Genome-wide identification of distinct target loci for de novo DNA methylation by DNMT3A and DNMT3B during mammalian development
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ABSTRACT:
PROVIDER: PRJNA436115 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11179756_1.fastq.gz | Fastqsanger.gz | |||
| SRR11179756_2.fastq.gz | Fastqsanger.gz | |||
| SRR11179757_1.fastq.gz | Fastqsanger.gz | |||
| SRR11179757_2.fastq.gz | Fastqsanger.gz | |||
| SRR11179758_1.fastq.gz | Fastqsanger.gz |
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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.
2020-05-13 | GSE111172 | GEO
Project description:Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterizations of DNMT3A and DNMT3B, here we uncovered distinct and interrelated modes-of-action underlying their CpG site and flanking sequence interaction. Strikingly, K777 of DNMT3B makes direct contacts with the DNA base at the +1 flank position of the cytosine methylation site, which contrasts with its counterpart in DNMT3A that forms base-specific contacts with the CpG site. Consequently, there is a divergent substrate and flanking sequence preference between DNMT3A and DNMT3B in vitro and in cells, thus providing an explanation for site-specific epigenomic alterations seen in ICF syndrome patients with DNMT3B mutations. Together, this study reveals crucial, yet complicated interplays of DNMT3s, DNA sequences and resultant methylation.
2020-06-18 | GSE145899 | GEO
Project description:Induced pluripotent stem cells (iPSCs) are generated from somatic cells by the transduction of defined transcription factors and involves dynamic changes in DNA methylation. While the reprogramming of somatic cells is accompanied by de-methylation of pluripotency genes, the functional importance of de novo DNA methylation has not been clarified. Here, using loss-of-function studies, we generated iPSCs from fibroblasts that were deficient in de novo DNA methylation mediated by Dnmt3a and Dnmt3b. These iPSCs reactivated pluripotency genes, underwent self-renewal and showed restricted developmental potential which was rescued upon re-introduction of Dnmt3a and Dnmt3b. We conclude that de novo DNA methylation by Dnmt3a and Dnmt3b is dispensable for nuclear reprogramming of somatic cells. RNA levels of Dnmt3ab deficient iPSC cell lines were compared to control iPSC cell lines
2011-05-16 | E-GEOD-28629 | biostudies-arrayexpress
Project description:Induced pluripotent stem cells (iPSCs) are generated from somatic cells by the transduction of defined transcription factors and involves dynamic changes in DNA methylation. While the reprogramming of somatic cells is accompanied by de-methylation of pluripotency genes, the functional importance of de novo DNA methylation has not been clarified. Here, using loss-of-function studies, we generated iPSCs from fibroblasts that were deficient in de novo DNA methylation mediated by Dnmt3a and Dnmt3b. These iPSCs reactivated pluripotency genes, underwent self-renewal and showed restricted developmental potential which was rescued upon re-introduction of Dnmt3a and Dnmt3b. We conclude that de novo DNA methylation by Dnmt3a and Dnmt3b is dispensable for nuclear reprogramming of somatic cells.
2011-05-16 | GSE28629 | GEO
Project description:DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) is essential for genome regulation and development. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-Å crystal structure of the DNMT3A-DNMT3L-DNA complex where two DNMT3A monomers simultaneously attack two CpG dinucleotides, with the target sites separated by fourteen base pairs within the same DNA duplex. The DNMT3A-DNA interaction involves a target recognition domain (TRD), a catalytic loop and DNMT3A homodimeric interface. A TRD residue Arg836 makes crucial contact with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Hematological cancer-associated somatic mutations at the substrate-binding sites decrease DNMT3A activity, induce CpG hypomethylation and promote transformation of hematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its etiologic link to human disease.
2018-01-01 | GSE99391 | GEO
Project description:De novo establishment of DNA methylation is accomplished by DNMT3A and DNMT3B. Here, we analyze de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from DNA-hypomethylated 2i/L ES cells with genetic ablation of Dnmt3a or Dnmt3b. We identify 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b knockout (KO) 2i-MEFs, respectively. We find that Dnmt3a is exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes, while Dnmt3b has a dominant role on the X chromosome. Consistent with this, tissue-specific DNA methylation at PcG target genes is substantially reduced in Dnmt3a KO embryos. Finally, we find that human patients with DNMT3 mutations exhibit reduced DNA methylation at regions that are hypomethylated in Dnmt3 KO 2i-MEFs. In conclusion, here we report 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.
| S-EPMC7314859 | biostudies-literature
Project description:DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) is essential for genome regulation and development. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-Å crystal structure of the DNMT3A-DNMT3L-DNA complex where two DNMT3A monomers simultaneously attack two CpG dinucleotides, with the target sites separated by fourteen base pairs within the same DNA duplex. The DNMT3A-DNA interaction involves a target recognition domain (TRD), a catalytic loop and DNMT3A homodimeric interface. A TRD residue Arg836 makes crucial contact with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Hematological cancer-associated somatic mutations at the substrate-binding sites decrease DNMT3A activity, induce CpG hypomethylation and promote transformation of hematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its etiologic link to human disease.
