimer complex of METTL3 and METTL14 bound with WTAP, and reversed by iron(II)- and α-ketoglutarate-dependent demethylases FTO and ALKBH5. This modification exhibits significant functional roles in various biological processes. The m6A modification as a RNA mark is recognized by reader proteins, such as YTH domain family proteins and HNRNPA2B1; m6A can also act as a structure switch to affect RNA-protein interactions for biological regulation. In Arabidopsis thaliana, the methyltransferase subunit MTA (the plant orthologue of human METTL3, encoded by At4g10760) was well characterized and FIP37 (the plant orthologue of human WTAP) was first identified as the interacting partner of MTA. Here we report the discovery and characterization of reversible m6A methylation mediated by AtALKBH10B (encoded by At4g02940) in A. thaliana, and noticeable roles of this RNA demethylase in affecting plant development and floral transition. Our findings reveal potential broad functions of reversible mRNA methylation in plants. m6A peaks were identified from wild type Columbia-0 and atalkbh10b-1 mutant in two biological replicates
2016-03-24 | E-GEOD-79523 | biostudies-arrayexpress
Project description:Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play important roles in virus control and it has been suggested that Dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the A. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the A. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus control.
2017-11-30 | GSE82063 | GEO
Project description:DNA methylation plays major roles in the epigenetic regulation of gene expression, transposon and transcriptional silencing, and DNA repair, with implications in developmental processes and phenotypic plasticity. Relevantly for arboreal species, DNA methylation constitute a regulative layer in cell wall dynamics associated with xylogenesis. The use of methyltransferase and/or demethylase inhibitors has been proven informative to shed light on the methylome dynamics behind the regulation of these processes. The present work employs the cytidine analog zebularine to inhibit DNA methyltransferases and induce DNA hypomethylation in Salix purpurea plantlets grown in vitro and in soil. An integrative approach has been adopted to highlight the effects of hypomethylation on proteomic dynamics, exposing the age-specific (three weeks of in vitro culture and one month of growth in soil) and tissue-specific (shoot and root) effects following exposure to zebularine. Significant proteomic shifts were revealed in the development from in vitro to in-soil culture and, whereas zebularine treatment decreased methylation in three-weeks roots, a functionally heterogeneous subset of protein entries was differentially accumulated in shoot samples, including entries associated with cell wall dynamics, tissue morphogenesis, and hormonal regulation. The identification of tissue-specific proteomic hallmarks in combination with hypomethylating agents provides new insights into the role of DNA methylation in early plant development in willow species.
2024-01-08 | PXD045653 | Pride