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Reduced representative bisulfite sequencing (RRBS) analysis for a genome-wide comparison of DNA methylation in wild-type, USP7-KO-1, DNMT3A/3B-DKO and DNMT3A/DNMT3B/USP7-TKO HeLa cell lines.

ABSTRACT: Reduced representative bisulfite sequencing (RRBS) analysis for a genome-wide comparison of DNA methylation in wild-type, USP7-KO-1, DNMT3A/3B-DKO and DNMT3A/DNMT3B/USP7-TKO HeLa cell lines.

PROVIDER: PRJNA561408 | ENA |

REPOSITORIES: ENA

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Reduced representative bisulfite sequencing (RRBS) analysis for a genome-wide comparison of DNA methylation in wild-type, USP7-KO-1, DNMT3A/3B-DKO and DNMT3A/DNMT3B/USP7-TKO HeLa cell lines.

Project description:To better characterize the effects of USP7 on DNA methylation, we performed reduced representative bisulfite sequencing (RRBS) analysis for a genome-wide comparison of DNA methylation in wild-type, USP7-KO-1, DNMT3A/3B-DKO and DNMT3A/DNMT3B/USP7-TKO HeLa cell lines. RRBS analysis showed increased DNA methylation in USK7-KO cells and loss of USP7 elevates DNA methylation on pre-existing sites and de novo methylation.

2020-09-07 | GSE136159 | GEO

RNA-seq of triple knockout (TKO) mouse embryonic stem cells: Dnmt1-/-, Dnmt3a-/-, Dnmt3b-/-.

Project description:TKO mouse embryonic stem cells were cultured in serum/LIF and 2i/LIF in order to investigate the role of methylation in the transcriptional regulation of ground state cells (Clone: TKO 121, Tsumura et.al. 2006).

2020-06-19 | GSE130686 | GEO

RNA-seq of triple knockout (TKO) mouse embryonic stem cells: Dnmt1-/-, Dnmt3a-/-, Dnmt3b-/-.

Project description:RNA-seq of triple knockout (TKO) mouse embryonic stem cells: Dnmt1-/-, Dnmt3a-/-, Dnmt3b-/-.

| PRJNA541006 | ENA

Expression data from mouse wild-type and Dnmt1-/-Dnmt3a-/-Dnmt3b-/- embryonic stem(ES) cells and nuclear transfer-trophoblastic stem(ntTS) cells, and wild-type trophoblastic stem(TS) cells, primitive endoderm cells, and extra-embryonic endoderm(XEN) cells

Project description:To investigate the role of DNA methylation in early embryogenesis, we created Dnmt1-/-Dnmt3a-/-Dnmt3b-/- triple knockout (TKO) embryos by the nuclear transfer (NT) technique using TKO ES cells as the nucleus donor, and found that the TKO NT embryos could develop into blastocysts. From the TKO NT blastocysts, we successfully established trophoblastic stem cells (ntTS cells), and these TKO ntTS cells could self-renew and contribute to the placenta in vivo. Expression profile analysis using microarray revealed that, although many genes were aberrantly expressed, the fundamental gene expression patterns of stem cells were retained in these TKO ntTS cells.

2010-09-03 | GSE20177 | GEO

Microarry from Treg with conditional knockout of Usp7

Project description:Microarry from Treg with conditional knockout of Usp7 RNA from three independent samples from FACS sorted CD4+YFP+ Treg of fl-Usp7/Foxp3cre mice, compared to Foxp3cre control (C57BL/6 background).

2015-09-15 | E-GEOD-72988 | biostudies-arrayexpress

YG001: A Usp7-Tip60 pathway regulates early adipogenesis

Project description:USP7, a dominant DUB activity in 3T3-L1 adipocytes and in mouse adipose tissue, increases Tip60 protein levels, and deubiquitinates Tip60 both in intact cells and in vitro. Treatment with a pan deubiquitinase (DUB) inhibitor, or knockdown of USP7, decreases adipogenesis. Transcriptome analysis reveals a common set of cell cycle genes to be co-regulated by both Tip60 and USP7. Knock down of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results therefore reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis Mature 3T3-L1 adipocytes were subjected to RNAi-mediated knock down with control(C), USP7(U)- or Tip60(T)-specific oligonucleotides. For this, 4 replicates of differentiated 3T3-L1 cells were transfected with Amaxa technology. Two days after transfection cells were washed twice with PBS twice and lysed in 0.5ml Trizol (Invitrogen). mRNA expression of Tip60 and USP7 was assessed by qRT-PCR. Amplified cRNA samples were labeled with either cy3 or cy5 and put on microarray together with and oppositely labeled common reference sample consisting of cRNA derived from undifferentiated 3T3-L1 cells.

