Distinct catalytic-dependent or -independent activities of DNMT1 in mouse embryonic stem cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE179006 | GEO | 2022/01/01
REPOSITORIES: GEO
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Project description:DNA methylation is an essential for genome stability and mammalian embryonic development. DNA methyltransferase 1 (DNMT1) maintains cellular DNA methylation profile and its loss causes embryonic lethality. In this study, immunofluorescence staining, which can compare the difference between sister chromatids, were performed in mouse embryonic stem cells (ESCs) and suggest that the timing and region of de novo methylation by DNMT3 are restricted in DNMT1-deficient ESCs. To reanalyze the timing of de novo methylation by DNMT3, we collected cells from G1 to early S phases (called G1), cells in the mid-S phase, and cells in late S, G2, and M phases (called G2) by FACS, respectively. Among them, two cell populations, G1 and G2, were used for Me/hMeDIP-seq. As a result, the 5mC and 5hmC were globally decreased in the G2 cells.
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Project description:Site-specific hypermethylation of tumor suppressor genes accompanied by genome-wide hypomethylation are epigenetic hallmarks of malignancy. However, molecular mechanisms that drive these linked changes in DNA methylation remain obscure. DNA methyltransferase 1 (DNMT1), the principle enzyme responsible for maintaining methylation patterns is commonly dysregulated in tumors. Replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in gain of DNMT1 function. However, earlier data suggested that RFTS is required for DNMT1 activity. Here, we determined cellular consequences of RFTS deletion from DNMT1 in immortalized human bronchial epithelial cells. Compared to full-length DNMT1, ectopic expression of DNMT1- ?RFTS caused greater malignant transformation and enhanced promoter methylation that silenced DAPK and DUOX1 gene expression and increased intensity of promoter methylation across the genome. Strikingly, DNMT1-?RFTS also produced genomic hypomethylation and demethylation of Satellite 2 repeat sequences. Because deletion of RFTS is sufficient to induce focal hypermethylation and global hypomethylation in parallel, evidence suggests that the RFTS domain is a target responsible for epigenetic changes in cancer. DNA samples from HBEC3 cells transfected with vector control and HBEC3 cells with full-length and RFTS-deleted DNMT1 were assessed for genome-wide methylation using the microarray-based high resolution HpaII tiny fragment enriched by ligation-mediated PCR (HELP) assay.
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Project description:The DNA methyltransferase activity of DNMT1 is vital for genomic maintenance of DNA methylation. We report here that DNMT1 function is regulated by O-GlcNAcylation, a protein modification that is sensitive to glucose levels, and that elevated O-GlcNAcylation of DNMT1 from high glucose environment leads to alterations to the epigenome. Using mass spectrometry and complementary alanine mutation experiments, we identified S878 as the major residue that is O-GlcNAcylated on DNMT1. Functional studies further revealed that O-GlcNAcylation of DNMT1-S878 results in an inhibition of methyltransferase activity, resulting in a general loss of DNA methylation that is preferentially at partially methylated domains (PMDs). This loss of methylation corresponds with an increase in DNA damage and apoptosis. These results establish O-GlcNAcylation of DNMT1 as a mechanism through which the epigenome is regulated by glucose metabolism and implicates a role for glycosylation of DNMT1 in metabolic diseases characterized by hyperglycemia.
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Project description:DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Somatic patterns of DNA methylation are largely static, apart from focal dynamics at gene regulatory elements. To further advance our understanding of the role of DNA methylation in human development and disease, we inactivated all three catalytically active DNA methyltransferases in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing. Disruption of DNMT3A or DNMT3B individually, as well as of both enzymes in tandem, creates viable, pluripotent cell lines with distinct effects on their DNA methylation landscape as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome the immediate lethality, we generated a doxycycline (DOX) responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1 mutant lines. However, DOX-mediated repression of the exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death, demonstrating that DNA methylation is essential for human ESCs cultured in standard conditions. In summary, our data provide a comprehensive characterization of DNMT mutant ESCs, including single base genome-wide maps of their targets. RRBS methylation profiling of a time course of DNMT1* withdrawal in human ES cells
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Project description:DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Somatic patterns of DNA methylation are largely static, apart from focal dynamics at gene regulatory elements. To further advance our understanding of the role of DNA methylation in human development and disease, we inactivated all three catalytically active DNA methyltransferases in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing. Disruption of DNMT3A or DNMT3B individually, as well as of both enzymes in tandem, creates viable, pluripotent cell lines with distinct effects on their DNA methylation landscape as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome the immediate lethality, we generated a doxycycline (DOX) responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1 mutant lines. However, DOX-mediated repression of the exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death, demonstrating that DNA methylation is essential for human ESCs cultured in standard conditions. In summary, our data provide a comprehensive characterization of DNMT mutant ESCs, including single base genome-wide maps of their targets. RRBS profiling of mouse ESCs (wild type and DNMT KOs)
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Project description:DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Somatic patterns of DNA methylation are largely static, apart from focal dynamics at gene regulatory elements. To further advance our understanding of the role of DNA methylation in human development and disease, we inactivated all three catalytically active DNA methyltransferases in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing. Disruption of DNMT3A or DNMT3B individually, as well as of both enzymes in tandem, creates viable, pluripotent cell lines with distinct effects on their DNA methylation landscape as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome the immediate lethality, we generated a doxycycline (DOX) responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1 mutant lines. However, DOX-mediated repression of the exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death, demonstrating that DNA methylation is essential for human ESCs cultured in standard conditions. In summary, our data provide a comprehensive characterization of DNMT mutant ESCs, including single base genome-wide maps of their targets. RRBS methylation profiling of DNMT3A/3B DKO human ES cells
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Project description:Site-specific hypermethylation of tumor suppressor genes accompanied by genome-wide hypomethylation are epigenetic hallmarks of malignancy. However, molecular mechanisms that drive these linked changes in DNA methylation remain obscure. DNA methyltransferase 1 (DNMT1), the principle enzyme responsible for maintaining methylation patterns is commonly dysregulated in tumors. Replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in gain of DNMT1 function. However, earlier data suggested that RFTS is required for DNMT1 activity. Here, we determined cellular consequences of RFTS deletion from DNMT1 in immortalized human bronchial epithelial cells. Compared to full-length DNMT1, ectopic expression of DNMT1- ΔRFTS caused greater malignant transformation and enhanced promoter methylation that silenced DAPK and DUOX1 gene expression and increased intensity of promoter methylation across the genome. Strikingly, DNMT1-ΔRFTS also produced genomic hypomethylation and demethylation of Satellite 2 repeat sequences. Because deletion of RFTS is sufficient to induce focal hypermethylation and global hypomethylation in parallel, evidence suggests that the RFTS domain is a target responsible for epigenetic changes in cancer.
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