Genome-wide DNA methylation reprogramming in porcine primordial germ cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Sus scrofa
SUBMITTER: Alfonso Gutiérrez Adán
PROVIDER: E-MTAB-9326 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Transcription of endogenous retroviruses (ERVs) is inhibited by de novo DNA methylation during gametogenesis, a process initiated after birth in oocytes and at ~E15.5 in prospermatogonia. Earlier in germline development however, the genome, including most retrotransposons, is progressively demethylated, with young ERVK and ERV1 elements retaining intermediate methylation levels. As DNA methylation reaches a low point in E13.5 primordial germ cells (PGCs) of both sexes, we determined whether retrotransposons are marked by H3K9me3 and H3K27me3 using a recently developed low input ChIP-seq method. Although these repressive histone modifications are predominantly found on distinct genomic regions in E13.5 PGCs, they concurrently mark partially methylated LTRs and LINE1 elements. Germline-specific conditional knock-out (KO) of the H3K9 methyltransferase SETDB1 yields a decrease of both histone marks and DNA methylation at H3K9me3 enriched retrotransposon families. Strikingly, Setdb1-KO E13.5 PGCs show concomitant de-repression of many marked ERVs, including IAP, ETn and ERVK10C elements and ERV-proximal genes, a subset in a sex-dependent manner. Furthermore, Setdb1 deficiency is associated with a reduced number of male PGCs and postnatal hypogonadism in both sexes. Taken together, these observations reveal that SETDB1 is an essential guardian against proviral expression prior to the onset of de novo DNA methylation in the germline. H3K9me3, H3K27me3 and expression profiles in Setdb1 WT, Het and KO male and female E13.5 PGCs.
2014-09-01 | E-GEOD-60377 | biostudies-arrayexpress
Project description:Gene-expression profile of human embryonic germ cells (EGCs), primordial germ cells (PGCs), embryonal carcinoma cells (ECCs), pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (IPSCs)
2018-06-28 | E-MTAB-6851 | biostudies-arrayexpress
Project description:Purpose: To investigate the effect of Tet1 depletion on global DNA methylation, we performed whole-genome bisulfite sequencing (WGBS). Methods: Starting with as little as 1400-5251 manually micro-dissected PGCs, we used an ultra-low input method, Tn5mC-seq. Results: We generated 945 million reads for Tet1Gt/Gt PGCs and 302 million reads for wild-type PGCs. We obtained 14-16 million CpG sites per genotype at 1.76-2.66x genome coverage, which enables a comprehensive view of genome-wide DNA methylation patterns in E13.5 PGCs. PGCs are almost completely unmethylated genome-wide. Loss of Tet1 led to a subtle increase of methylation level in various genomic elements including promoters, exons, introns and repetitive elements in Tet1Gt/Gt PGCs (p<0.01). Local analysis identified 4,337 differentially methylated regions (DMRs) between Tet1-/- PGCs and wild-type cells. These DMRs are associated with 5,261 genes, among which 271 genes also exhibited differential gene expression and enriched for the cell cycle pathway (FDR=0.02). Conclusions: This result revealed that demethylation of certain set of cell cycle genes is largely abolished in the Tet1-/- PGCs. Genome-wide methylation profiles of primordial germ cells derived from the wild type (WT) and Tet1-null female embryos at E13.5 were generated by whole genome bisulfite sequencing using Illumina Hiseq.
2012-11-20 | E-GEOD-41912 | biostudies-arrayexpress
Project description:Pluripotency can be induced in somatic cells by ectopic expression of defined transcription factors, however the identity of epigenetic regulators driving the progression of cellular reprogramming requires further investigation. Here we uncover a non-redundant role for the JmjC-domain-containing protein histone H3 methylated Lys 27 (H3K27) demethylase Utx, as a critical regulator for the induction, but not for the maintenance, of primed and naM-CM-/ve pluripotency in mice and in humans. Utx depletion results in aberrant H3K27me3 repressive chromatin demethylation dynamics, which subsequently hampers the reactivation of pluripotency promoting genes during reprogramming. Remarkably, Utx deficient primordial germ cells (PGCs) display a cell autonomous aberrant epigenetic reprogramming in vivo during their embryonic maturation, resulting in the lack of functional contribution to the germ-line lineage. H3K27me3 and H3K4me3 were measured genome-wide in the following cell types: Utx+/Y (WT) and Utx-/Y (KO) mouse ES cells and mouse embryonic fibroblast (MEF) before and after DOX induction (initiating reprogramming by OSKM factors).
