Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE145899 | GEO | 2020/06/18
REPOSITORIES: GEO
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Project description:DNA methyltransferases DNMT3A- and DNMT3B-mediated de novo DNA methylation critically regulates epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote macro-oligomer formation, leading to aberrant DNA methylation that in turn contributes to pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the hotspot mutation-induced functional mis-regulation of DNMT3A remains unclear. Here, we report the crystal structure of DNMT3A methyltransferase (MTase) domain, revealing a molecular basis for its DNMT3B-distinct oligomerization behavior. Introducing DNMT3B-converting mutations to DNMT3A R882 mutants also led to structure determination of R882H- and R882C-mutated DNMT3A, which show enhanced intermolecular contacts than wild-type DNMT3A. Consistently, our in vitro and genomic DNA methylation analyses reveal that the DNMT3B-converting mutations eliminate the gain-of-function effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutation-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy.
2023-12-31 | GSE226062 | GEO
Project description:DNA methyltransferases DNMT3A- and DNMT3B-mediated de novo DNA methylation critically regulates epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote macro-oligomer formation, leading to aberrant DNA methylation that in turn contributes to pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the hotspot mutation-induced functional mis-regulation of DNMT3A remains unclear. Here, we report the crystal structure of DNMT3A methyltransferase (MTase) domain, revealing a molecular basis for its DNMT3B-distinct oligomerization behavior. Introducing DNMT3B-converting mutations to DNMT3A R882 mutants also led to structure determination of R882H- and R882C-mutated DNMT3A, which show enhanced intermolecular contacts than wild-type DNMT3A. Consistently, our in vitro and genomic DNA methylation analyses reveal that the DNMT3B-converting mutations eliminate the gain-of-function effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutation-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy.
2023-12-31 | GSE225828 | 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: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: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: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
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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
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Project description:We quantified the targets and kinetics of DNA methylation acquisition in mouse embryos, and determined the contribution of the de novo methyltransferases DNMT3A and DNMT3B to this process. We provide single-base maps of cytosine methylation by RRBS from the blastocysts to post-implantation stages and in embryos lacking DNMT3A or DNMT3B activity, and performed RNA-Seq in embryos lacking DNMT3B activity.
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Project description:Mammalian de novo DNA methyltransferases (DNMT) are responsible for the establishment of cell-type-specific DNA methylation in healthy and diseased tissues. Through genome-wide analysis of de novo methylation activity in murine stem cells we uncover that DNMT3A prefers to methylate CpGs followed by cytosines or thymines, while DNMT3B predominantly methylates CpGs followed by guanines or adenines. These signatures are further observed at non-CpG sites, resembling methylation context observed in specialised cell types, including neurons and oocytes. We further show that these preferences result from structural differences in the catalytic domains of the two de novo DNMTs and are not a consequence of differential recruitment to the genome. Molecular dynamics simulations suggest that, in case of human DNMT3A, the preference is due to favourable polar interactions between the flexible Arg836 side chain and the guanine that base-pairs with the cytosine following the CpG. By exchanging arginine to a lysine, the corresponding sidechain in DNMT3B, the sequence preference is reversed, confirming the requirement for arginine at this position. This context-dependent de novo DNA methylation by DNMT3A and DNMT3B, provides additional insights into the complex regulation of methylation patterns.
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