Cohesin regulates gene expression through spatial clustering of enhancer elements
Ontology highlight
ABSTRACT: H3K27Ac ChIP-seq in wild type and cohesin-deficient thymocytes Rad21 was deleted in CD4+ CD8+ double positive (DP) thymocytes by crossing a Rad21 floxed allele with a Cd4-driven Cre transgene. DP positive thymocytes were FACS-sorted from control and Rad21-/- littermates, which were then used to perform chromatin immunoprecipitation for histone H3 acetylated on lysine 27 (H3K27Ac).
Project description:T cell-specific deletion of PTEN induces premalignancy in CD4+ CD8+ (DP) immature T cells in the thymus, which progresses to the development of mature CD4+ T cell lymphomas in the lymph nodes and spleen. As part of a screen to identify factors that inhibit progression to malignancy, we compared miRNA expression in premalignant PTEN-deficient DP thymocytes versus wild-type controls. DP thymocytes were collected by cell sorting from three 9-week-old, premalignant T cell-specific PTEN-deficient mice (tPTEN-/-) and three littermate controls. miRNA expression was assessed relative to a reference pool generated from an equal mixture of all samples.
Project description:We performed ChIP-Seq for hallmark TFs (Ets1, Runx1), histone modification marks (H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3), total RNA Pol II, short RNA-Seq as well as nucleosome mapping mainly in murine Rag2 -/- thymocytes. We also performed ChIP-Seq for E47 as well as nucleosome mapping, gene expression microarray analysis in CD4+ CD8+ WT and Ets1-/- DP thymocytes. Overall, we find a key role for the transcription factor Ets1, contributing towards alpha beta T cell lineage commitment via differential transactivation of stage-specific genes orchestrated by dynamic, co-association -mediated chromatin remodeling, as well as transcription dependent generation of a specialized chromatin structure at the TCR beta locus. Genome-wide analysis via ChIP-Seq for Ets1, Runx1, total RNA Pol II binding, H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3, short RNA-Seq, Mnase-Seq in murine Rag2 -/- thymocytes, ChIP-Seq for E47, Mnase-Seq and gene expression microarray analysis in DP thymocytes Gene expression analysis of Ets1-/- CD4+ CD8+ thymocytes
Project description:While histone H3 lysine 27 trimethylation (H3K27Me3) is associated with gene silencing, whether H3K27Me3 demethylation affects transcription and cell differentiation in vivo has remained elusive. To investigate this, we conditionally inactivated the two H3K27Me3 demethylases, Jmjd3 and Utx, in non-dividing intrathymic CD4+ T cell precursors. We show that both enzymes redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1, encoding a sphingosine-phosphate receptor required for thymocyte egress. Floxed alleles of the genes encoding Utx and Jmjd3 (Kdm6a and Kdm6b, respectively) were deleted in double positive (DP) thymocytes carrying a CD4 Cre transgene. Genome-wide H3K27Me3 ChipSeq was performed on (i) pre-selection (CD69lo) DP thymocytes from wild-type mice carrying an endogenous polyclonal TCR repertoire, (ii) mature (TCRhi CD24lo) CD4 SP thymocytes from wild type (Wt), Jmjd3KO, UtxKO and dKO mice carrying an endogenous polyclonal TCR repertoire and (iii) mature (Va2hi CD24lo) CD4 SP thymocytes from wild type and dKO mice carrying the OTII TCR transgene.
Project description:We performed ChIP-Seq for hallmark TFs (Ets1, Runx1), histone modification marks (H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3), total RNA Pol II, short RNA-Seq as well as nucleosome mapping mainly in murine Rag2 -/- thymocytes. We also performed ChIP-Seq for E47 as well as nucleosome mapping, gene expression microarray analysis in CD4+ CD8+ DP thymocytes. Overall, we find a key role for the transcription factor Ets1, contributing towards alpha beta T cell lineage commitment via differential transactivation of stage-specific genes orchestrated by dynamic, co-association -mediated chromatin remodeling, as well as transcription dependent generation of a specialized chromatin structure at the TCR beta locus. Genome-wide analysis via ChIP-Seq for Ets1, Runx1, total RNA Pol II binding, H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3, short RNA-Seq, Mnase-Seq in murine Rag2 -/- thymocytes, ChIP-Seq for E47, Mnase-Seq and gene expression microarray analysis in DP thymocytes This Series represents Mnase-Seq data.
