Transcriptome analysis in DNMT3A and 3B mutant female human pluriopotent stem cells.
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE160454 | GEO | 2021/07/15
REPOSITORIES: GEO
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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: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.
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Project description:DNA methylation plays essential roles in mammalian development. During post-implantation development, de novo establishment of DNA methylation is accomplished by DNA methyltransferases, in particular DNMT3A and DNMT3B. At present, the distinct functions of these two enzymes remain largely elusive. To comprehensively identify the target sites for de novo DNA methylation by the DNMT3 enzymes, we took advantage of female mouse ES cells established in the presence of MEK and Gsk3 inhibitors, which lack most DNA methylation (2i/L ES cells), in combination with genetic ablation of Dnmt3a or Dnmt3b. We analyzed de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from Dnmt3 knockout (KO) 2i/L ES cells. Both Dnmt3a and Dnmt3b KO 2i-MEFs exhibited a modest but global reduction in CpG methylation, which was particularly notable on the X chromosome in Dnmt3b KO cells. Although most genes were methylated similarly in both knockouts, we identified 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b KO 2i-MEFs, respectively. Notably, Dnmt3a was exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes. Consistent with this, tissue-specific DNA methylation at PcG target developmental genes was substantially reduced in Dnmt3a KO embryos. Finally, we found that human patients with DNMT3A mutant acute myeloid leukemia (AML) or harboring DNMT3B mutation associated with immunodeficiency, centromere, and facial anomalies (ICF) syndrome exhibited reduced CpG methylation at regions that were hypomethylated in Dnmt3a or Dnmt3b KO 2i-MEFs, respectively. Collectively, our findings in DNA-hypomethylated 2i/L ES cells revealed a set of unique de novo DNA methylation target sites for both DNMT3 enzymes during mammalian development that overlap with hypomethylated sites in human patients.
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