EM-seq of TET1 knockout, catalytic mutant and wild type mESCs
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina NovaSeq 6000
ORGANISM(S): Mus musculus
SUBMITTER: Sebastian Bultmann
PROVIDER: E-MTAB-10933 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Investigation of TET1 non-catalytic functions in mESCs cultured in serum + LIF (primed)
2022-07-29 | E-MTAB-10937 | biostudies-arrayexpress
Project description:Precise regulation of DNA methylation in mammals is critical for genome stability and epigenetic regulation. The discovery of the ten-eleven translocation (TET) proteins catalyzing the oxidation from 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized the perspective on the complexity and regulation of DNA modifications. Despite accumulating knowledge about the role of TET1, it remains unclear to what extent these can be attributed to its catalytic activity. Here, we use genome engineering and quantitative multi-omics approaches to dissect the role and mechanism of TET1 in mESCs. Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and as a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. Moreover, we show that the establishment of H3K9me3 and H4K20me3 at ERV1, ERVK, and ERVL is mediated by TET1 independent of DNA demethylation. We provide evidence that repression of endogenous retroviruses depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.
2022-08-31 | PXD028850 | Pride
Project description:Human preimplantation embryo development involves complex dynamic cellular and molecular events that lead to the establishment of the three lineages of the blastocyst â the trophectoderm, primitive endoderm and epiblast. Owing to limited resources of biological specimens, our understanding of how the earliest lineage commitments are regulated is limited. Here, we examined role for MCRS1, TET1, and THAP11 in inducing naive pluripotency in human embryonic stem cells in vitro. Human embryonic stem cells (H9) were nucleofected with piggybac constructs that encode for three genes (MCRS1, THAP11, TET1) followed by neomycin selection for 2 weeks to ensure stable integration. Overexpression of genes can be induced by doxycycline (dox) administration to the culture medium. Prior to dox treatment cells were cultured in conventional feeder-free conditions and then transferred to a feeder layer. Culture medium was switched from mTeSR to W8. Administration of dox and media change into 2i/LIF (2 inhibitors against MEK and GSK3 pathway + leukemia inhibitory factor) induces the overexpression of piggybac constructs and over time transitions primed pluripotent hESCs into a naive pluripotent state. Both pluripotent states were examined with microarrays in three biological replicates for each condition. MCRS1 = microspherule protein 1; TET1 = tet methylcytosine dioxygenase 1; THAP11 = THAP domain containing 11
2016-08-31 | E-MTAB-4567 | biostudies-arrayexpress
Project description:Enzymes catalyzing the methylation of the 5-position of cytosine (mC) have essential roles in regulating gene expression, genome stability, and maintaining cellular identity. Recently Tet1, which is highly expressed in embryonic stem (ES) cells, was found to oxidize the methyl group of mC converting it to 5-hydroxymethyl cytosine (hmC)3. Here, we present the genome-wide mapping of Tet1 and hmC in mouse ES cells. We show that Tet1 binds throughout the genome with the majority of binding sites located at transcription start sites (TSSs) and within genes. Similar to Tet1 and mC, also hmC is found throughout the genome and in particular in gene bodies. However, in contrast to mC, hmC is enriched at TSSs. Tet1 and hmC are associated with genes critical for the control of development and differentiation, which become methylated during differentiation. Surprisingly our results also suggest that Tet1 has a role in transcriptional repression. We show that Tet1 binds to a significant proportion of target genes that are positive for the Polycomb repressive histone mark H3K27me3, and that downregulation of Tet1 also leads to increased expression of a group of Tet1 target genes. In agreement with a potential repressive function, we show that Tet1 associates with the Sin3A co-repressor complex, which also co-localises with Tet1 throughout the genome. We propose that Tet1 fulfils dual functions in transcriptional regulation, where it fine-tunes DNA methylation and associates with the Sin3A co-repressor complex to prevent transcriptional activation. [GSM611209-GSM611217] Control (shScr) or two different Tet1 knockdown (shTet1#4 or shTet1#5) mouse ES cells were used. Each experiment was performed in triplicates. [GSM675884-GSM675889] Control (shScr) or Sin3A knockdown (shSin3A) mouse ES cells were used.Each experiment was performed in triplicates.
