Bisulfite-Seq profiling of undifferentiated ESCs, and in vitro derived primordial germ cells (iPGCs)
Ontology highlight
ABSTRACT: 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
Project description:In plants, RNA polymerase II (Pol II) transcription of inverted DNA repeats produces hairpin RNAs that are processed by several DICER-LIKE enzymes into siRNAs that are 21-24-nt in length. When targeted to transcriptional regulatory regions, the 24-nt size class can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS). In a forward genetic screen to identify mutants defective in RdDM of a target enhancer leading to TGS of a downstream GFP reporter gene in Arabidopsis thaliana, we recovered a structurally mutated silencer locus, named SM-NM-^T35S, in which the 35S promoter driving transcription of an inverted repeat of target enhancer sequences had been specifically deleted. Although Pol II-dependent, hairpin-derived 21-24-nt siRNAs were no longer generated at the newly created SM-NM-^T35S locus, the GFP reporter gene was nevertheless still partially silenced. Silencing was associated with methylation in a short tandem repeat in the upstream target enhancer and with low levels of 24-nt tandem repeat siRNAs. Introducing an nrpd1 mutation into the SM-NM-^T35S line fully released GFP silencing and eliminated both the tandem repeat methylation and associated 24-nt siRNAs, demonstrating their dependence on Pol IV. Deletion of the 35S promoter thus revealed a Pol IV-dependent pathway of 24-nt siRNA biogenesis that was previously inhibited or masked by the Pol II-dependent pathway in wild-type plants. Both Pol II- and Pol IV-dependent siRNAs accrued predominantly from cytosine (C)-containing segments of the tandem repeat monomer, suggesting that the local base composition influenced siRNA accumulation. Preferential accumulation of siRNAs at C-containing sequences was also observed at an endogenous tandem repeat comprising discrete C-rich and AT-rich sections. Our studies illuminate the potential complexity of siRNA generation at repeat-containing loci and show that Pol IV can act in siRNA biogenesis in the absence of a conventional Pol II promoter. Examination of whole-genome DNA methylation status in transgenic T+S Arabidopsis plant
Project description:We performed WGBS analyses on 6 human fetal samples at 53-137 days of development, 4 female and 2 male. We show that methylation reprogramming in the human germline is global yet incomplete with exons, 3’UTRs and human-specific transposons remaining methylated. Whole Genome Bisulfite-Seq of cKIT+ cells analyzed from 4 biological samples for fetal ovaries from 57-113 days of development and 2 samples for fetal testes at 59 and 137 days of development.
Project description:Analysis of DNA methylation in the bed nucleus of the stria terminalis/preoptic area and striatum in response to perinatal testosterone exposure. The hypothesis tested was that treatment of females with testosterone on the day of birth would lead to masculinization of the methylome in adulthood. There were three experimental groups: males, females, and females treated with T on the day of birth. The methylation patterns in each group was determined using reduced representation bisulfite sequencing. Two brain regions and two time points (day 4 and day 60) were surveyed in each group. Each biological replicate is a pool of tissue from three animals.
Project description:Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytsosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), while some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naïve ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR-domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of PRDM14 expression rapidly removed the 5mC associated with transient elevation of 5hmC at pluripotency-associated genes, germline-specific genes, and imprinted loci but not across the entire genome, which resemble second wave of DNA demethylation in gonadal PGCs. PRDM14 physically interacts with TET1/TET2 and enhances the recruitment of TET1/TET2 at target loci. Knockdown of Tet1/Tet2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors against APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 normally takes place in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs. To investigate the function of TET1/TET2 in transcriptional regulation by PRDM14 in ESCs, we exploited microarray analysis using total mRNA derived from Scramble, Scramble + PRDM14, Tet1/Tet2 KD, Tet1/Tet2 KD + PRDM14 mouse ESC.
Project description:We investigated the ascomycete truffle Tuber melanosporum exploits DNA methylation and transcription to cope with the more than 45,000 repeated elements that are present in its genome. Whole-genome bisulfite sequencing and RNA-sequencing, were performed on different developmental stages of this symbiotic hypogeous fungus -fruitbody (FB), free-living mycelium (FLM), and ectomycorrhiza. Examination of DNA methylation and transcription of truffle in its free living mycelium (FLM), fruit body (FB), and ectomycorrhizal root tips (ECM)
Project description:Dissection of catalytic and non-catalytic functions of Tet1 and Tet2. Transcriptome profiling of wild type, Tet1 catalytic mutants, Tet2 and Tet1/2 catalytic mutant catalytic during EpiLC differentiation.
Project description:This study uses whole methylome sequencing to characterize the methylomes of mouse embryonic fibroblasts (MEF's). Two conditions were analyzed, MEF cells with intact TET1/TET2 enzymes (WT) and MEF cells with TET1/TET2 knocked out (DKO). Our results identify sets of differentially methylated genes which are correlated with TET1/TET2 induced expression changes of the corresponding genes. Whole methylome analysis of M. musculus MEF cells. Two conditions were sequenced and analyzed, the first is wild type (WT), the second (DKO) corresponds to knock-out of TET1 and TET2 enzymes.
Project description:We report that full length TET1 (TET1-FL) overexpression fails to induce global DNA demethylation in HEK293T cells. The preferential binding of TET1-FL to hypomethylated CpG islands (CGIs) through its CXXC domain leads to its inhibited 5-hydroxymethylcytosine (5hmC) production as methylation level increases. TET1-FL-induced 5hmC accumulates at CGI edges, while TET1 knockdown induces methylation spreading from methylated edges into hypomethylated CGIs. However, TET1 can regulate gene transcription independent of its dioxygenase catalytic function. Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically maintains the DNA hypomethylation state of CGIs in adult cells. hMeDIP-seq analysis of genomic 5-hydroxymethylcytosine in HEK293T cells overexpressing mTET1-CD, TET1-CD, mTET1-FL, or TET1-FL
Project description:Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several transgenic rice lines. We found that all tested transgenic plants had significant losses of methylation compared to untransformed plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability. Whole genome methylation maps of rice were generated using BS-seq (Hume Stroud, Suhua Feng, Steve Jacobsen at UCLA). Whole genome expression maps of rice were generated using mRNA-seq and smRNA (Stacey Simon, Blake Meyers at Univ of Delaware).