2013-08-28 | E-GEOD-28587 | biostudies-arrayexpress

USP7 cooperates with SCML2 to regulate the activity of PRC1

Project description:In this experiment, we sought to identify the distribution of USP7 on chromatin relative to other PRC1 components ChIP-seq for USP7 in 293T-REx cells

2015-06-10 | E-GEOD-61048 | biostudies-arrayexpress

Active DNA Demethylation by DNMT3A and DNMT3B - Mechanistic Aspect and In Vivo Evidence

Project description:The DNA methylation program is at the bottom layer of the epigenetic regulatory cascade of vertebrate development. While the methylation at C-5 position of the cytosine (C) residues on the vertebrate genomes is achieved through the catalytic activities of the DNA methyltransferases (DNMTs), the conversion of the methylated cytosine (5mC) could be accomplished by the combined actions of the TET enzyme and DNA repair. Interestingly, it has been found recently that the mouse and human DNMTs also possess active DNA demethylation activity in vitro in a Ca2+- and redox condition-dependent manner. We report here the study of tracking down the fate of the methyl group removed from 5mC on DNA during in vitro demethylation reaction by mouse de novo DNMTs, i.e. DNMT3A and DNMT3B. Remarkably, the methyl group becomes covalently linked to the catalytic cysteines utilized by the two de novo DNMTs in their DNA methylation reactions. Thus, the forward and reverse reactions of DNA methylations by the DNMTs may utilize the same cysteine residue(s) as the active site despite of their distinctive pathways. Secondly, we demonstrate that active DNA demethylation of a heavily methylated GFP reporter plasmid by ectopically expressed DNMT3A or DNMT3B occurs in vivo in transfected human HEK 293 cells in culture. Furthermore, the extent of DNA demethylation by the DNMTs in this cell-based system is affected by Ca2+ homeostasis as well as by mutation of their putative active cysteines. These findings substantiate the roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation in vitro as well as in a cellular context.

2022-02-24 | PXD003401 | Pride

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

Dnmt3a and Dnmt3b-mediated decommissioning of fetal enhancers linked to kidney disease

Project description:Cytosine methylation is an epigenetic mark that dictates cell fate and response to stimuli. The timing and establishment of methylation logic during kidney development remains unknown. DNA methyltransferase 3a and 3b are the enzymes capable of establishing de novo methylation. We generated mice with genetic deletion of Dnmt3a and Dnmt3b in nephron progenitor cells (Six2Cre Dnmt3a/3b) and kidney tubule cells (KspCre Dnmt3a/3b). We characterized KspCre Dnmt3a/3b mice at baseline and after injury. Unbiased omics profiling, such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing and RNA sequencing were performed on whole-kidney samples and isolated renal tubule cells. KspCre Dnmt3a/3b mice showed no obvious morphologic and functional alterations at baseline. Knockout animals exhibited increased resistance to cisplatin-induced kidney injury, but not to folic acid–induced fibrosis. Whole-genome bisulfite sequencing indicated that Dnmt3a and Dnmt3b play an important role in methylation of gene regulatory regions that act as fetal-specific enhancers in the developing kidney but are decommissioned in the mature kidney. Loss of Dnmt3a and Dnmt3b resulted in failure to silence developmental genes. We also found that fetal-enhancer regions methylated by Dnmt3a and Dnmt3b were enriched for kidney disease genetic risk loci. Methylation patterns of kidneys from patients with CKD showed defects similar to those in mice with Dnmt3a and Dnmt3b deletion. Our results indicate a potential locus-specific convergence of genetic, epigenetic, and developmental elements in kidney disease development.

2020-03-03 | GSE134267 | GEO

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