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

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 DNMT3A/3B DKO human ES cells

ORGANISM(S): Homo sapiens

SUBMITTER: Rahul Karnik

PROVIDER: E-GEOD-63280 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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

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 a time course of DNMT1* withdrawal in human ES cells

2015-03-30 | E-GEOD-63279 | biostudies-arrayexpress

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

2015-04-13 | E-GEOD-67811 | biostudies-arrayexpress

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.

2013-06-12 | E-GEOD-47088 | biostudies-arrayexpress

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

2015-03-30 | GSE63280 | GEO

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]

2015-03-30 | GSE63278 | GEO

Single base resolution analysis of 5-formyl and 5-carboxyl cytosine reveals the actual promoter DNA methylation dynamics in embryonic stem cells [MAB-Seq]

Project description:DNA methylation is catalysed by DNA methyltransferases (DNMTs) and is necessary for a correct embryonic development. On the other hand, the DNA demethylation is mediated by the Ten Eleven Translocation (Tet) proteins through oxidation of 5-methyl cytosine (5mC) to 5-hydroxyl (5hmC), 5-formyl (5fC) and 5-carboxyl (5caC) cytosine, and by the Thymine-DNA glycosylase (TDG) that excises the 5fC and 5caC. In embryonic stem cells (ESCs), gene promoters are maintained in an hypomethylated state, but the dynamics of this phenomenon still remains unknown. Here we present a genome-wide approach, named methylation-assisted bisulfite sequencing (MAB-Seq) that enables single-base resolution mapping of 5fC and 5caC and measuring of their relative abundance. Application of this method to mouse ESCs exposed the presence of 5fcaC residues on the hypomethylated promoters of the expressed genes, revealing an active DNA demethylation mechanism since the loss of TDG leads to an increase of 5fC/5caC. We also show that TDG is actually bound on these regions and that co-localizes and interacts with Tet1. We moreover demonstrate, by reduced representation of bisulfite sequencing (RRBS), that active promoters are actually demethylated by a Tet-dependent mechanism and that Dnmt1 and Dnmt3a are responsible of this DNA methylation. Our work shows the whole-genome map of 5fC and 5caC at single base resolution in ESCs, it demonstrates in detail the DNA methylation dynamics occurring on expressed gene promoters and identifies the key players of this mechanism. Furthermore, we provide a new tool (MAB-Seq) that can be broadly used in all biological contexts for epigenetics study involving identification and quantification of 5fC and 5caC at single base resolution. Methylation-assisted bisulfite sequencing (MAB-Seq) of E14 embryonic stem cells (ESCs), Biotag ChIP-Seq of Tdg and Reduced representation Bisulfite Sequencing (RRBS) in E14 ESCs.

2015-02-10 | E-GEOD-55658 | biostudies-arrayexpress

Expression data from wild type and globally hypomethylated mouse embryonic stem cells (ESCs) during their differentiation as embyroid bodies

2013-02-05 | GSE36679 | GEO

Expression data from wild type and globally hypomethylated mouse embryonic stem cells (ESCs) during their differentiation as embyroid bodies

Project description:To analyze the role of DNA methylation during differentiation, we performed genome-wide expression analysis of undifferentiated wild type, dnmt1-/- and triple knock out (TKO; dnmt1-/-, dnmt3a-/-, dnmt3b-/-) ESCs as well as respective embryoid bodies (EBs) at two stages of differentiation We generated EBs from wild type, dnmt1-/- and TKO ESCs using the hanging drop method and performed affymetrix microarray expression analysis at three time points; i) in the undifferentiated ESC state (d0), ii) early EB differentiation (d4) and iii) later EB differentiation stage (d16).

2013-02-05 | E-GEOD-36679 | biostudies-arrayexpress

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