2012-05-30 | E-GEOD-37821 | biostudies-arrayexpress
Project description:Background: Genes, RNAs, and proteins play important roles during germline development. However, the functions of non-coding RNAs (ncRNAs) on germline development remain unclear in avian species. Recent high-throughput techniques have identified several classes of ncRNAs, including micro RNAs (miRNAs), small-interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs). These ncRNAs are functionally important in the genome, however, the identification and annotation of ncRNAs in a genome is challenging. The aim of this study was to identify different types of small ncRNAs particularly piRNAs, and the role of piRNA pathway genes in the protection of chicken primordial germ cells (PGCs). Results: At first, we performed next-generation sequencing to identify ncRNAs in chicken PGCs, and we performed ab initio predictive analysis to identify putative piRNAs in PGCs. Then, we examined the expression of three repetitive sequence-linked piRNAs and 14 genic-transcript-linked piRNAs along with their linked genes using real-time PCR. All piRNAs and their linked genes were highly expressed in PGCs. Subsequently, we knocked down two known piRNA pathway genes of chicken, PIWI-like protein 1 (CIWI) and 2 (CILI), in PGCs using siRNAs. After knockdown of CIWI and CILI, we examined their effects on the expression of six putative piRNA-linked genes and DNA double-strand breakage in PGCs. The knockdown of CIWI and CILI upregulated chicken repetitive 1 (CR1) element and RAP2B, a member of RAS oncogene family, and increased DNA double-strand breakage in PGCs. Conclusions: Our results increase the understanding of PGC-expressed ncRNAs and the role of piRNA pathway genes in the protection of germ cells. Small ncRNA expression profiling in SSEA-1 positive PGCs, SSEA-1 negative gonadal stromal cells (GSCs), Stage X blastoderms, and chicken embryonic fibroblast (CEFs)
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Project description:We report that in vitro derived PGCs undergo genome-wide DNA demethylation and that this demethylation does not require Tet1/Tet2 Examination of methylation in ESCs, iPGCs, and Tet2-/- iPGCs depleted of Tet1 by shRNA lentiviruses
2013-02-14 | E-GEOD-44092 | biostudies-arrayexpress
Project description:In mammals, sex differentiation of primordial germ cells (PGCs) is determined by extrinsic cues from the environment1. In female PGCs, expression of Stimulated by retinoic acid 8 (Stra8) and meiosis are induced in response to retinoic acid (RA) provided by the mesonephroi2-4. Given the widespread role of RA signaling during development8,9, the molecular mechanism specifying the competence of PGCs to timely express Stra8 and enter meiosis are unknown2,10. Here we identify gene dosage dependent roles in PGC development for Ring1 and Rnf2, two central components of the Polycomb Repressive Complex 1 (PRC1)11,13. Both paralogs are essential for PGC development between day 10.5 and 11.5 of gestation. Rnf2 is subsequently required in female PGCs for maintaining high levels of Oct4 and Nanog expression6, and for preventing premature induction of meiotic gene expression and entry into meiotic prophase. Chemical inhibition of RA signaling partially suppresses precocious Oct4 down-regulation and Stra8 activation in Rnf2-deficient female PGCs. Chromatin immunoprecipitation analyses show that Stra8 is a direct target of PRC1 and PRC2 in PGCs. These data demonstrate the importance of PRC1 gene dosage in PGC development and in coordinating the timing of sex differentiation of female PGCs by antagonizing extrinsic RA signaling. Gene expression of mouse primordial germ cells was analysed using Affymetrix microarray. Primodial germ cells were purified from embryos carrying Oct4(deltaPE)-GFP transgene by FACS.
2013-03-14 | E-GEOD-42782 | biostudies-arrayexpress
Project description:Primordial germ cells (PGCs) are the embryonic precursors to egg and sperm. When removed from the embryonic gonad, PGCs can give rise to embryonic germ cell lines (EGs), pluripotent stem cells that display most of the characteristics of embryonic stem cells (ESCs) including the ability to form teratomas and to contribute to chimera formation. In mice, EG cells can be derived between E8.5 up to E12.5 of embryonic development, at which point the PGCs undergo sexual differentiation and in the male transition into unipotent gonocytes. Dazl, a germ cell-specific RNA-binding protein, is specifically expressed in developing PGCs and is required for proper germ cell development. Dazl knockout mice are infertile, but the molecular mechanisms underlying this phenotype are still unknown. Here we demonstrate that Dazl localizes in granular structures in mammalian PGCs but not in ESCs. We demonstrate Dazl plays a central role in a large mRNA/protein interactive network that includes members of Fragile-X family RNA-binding proteins. We demonstrate that Dazl and Fxr1 play a central role in these granules and directly regulate the translation of specific core pluripotency factors, including Sox2 and Suz12. Global gene expression changes during in vitro germ cell differentiation from murine ES cells and comparison to in vivo germ cells. In vitro primordial germ cells differentiated from Stella-GFP ES cells and Dazl-GFP ES cells. The GFP-positive cells were isolated by FACS analysis. Dazl-GFP+ cells were isolated from transgenic E13.5 embryonic gonads and P7-P11 juvenile mouse testes. The global gene expression profiles were analyzed by Agilent Mouse Whole Genome 4X44K one-color microarrays. Two replicates per condition.
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