2018-01-01 | GSE99390 | GEO
Project description:The DNA methylation program is at the bottom layer of the epigenetic regulatory cascade of vertebrate development. While the methylation at C-5 position of the cytosine (C) residues on the vertebrate genomes is achieved through the catalytic activities of the DNA methyltransferases (DNMTs), the conversion of the methylated cytosine (5mC) could be accomplished by the combined actions of the TET enzyme and DNA repair. Interestingly, it has been found recently that the mouse and human DNMTs also possess active DNA demethylation activity in vitro in a Ca2+- and redox condition-dependent manner. We report here the study of tracking down the fate of the methyl group removed from 5mC on DNA during in vitro demethylation reaction by mouse de novo DNMTs, i.e. DNMT3A and DNMT3B. Remarkably, the methyl group becomes covalently linked to the catalytic cysteines utilized by the two de novo DNMTs in their DNA methylation reactions. Thus, the forward and reverse reactions of DNA methylations by the DNMTs may utilize the same cysteine residue(s) as the active site despite of their distinctive pathways. Secondly, we demonstrate that active DNA demethylation of a heavily methylated GFP reporter plasmid by ectopically expressed DNMT3A or DNMT3B occurs in vivo in transfected human HEK 293 cells in culture. Furthermore, the extent of DNA demethylation by the DNMTs in this cell-based system is affected by Ca2+ homeostasis as well as by mutation of their putative active cysteines. These findings substantiate the roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation in vitro as well as in a cellular context.
2022-02-24 | PXD003401 | Pride
Project description:DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) is essential for genome regulation and development. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-Å crystal structure of the DNMT3A-DNMT3L-DNA complex where two DNMT3A monomers simultaneously attack two CpG dinucleotides, with the target sites separated by fourteen base pairs within the same DNA duplex. The DNMT3A-DNA interaction involves a target recognition domain (TRD), a catalytic loop and DNMT3A homodimeric interface. A TRD residue Arg836 makes crucial contact with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Hematological cancer-associated somatic mutations at the substrate-binding sites decrease DNMT3A activity, induce CpG hypomethylation and promote transformation of hematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its etiologic link to human disease.
2018-01-01 | GSE93727 | GEO
Project description:Precise deposition of CpG methylation is critical for mammalian development and tissue homeostasis and is often dysregulated in human diseases. The localization of de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) is facilitated by PWWP domain recognition of histone H3 lysine 36 (H3K36) methylation (Baubec et al. 2015, Weinberg et al. 2019) and is normally excluded from CpG islands (CGIs) (Wu et al. 2010). However, CpG methylation of CGIs that are regulated by Polycomb repressive complexes (PRCs) has been observed during embryogenesis (Chen et al. 2019), cellular differentiation (Mohn et al. 2008), and cancer progression (Ohm et al. 2007, Schlesinger et al. 2007, Widschwendter et al. 2007), suggesting that an uncharacterized mechanism exists to compete for de novo DNMT recruitment. Here we report that DNMT3A PWWP domain mutations recently identified in paragangliomas (Remacha et al. 2018) and microcephalic dwarfism (Heyn et al. 2019) promote localization of DNMT3A to CGIs in a PRC1-dependent manner. Genome-wide analysis shows that DNMT3A PWWP mutants redistribute to regions containing ubiquitylation of histone H2A at lysine 119 (H2AK119Ub) deposited by PRC1, irrespective of the levels of PRC2-catalyzed tri-methylation of histone H3 at lysine 27 (H3K27me3). DNMT3A, but not DNMT3B, is capable of directly interacting with H2AK119Ub-modified nucleosomes through a putative amino-terminal ubiquitin-dependent recruitment (UDR) region, which serves as an alternative form of genomic targeting in cells upon loss of PWWP reader function. Ablation of PRC1 abrogates localization of DNMT3A PWWP mutants to CGIs and prevents aberrant hypermethylation at these sites. Our study implies that a balance between DNMT3A recruitment by distinct reader domains guides de novo CpG methylation and may underlie the abnormal DNA methylation landscapes observed in human cancers and developmental disorders.
2021-05-13 | GSE147879 | GEO