De novo Assembly and Delivery of Synthetic Megabase-Scale Human DNA into Mouse Early Embryos
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus Saccharomyces cerevisiae
PROVIDER: GSE250094 | GEO | 2025/04/16
REPOSITORIES: GEO
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Project description:DNA methylation is a central epigenetic modification that has essential roles in cellular processes including chromatin structure, gene regulation, development and disease. The de novo DNA methyltransferases are responsible for the generation of genomic methylation patterns, but the underlying mechanisms are still poorly understood. Here, we show that phosphorylation of DNMT3A by the CK2 protein kinase regulates the establishment of DNA methylation patterns. We find that DNMT3A is phosphorylated by CK2 at two key residues located near its PWWP domain. We observed that, through phosphorylation of these residues, CK2 negatively regulates DNMT3AM-bM-^@M-^Ys ability to methylate DNA and consistent with this, CK2 was found to decrease overall genomic level of 5-methylcytosine. Further, genome-wide DNA methylation analysis in CK2-depleted cells revealed that CK2 affects primarily CpG methylation of several heterochromatin repeats as well as Alu elements. Along these lines, we found that CK2-mediated phosphorylation of DNMT3A was required for its proper heterochromatin localization. Our results define phosphorylation as a new mode of regulation of de novo DNA methyltransferase function. These findings further uncover a previously unrecognized mechanism for the regulation of methylation at repetitive elements. They shed new light into the origin of DNA methylation patterns. Bisulphite converted DNA from 6 samples were hybridised to the Illumina Infinium 27K Human Methylation Beadchip v1.2
2014-07-17 | E-GEOD-26164 | biostudies-arrayexpress
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
2015-01-20 | E-GEOD-57412 | biostudies-arrayexpress
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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Project description:DNA methylation serves a stable gene regulatory function in mature somatic cells. In the germ line and during early embryogenesis, however, DNA methylation undergoes global erasure and re-establishment to support germ cell and embryonic development. While de novo DNA methylation during male germ cell development is essential for setting genomic imprints, possible other intergenerational roles for paternal DNA methylation following fertilization are unknown. To address this question, we reduced the level of DNA methylation in developing male germ cells through conditional gene deletion of the de novo DNA methyltransferases DNMT3A and DNMT3B in undifferentiated spermatogonia. Mutant male germ cells nevertheless completed their differentiation to sperm. We observed that DNMT3A serves a largely maintenance-like methylation function at many intragenic sites in undifferentiated spermatogonia while DNMT3B catalyzes de novo methylation during spermatogonial differentiation. In spermatogonia, the acquisition of DNA methylation and deposition of H3K4me3 occur mutually exclusive. Failing de novo DNA methylation in spermatogonia leads to increased nucleosome occupancy in mature sperm at sites with high CpG content, reinforcing the model that DNA methylation constrains nucleosome retention in sperm. To assess the impact of altered sperm chromatin in the formation of embryonic chromatin, we measured H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a highly sensitive transposon-based tagging assay for modified chromatin. Our data show that reduced DNA methylation in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently from paternal inheritance of sperm born H3K4me3. Together, this study provides first evidence that paternally inherited DNA methylation directs chromatin formation during early embryonic development.
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Project description:DNA methylation serves a stable gene regulatory function in mature somatic cells. In the germ line and during early embryogenesis, however, DNA methylation undergoes global erasure and re-establishment to support germ cell and embryonic development. While de novo DNA methylation during male germ cell development is essential for setting genomic imprints, possible other intergenerational roles for paternal DNA methylation following fertilization are unknown. To address this question, we reduced the level of DNA methylation in developing male germ cells through conditional gene deletion of the de novo DNA methyltransferases DNMT3A and DNMT3B in undifferentiated spermatogonia. Mutant male germ cells nevertheless completed their differentiation to sperm. We observed that DNMT3A serves a largely maintenance-like methylation function at many intragenic sites in undifferentiated spermatogonia while DNMT3B catalyzes de novo methylation during spermatogonial differentiation. In spermatogonia, the acquisition of DNA methylation and deposition of H3K4me3 occur mutually exclusive. Failing de novo DNA methylation in spermatogonia leads to increased nucleosome occupancy in mature sperm at sites with high CpG content, reinforcing the model that DNA methylation constrains nucleosome retention in sperm. To assess the impact of altered sperm chromatin in the formation of embryonic chromatin, we measured H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a highly sensitive transposon-based tagging assay for modified chromatin. Our data show that reduced DNA methylation in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently from paternal inheritance of sperm born H3K4me3. Together, this study provides first evidence that paternally inherited DNA methylation directs chromatin formation during early embryonic development.
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Project description:DNA methylation serves a stable gene regulatory function in mature somatic cells. In the germ line and during early embryogenesis, however, DNA methylation undergoes global erasure and re-establishment to support germ cell and embryonic development. While de novo DNA methylation during male germ cell development is essential for setting genomic imprints, possible other intergenerational roles for paternal DNA methylation following fertilization are unknown. To address this question, we reduced the level of DNA methylation in developing male germ cells through conditional gene deletion of the de novo DNA methyltransferases DNMT3A and DNMT3B in undifferentiated spermatogonia. Mutant male germ cells nevertheless completed their differentiation to sperm. We observed that DNMT3A serves a largely maintenance-like methylation function at many intragenic sites in undifferentiated spermatogonia while DNMT3B catalyzes de novo methylation during spermatogonial differentiation. In spermatogonia, the acquisition of DNA methylation and deposition of H3K4me3 occur mutually exclusive. Failing de novo DNA methylation in spermatogonia leads to increased nucleosome occupancy in mature sperm at sites with high CpG content, reinforcing the model that DNA methylation constrains nucleosome retention in sperm. To assess the impact of altered sperm chromatin in the formation of embryonic chromatin, we measured H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a highly sensitive transposon-based tagging assay for modified chromatin. Our data show that reduced DNA methylation in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently from paternal inheritance of sperm born H3K4me3. Together, this study provides first evidence that paternally inherited DNA methylation directs chromatin formation during early embryonic development.
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Project description:DNA methylation serves a stable gene regulatory function in mature somatic cells. In the germ line and during early embryogenesis, however, DNA methylation undergoes global erasure and re-establishment to support germ cell and embryonic development. While de novo DNA methylation during male germ cell development is essential for setting genomic imprints, possible other intergenerational roles for paternal DNA methylation following fertilization are unknown. To address this question, we reduced the level of DNA methylation in developing male germ cells through conditional gene deletion of the de novo DNA methyltransferases DNMT3A and DNMT3B in undifferentiated spermatogonia. Mutant male germ cells nevertheless completed their differentiation to sperm. We observed that DNMT3A serves a largely maintenance-like methylation function at many intragenic sites in undifferentiated spermatogonia while DNMT3B catalyzes de novo methylation during spermatogonial differentiation. In spermatogonia, the acquisition of DNA methylation and deposition of H3K4me3 occur mutually exclusive. Failing de novo DNA methylation in spermatogonia leads to increased nucleosome occupancy in mature sperm at sites with high CpG content, reinforcing the model that DNA methylation constrains nucleosome retention in sperm. To assess the impact of altered sperm chromatin in the formation of embryonic chromatin, we measured H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a highly sensitive transposon-based tagging assay for modified chromatin. Our data show that reduced DNA methylation in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently from paternal inheritance of sperm born H3K4me3. Together, this study provides first evidence that paternally inherited DNA methylation directs chromatin formation during early embryonic development.
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