Project description:We performed ChIP-Seq for hallmark TFs (Ets1, Runx1), histone modification marks (H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3), total RNA Pol II, short RNA-Seq as well as nucleosome mapping mainly in murine Rag2 -/- thymocytes. We also performed ChIP-Seq for E47 as well as nucleosome mapping, gene expression microarray analysis in CD4+ CD8+ DP thymocytes. Overall, we find a key role for the transcription factor Ets1, contributing towards alpha beta T cell lineage commitment via differential transactivation of stage-specific genes orchestrated by dynamic, co-association -mediated chromatin remodeling, as well as transcription dependent generation of a specialized chromatin structure at the TCR beta locus. Genome-wide analysis via ChIP-Seq for Ets1, Runx1, total RNA Pol II binding, H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3, short RNA-Seq, Mnase-Seq in murine Rag2 -/- thymocytes, ChIP-Seq for E47, Mnase-Seq and gene expression microarray analysis in DP thymocytes Genome-wide analysis via ChIP-Seq for Ets1, Runx1, total RNA Pol II binding, H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3, short RNA-Seq, Mnase-Seq in murine Rag2 -/- thymocytes, ChIP-Seq for E47, Mnase-Seq and gene expression microarray analysis in DP thymocytes This Series represents gene expression microarray data.
Project description:Genomewide microaarray analysis of murine DP thymocytes to determine the genes whose expression was altered by FLVCR loss Gene signatures from Flvcr+/+ DP thymocytes was compared to gene signatures from Flvcr-/- DP thymocytes Total RNA obtained from sort-purified Flvcr-/- and Flvcr+/+ DP thymocytes
Project description:We performed paired-end RNA-seq to compare the transcriptome of DP thymocytes that ectopically express Lin28 in vivo versus untransduced (GFP-ve) DP thymocytes. Transduced (GFP+) and untransduced (GFP-) CD4+ CD8+ CD3- thymocytes were sorted and pooled from three recipients of hematopoietic stem and progenitor cells transduced with Lin28-RV six weeks post-reconstitution. Total RNA was extracted and paired-end library construction and sequencing was performed on oligo-dT purified RNA.
Project description:Comparing the mRNA expression profiles of c-Myb deficient and c-Myb sufficient Tcra-/- DP thymocytes. Results provide insight into the role of c-Myb in the regulation of survival and differentiation during the pre-selection DP stage where c-Myb expression is abundant during T cell development. DP thymocytes were purified from four c-Myb deficient and four c-Myb sufficient mice over magnetic columns. RNA from each biological replicate was individually hybridized onto a total of eight MOE430 2.0 Chips.
Project description:We performed ChIP-Seq for hallmark TFs (Ets1, Runx1), histone modification marks (H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3), total RNA Pol II, short RNA-Seq as well as nucleosome mapping mainly in murine Rag2 -/- thymocytes. We also performed ChIP-Seq for E47 as well as nucleosome mapping, gene expression microarray analysis in CD4+ CD8+ DP thymocytes. Overall, we find a key role for the transcription factor Ets1, contributing towards alpha beta T cell lineage commitment via differential transactivation of stage-specific genes orchestrated by dynamic, co-association -mediated chromatin remodeling, as well as transcription dependent generation of a specialized chromatin structure at the TCR beta locus. Genome-wide analysis via ChIP-Seq for Ets1, Runx1, total RNA Pol II binding, H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3, short RNA-Seq, Mnase-Seq in murine Rag2 -/- thymocytes, ChIP-Seq for E47, Mnase-Seq and gene expression microarray analysis in DP thymocytes This Series represents ChIP-Seq data.
Project description:The RNase III enzyme dicer is essential for the processing of microRNAs (miRNAs) and small interfering RNAs (siRNAs) from double-stranded RNA precursors. miRNAs and siRNAs regulate chromatin structure, gene transcription, mRNA stability and translation in a wide range of organisms. To provide a model system to explore the role of dicer-generated RNAs in the differentiation of mammalian cells in vivo, we have generated a conditional dicer allele. Deletion of dicer at an early stage of T cell development compromised the survival of αβ lineage cells, while the numbers of γδ-expressing thymocytes were not affected. In developing thymocytes, dicer was not required for the maintenance of transcriptional silencing at pericentromeric satellite sequences (constitutive heterochromatin), the maintenance of cytosine DNA methylation and X chromosome inactivation in female cells (facultative heterochromatin) and the stable shutdown of a developmentally regulated gene (developmentally regulated gene silencing). Most remarkably, given that one-third of mammalian mRNAs are putative miRNA targets, dicer appears to be dispensable for CD4/8 lineage commitment, a process where epigenetic regulation of lineage choice has been well documented. Thus, although dicer appears critical for the development of the early embryo, it may have limited impact on the implementation of lineage-specific gene expression programs. LckCre was used to delete a floxed Dicer1 allele from developing thymocytes (Cobb BS, Nesterova TB, Thompson E, Hertweck A, O'Connor E, Godwin J, Wilson CB, Brockdorff N, Fisher AG, Smale ST and Merkenschlager M. T cell lineage choice and differentiation in the absence of the RNAse III enzyme dicer. J. Exp. Med. 2005 201: 1367-1373). Thymocytes were stained for CD4 and CD8, and double-positive (DP) thymocytes were sorted by flow cytometry. Three biological replicates of Dicer lox/lox control samples and LckCre Dicer lox/lox KO samples were analysed.