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Project description:Loss of Tet1 expression causes global 5mC and 5hmC changes in stem and progenitor cells in mice and enhanced pro-B cell self-renewal, increased DNA damage and B-lymphomageneis. In this study we performed whole transciptome analysis using RNA-sequencing in purified long-term HSCs and MPPs. These results revealed that genes regulated byTet1 included Histones, DNA repair enzymes and B-lineage specific factors. Purified long-term HSCs and MPPs from WT and Tet1 KO mice were used for RNA isolation. RNA was extracted using RNeasy kit (Qiagen) and PolyA selection using oligo-dT beads (Life Technologies) was performed according to the manufacturer’s instructions. Libraries were generated as described before, including end-repair, A-tailing, adapter (Illumina Truseq system) ligation and PCR amplification. RNA libraries were then sequenced on the Illumina HiSeq 2000 using 50bp paired-end reads. Transcriptome profiling of LT-HSC and MPP cells in WT and Tet1 KO mice
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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.
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Project description:We performed lineage tracing experiments using VE-Cadherin-Cre;LoxP-tdTomato mice. In these mice, endothelial cells (ECs) and their progeny are permanently marked by tdTomato fluorescence. We found that a substantial subset of stromal cells is derived from ECs, as indicated by their tdTomato expression. These findings support the notion that endothelial to mesenchymal transition (EndoMT) contributes to hematopoietic bone marrow niche formation in mice. Here we sought to determine the transcriptomic differences between endothelial-derived (tdTomato-positive) and non-endothelial-derived (tdTomato-negative) bone marrow stromal cells (BMSCs) and osteo/chondrolineage progenitor cells (OLCs). Murine niche populations were obtained from collagenased bone fraction of VE-Cadherin-Cre;LoxP-tdTomato mice at 3 weeks (n=2) or 11 weeks (n=2) of age. BMSCs (CD45-TER119-CD31-CD144-SCA-1+ CD51+ cells) and OLCs (CD45-TER119-CD31-CD144-Sca1-CD51+ cells) were FACS-purified and sequenced.
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Project description:The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem (ES) cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence, and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres, and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells. Crosslinking ChIP-seq: Examination of 1 histone variant (H3.3), 2 histone modifications, and Serine-5 phosphorylated RNA polymerase in 2 different cell types (H3.3-HA ES samples 1-4, and H3.3-HA NPC samples 7-10). Examination of 1 histone variant (H3.2), and one histone modification (H3K36me3) in 2 different cell types (H3.2-HA ES samples 5-6, and H3.2-HA NPC samples 11-12). Examination of 1 histone variant (H3.3), input control, and one histone modification (H3K36me3) in one cell type (H3.3-HA hybrid ES, samples 13-15). Examination of 1 histone variant (H3.1S31), input control, and one histone modification (H3K36me3) in one cell type (H3.1S31-HA hybrid ES, samples 16-18). Native ChIP-seq: Examination of 1 histone variant (H3.3), input control, and one histone modification (H3K4me3) in one cell type (H3.3-HA ES, samples 19-21). Examination of 1 histone variant (H3.2), input control, and two histone modifications (H3K4me3 and H3K27me3) in one cell type (H3.2-HA ES, samples 22-25). Examination of 1 histone variant (H3.3), input control, and two histone modifications (H3K4me1 and H3K36me3) in one cell type (H3.3-EYFP ES, samples 26-29). Examination of 1 histone variant (H3.3), input control, and two histone modifications (H3K4me1 and H3K36me3) in one cell type (Hira -/- H3.3-EYFP ES, samples 30-33). Examination of 1 histone variant (H3.3) and input control in one cell type (Atrxflox H3.3-EYFP ES, samples 34-37). Examination of HA antibody background in one cell type (wild-type ES, sample 38).
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