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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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DNA methylation analysis and expression profiling during murine embryogenesis and in embryos deficient for DNMT3A or DNMT3B

ABSTRACT: 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. We sequenced RRBS libraries prepared from genomic DNA isolated from embryos at consecutive stages of development between E3.5 and E11.5,and adult differentiated cells (sperm, liver). We performed RRBS on blastocysts at E3.5/E4.5, dissected epiblasts at E5.5/E6.5/E7/5, whole embryos at E8.5/E10.5 and limbs at E11.5. RRBS experiments in Dnmt3a-/- and Dnmt3b-/- embryos were performed in biological duplicates on individual embryos. We sequenced RNA-Seq libraries prepared from total RNAs of three WT and Dnmt3b-/- littermate embryos collected at E8.5.

ORGANISM(S): Mus musculus

SUBMITTER: Michael Weber

PROVIDER: E-GEOD-60334 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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DNA methylation analysis and expression profiling during murine embryogenesis and in embryos deficient for DNMT3A or DNMT3B

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.

2014-11-14 | GSE60334 | GEO

DNA methylation analysis by RRBS in germ cells of mice exposed to Vinclozolin

Project description:We provide quantitative maps of cytosine methylation at single base resolution by RRBS in E13.5 primordial germ cells and adult sperm purified from mice exposed to Vinclozolin. Pregnant F0 female mice were exposed to two doses (a low dose VD1=1 mg/kg bw/d and a high dose VD2=100mg/kg bw/d) of Vinclozolin in the drinking water during pregnancy. We isolated primordial germ cells at E13.5 and mature sperm from adult F1 males obtained from control and exposed mothers. For PGCs, we sequenced RRBS libraries prepared from one pool isolated from F1 embryos exposed to the low dose, one pool isolated from F1 embryos exposed to the high dose, and two control pools isolated from unexposed embryos. For sperm, we sequenced three RRBS libraries prepared from a pool of sperm isolated from F1 males exposed to the high dose, and five RRBS libraries prepared from control pools isolated from unexposed animals.

2015-06-22 | E-GEOD-65784 | biostudies-arrayexpress

Human ART culture conditions prime mouse embryos for postimplantation development

Project description:With the increasing use of Assisted Reproductive Technologies (ART) for treatment of human infertility, there is an increasing requirement for embryo culture conditions that perform as similar to nature as possible. How good the match, however, cannot be tested experimentally in human. We solved the central question of how well ART culture protocols prepare embryos for postimplantation development, under the provisions of the 'mouse embryo assay' (MEA). Our side-by-side comparison of 8 conditions [i.e., 3 culture conditions (KSOM, HTF and ISDM1) plus the in vivo system in two different mouse strains (B6 and CD1)] shows that mouse embryos cultured under ART conditions are differentially primed for postimplantation development, and that certain ART protocols outperform the oviduct. The distinct performances of blastocysts formed in ART vs. oviduct do not correlate with any significant transcriptome changes, whereas protein analysis by immunoconfocal microscopy reveals differences in the allocation of embryonic cells to the three germ layers of blastocysts. We conclude that in vitro technology is not always a defective copy of nature, and that the choice of ART protocol primes the embryos for subsequent development. 22 samples were analyzed. B6KSOM: Mouse B6 background, E3.5 blastocysts in KSOM medium, 3 biological rep B6HTF: Mouse B6 background, E3.5 blastocysts in HTF medium, 3 biological rep B6ISM1: Mouse B6 background, E3.5 blastocysts in ISM1 medium, 3 biological rep B6vivo: Mouse B6 background, E3.5 blastocysts in vivo, 3 biological rep CD1KSOM: Mouse CD1 background, E3.5 blastocysts in KSOM medium, 1 biological rep CD1HTF: Mouse CD1 background, E3.5 blastocysts in HTF medium, 3 biological rep CD1ISM1: Mouse CD1 background, E3.5 blastocysts in ISM1 medium, 3 biological rep CD1vivo: Mouse CD1 background, E3.5 blastocysts in vivo, 3 biological rep

2012-05-02 | E-GEOD-31414 | biostudies-arrayexpress

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

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

Developmental Genome-Wide DNA Methylation Asymmetry Between Mouse Placenta and Embryo

Project description:In early embryos, DNA methylation is remodelled to initiate the developmental program. For mostly unknown reasons, methylation marks are acquired unequally between embryonic and placental cells. To better understand this, we generated high-resolution maps of DNA methylation in mouse mid-gestation (E10.5) embryo and placenta. We uncovered specific subtypes of differentially methylated regions (DMRs) that contribute directly to the developmental asymmetry existing between mid-gestation embryo and placenta. We show that the asymmetry between embryonic and placental DNA methylation patterns occurs rapidly during the acquisition of marks in the post-implanted conceptus (E3.5-E6.5), and that these patterns are long-lasting across subtypes of DMRs throughout prenatal development and in somatic tissues. We reveal that at the peri-implantation stages, the de novo methyltransferase activity of DNMT3B is the main driver of methylation marks on asymmetric DMRs, and that DNMT3B can largely compensate for lack of DNMT3A in the epiblast and extraembryonic ectoderm, whereas DNMT3A can only partially palliate for the absence of DNMT3B. However, as development progresses and as DNMT3A becomes the principal de novo methyltransferase, the compensatory DNA methylation mechanism on DMRs becomes less effective.

2020-02-18 | GSE95610 | GEO

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

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

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

Increased DNA Methylation of Dnmt3b-Targets Impairs Leukemogenesis (RRBS)

Project description:The de novo DNA methyltransferases Dnmt3a and Dnmt3b are of crucial importance in hematopoietic stem cells, and Dnmt3b has recently been shown to play a role in genic methylation. Forced Dnmt3b expression induced widespread DNA hypermethylation in myc-bcl2 induced leukemias, especially at promoters and gene bodies of stem cell-related genes. MLL-AF9 induced leukemogenesis showed much less pronounced DNA hypermethylation upon Dnmt3b expression. Nonetheless, leukemogenesis was delayed in both models with a shared core set of DNA hypermethylated regions and suppression of stem cell-related genes. Our findings indicate a critical role for Dnmt3b-mediated DNA methylation in leukemia development and maintenance of LSC function. To investigate how Dnmt3b-mediated DNA methylation affects leukemogenesis, we analyzed leukemia development under conditions of high and physiological methylation levels in a tetracycline-inducible knockin mouse model. High expression of Dnmt3b slowed leukemia development in serial transplantations and impaired leukemia stem cell (LSC) function.

2016-01-11 | E-GEOD-71039 | biostudies-arrayexpress

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

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

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