Structural basis for DNMT3A-mediated de novo DNA methylation (eRRBS)
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE99390 | GEO | 2018/01/01
SECONDARY ACCESSION(S): PRJNA388378
REPOSITORIES: GEO
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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: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:DNA Methyltransferase 3A (DNMT3A) is frequently mutated in various hematopoietic malignancies; however, the underlying oncogenic mechanisms remain elusive. Here, we report that DNMT3A mutational âhotspotâ at Arg882 (DNMT3A-R882H) cooperates with constitutively activated RAS in transforming murine hematopoietic stem/progenitor cells (HSPCs) ex vivo and inducing acute leukemias in vivo. DNMT3A-R882H potentiates aberrant transactivation of âstemnessâ gene expression programs, notably transcription factors Meis1, Hox-A, Mn1 and Mycn. Mechanistically, R882-mutated DNMT3A directly binds to cis-regulatory elements of these genes and induces focal CpG hypomethylation reminiscent of what was seen in human leukemias bearing DNMT3A R882 mutation. Furthermore, DNMT3A-R882H induced DNA hypomethylation facilitates gene enhancer/promoter activation and recruitment of Dot1l-associated transcription elongation machineries. Inactivation of Dot1l represses DNMT3AR882H-mediated stem cell gene dysregulation and acute leukemogenicity. In this dataset, we provided enhanced Reduced Representation Bisulfite Sequencing (eRRBS) DNA methylome profiling data showing effect of DNMT3A R882H mutation or WT expression on hematopoietic stem/progenitor cells with NRAS G12D co-transduction. eRRBBs DNA methylome analysis of Lin- enriched hematopoietic stem/progenitor cells with retroviral infection of NRAS G12D alone (EV-RAS), DNMT3A R882H with NRAS G12D (RH-RAS) or DNMT3A WT with NRAS G12D (WT-RAS) at day 16 post-transduction.
2016-06-23 | E-GEOD-71473 | biostudies-arrayexpress
Project description:DNA Methyltransferase 3A (DNMT3A) is frequently mutated in various hematopoietic malignancies; however, the underlying oncogenic mechanisms remain elusive. Here, we report that DNMT3A mutational ‘hotspot’ at Arg882 (DNMT3A-R882H) cooperates with constitutively activated RAS in transforming murine hematopoietic stem/progenitor cells (HSPCs) ex vivo and inducing acute leukemias in vivo. DNMT3A-R882H potentiates aberrant transactivation of ‘stemness’ gene expression programs, notably transcription factors Meis1, Hox-A, Mn1 and Mycn. Mechanistically, R882-mutated DNMT3A directly binds to cis-regulatory elements of these genes and induces focal CpG hypomethylation reminiscent of what was seen in human leukemias bearing DNMT3A R882 mutation. Furthermore, DNMT3A-R882H induced DNA hypomethylation facilitates gene enhancer/promoter activation and recruitment of Dot1l-associated transcription elongation machineries. Inactivation of Dot1l represses DNMT3AR882H-mediated stem cell gene dysregulation and acute leukemogenicity. In this dataset, we provided enhanced Reduced Representation Bisulfite Sequencing (eRRBS) DNA methylome profiling data showing effect of DNMT3A R882H mutation or WT expression on hematopoietic stem/progenitor cells with NRAS G12D co-transduction.
2016-06-23 | GSE71473 | 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: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
Project description:This SuperSeries is composed of the SubSeries listed below. DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined chromosomal binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects chromosomal binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity. Refer to individual Series
2015-01-22 | E-GEOD-57413 | biostudies-arrayexpress
Project description:DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined chromosomal binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects chromosomal binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity. Whole-genome bisulfite sequencing for Dnmt1,3a,3b-triple-KO ES cells expressing DNMT3A2 or DNMT3B1 and for Dnmt1,3a,3b,Setd2-KO ES cells expressing DNMT3B1
2015-01-20 | E-GEOD-57411 | biostudies-arrayexpress
Project description:DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined chromosomal binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects chromosomal binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity. Genome-wide binding analysis for biotin-tagged DNMT3A2 and DNMT3B and variants in wild type ES, wild type neuroprogenitor cells, ES cells triple-KO for Dnmt1,3a,3b and ES cell mutant for Setd2
2015-01-20 | E-GEOD-57412 | biostudies-arrayexpress
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