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Targeted disruption of DNMT1, 3A and 3B in human embryonic stem cells [DNMT1]

ABSTRACT: 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 a time course of DNMT1* withdrawal in human ES cells

ORGANISM(S): Homo sapiens

SUBMITTER: Rahul Karnik

PROVIDER: E-GEOD-63279 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Targeted disruption of DNMT1, 3A and 3B in human embryonic stem cells [DNMT3A/3B DKO]

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

2015-03-30 | E-GEOD-63280 | biostudies-arrayexpress

RRBS profiling of mouse ESCs (wild type and DNMT KOs)

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DNA methylation of hematopoietic stem cells during ontogeny and after enforced proliferative history

Project description:Genome-scale DNA methylation profiles at nucleotide resolution covering the vast majority of CpG islands and a representative sampling of conserved non-coding elements, transposons and other genomic features generated using high-throughput reduced representation bisulfite sequencing (RRBS) for murine hematopoietic stem cells during ontongeny and after enforced prolferative history. Analysis of hematopoietic stem cell methylation during ontogeny and after undergoing enforced proliferative history

2013-02-14 | E-GEOD-44117 | biostudies-arrayexpress

Identification of novel imprinted differentially methylated regions by global analysis of human parthenogenetic induced pluripotent stem cells

Project description:Parental imprinting is an epigenetic phenomenon by which genes are expressed in a monoallelic fashion, according to their parent-of-origin. DNA methylation is considered the hallmark mechanism regulating parental imprinting. To identify imprinted differentially methylated regions (DMRs), we compared the DNA methylation status between multiple normal and parthenogenetic human pluripotent stem cells (PSCs) by performing reduced representation bisulfite sequencing. Our analysis identified over twenty previously unknown imprinted DMRs in addition to the known DMRs. These include DMRs in loci associated with human disorders, and a class of intergenic DMRs that do not seem to be related to gene expression. Furthermore, the study showed some DMRs to be unstable, liable to differentiation or reprogramming. A comprehensive comparison between mouse and human DMRs identified almost half of the imprinted DMRs to be species-specific. Taken together our data map novel DMRs in the human genome, their evolutionary conservation, and relation to gene expression. RRBS profiles were generated from 6 parthenogenetic iPSC lines (hiPS A11, hiPS A20, hIPS A26, hiPS B34, hiPS B36, hiPS B41) and fibroblasts from the 2 parents whose ovarian teratomas were used to derive the iPSC lines, for a total of 8 samples.

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Targeted disruption of DNMT1, 3A and 3B in human embryonic stem cells [DNMT1]

2015-03-30 | GSE63279 | GEO

Targeted disruption of DNMT1, 3A and 3B in human embryonic stem cells [WGBS]

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Targeted disruption of DNMT1, 3A and 3B in human embryonic stem cells [DNMT3A/3B DKO]

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Distinct catalytic-dependent or -independent activities of DNMT1 in mouse embryonic stem cells

Project description:DNMT1 maintains DNA methylation profile and regulates gene expression. Although DNMT1 interacts with other proteins that regulate gene expressions, it remains unclear how the protein-protein interaction or the catalytic activity of DNMT1 itself affect gene expression and DNA methylation pattern. In this study, immunofluorescence staining showed abnormal DNA methylation pattern in chromosome arms of DNMT1-deficient (1KO) mouse embryonic stem cells (ESCs), and we hypothesized that DNMT1 helps methylation activity of DNMT3. To validate this hypothesis, we generate transgenic 1KO ESCs expressing catalytic inactive DNMT1 (1KO+1CI). Here, we used RNA-seq to analyze the effect of catalytic-dependent or -independent DNMT1 activity on gene expression in ESCs or epiblast-like cells (EpiLCs) induction.

2022-01-01 | GSE179006 | GEO

Dnmt1-Independent CG Methylation Contributes to Nucleosome Positioning in Diverse Eukaryotes

Project description:Dnmt1 epigenetically propagates symmetrical CG methylation in many eukaryotes. Their genomes are typically depleted of CG dinucleotides because of imperfect repair of deaminated methylcytosines. Here, we extensively survey diverse species lacking Dnmt1 and show that, surprisingly, symmetrical CG methylation is nonetheless frequently present and catalyzed by a different DNA methyltransferase family, Dnmt5. Numerous Dnmt5-containing organisms that diverged more than a billion years ago exhibit clustered methylation, specifically in nucleosome linkers. Clustered methylation occurs at unprecedented densities and directly disfavors nucleosomes, contributing to nucleosome positioning between clusters. Dense methylation is enabled by a regime of genomic sequence evolution that enriches CG dinucleotides and drives the highest CG frequencies known. Species with linker methylation have small, transcriptionally active nuclei that approach the physical limits of chromatin compaction. These features constitute a previously unappreciated genome architecture, in which dense methylation influences nucleosome positions, likely facilitating nuclear processes under extreme spatial constraints. DNA methylation, RNA and nucleosome sequencing data for diverse eukaryotes

2014-03-13 | E-GEOD-46692 | biostudies-arrayexpress

DNMT1 IP-MS alalysis to identify ubiquitin-dependent interacting partners

Project description:Stable inheritance of DNA methylation is critical for maintaining the differentiated phenotypes in multicellular organisms. However, the molecular basis ensuring high fidelity of maintenance DNA methylation is largely unknown. Here, we demonstrate that two distinct modes of DNMT1 recruitment, one is DNA replication-coupled and the other is uncoupled mechanism, regulate the stable inheritance of DNA methylation. PCNA-associated factor 15 (PAF15) represents a primary target of UHRF1 and undergoes dual mono-ubiquitylation (PAF15Ub2) on chromatin. PAF15Ub2 specifically interacts with DNMT1 and controls the recruitment of DNMT1 in a DNA replication-coupled manner. Thus, loss of PAF15Ub2 results in impaired DNA methylation at sites replicating during early S phase. In contrast, outside of S phase or when PAF15 ubiquitylation is perturbed, UHRF1 ubiquitylates histone H3 to promote DNMT1 recruitment. Together, we identify replication-coupled and uncoupled mechanisms of maintenance DNA methylation, both of which collaboratively ensure the stable DNA methylation.

2020-02-25 | PXD015282 | Pride

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