ChIP-seq of T/Brachyury in mouse embryonic stem cells in the process of disruption of naive pluripotency caused by T induction
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ABSTRACT: ChIP-seq to clarify the molecular mechanism by which a mesodermal transcription factor, T/Brachyury, disrupts the defined naive pluripotency.
Project description:E2F2 is essential for the maintenance of T lymphocyte quiescence. To identify the full set of E2F2 target genes, and to gain further understanding of the role of E2F2 in transcriptional regulation, we have performed ChIP-chip analyses across the genome of lymph node-derived T lymphocytes. Here we show that during quiescence, E2F2 binds the promoters of a large number of genes involved in DNA metabolism and cell cycle regulation, concomitant with their transcriptional silencing. We performed 3 ChIP-chip experiments with an antibody against E2F2 and another 3 ChIP-chip experiments with an antibody against SV40TAg (irrelevant antibody).
Project description:Germ cells ensure reproduction and heredity in metazoans. Primordial germ cells (PGCs) in mice are induced in pluripotent epiblasts by BMP4 and WNT3, yet their mechanism of action remains unclear. Here, using in vitro PGC specification system, we show that WNT3 induces many transcription factors associated with mesoderm in epiblast-like cells (EpiLCs) through b-CATENIN. Among these, T (BRACHYURY), a classical and conserved mesodermal factor, was essential for robust activation of Blimp1 and Prdm14, two of the germline determinants. T, but not SMAD1 or b-CATENIN/TCF1, binds distinct regulatory elements of both Blimp1 and Prdm14, and without BMP4 and WNT3, directly up-regulates these genes, consequently delineating downstream PGC program. Without BMP4, a program induced by WNT3 prevents T from activating Blimp1 and Prdm14, demonstrating that BMP4 is permissive for PGC specification. These findings establish a fundamental role of a mesodermal transcription factor in PGC specification, a potentially evolutionarily conserved mechanism across metazoans. Genome target search for transcription factors, SMAD1, T, and TCF1 on PGC-like cells
Project description:The distribution of histone variants H2Abbd and macroH2A in 13 regions of the HG18 assembly have been studied using a variant of the ChIP-on-Chip technique. HeLa S3 cell lines expressing tagged histones H2A, H2Abbd or macroHA were obtained using retroviral transfer. DNA fractions associated with tagged histones were isolated using a two-step purification procedure that involved affinity chromatography on a column with anti-FLAG antibodies, followed by affinity chromatography on a Ni-agarose column. The obtained genomic DNA samples were analyzed by hybridization with custom NimbleGene genomic microarrays. Two samples. Test sample 1 is HeLa S3 cells expressing epitope-tagged histone H2Abbd and test sample 2 is HeLa S3 cells expressing epitope-tagged histone macroH2A . The control for both test sample 1 and test sample 2 is HeLa S3 expressing epitope-tagged histone H2A. Two copies of each probe per array were made.
Project description:Genomewide mapping of D. melanogaster Tramtrack69 protein binding at 6-8hrs after egg laying. Two different rabbit antibodies were used to precipitate the Tramtrack69 protein isoform in 3 biological replicates. Additionally a rabbit preimmune-serum was used as a control for every precipitation. The enriched DNA was hybridized to high density Affymetrix GeneChip Drosophila Tiling 1.0R array.
Project description:ChIP-seq of ASY1 was carried out on meiotic-stage floral buds of Arabidopsis using an a-ASY1 antibody. The experiment aims to determine the genome-wide profile of ASY1. ASY1 is a component of the chromosome axis and is expressed exclusively during meiosis. Two negative controls were used to test the specificity of the ChIP experiment. First, ChIP-seq using the pre-immune on floral buds was carried out. Second, ChIP-seq using an a-ASY1 antibody was performed on leaf tissue where ASY1 is not expressed.
Project description:In Drosophila melanogaster, two chromosome-specific targeting and regulatory systems have been described. The male-specific lethal (MSL) complex supports dosage compensation by stimulating gene expression from the male X-chromosome and the protein Painting of fourth (POF) specifically targets and stimulates expression from the heterochromatic 4th chromosome. The targeting sites of both systems are well characterized, but the principles underlying the targeting mechanisms have remained elusive. Here we present an original observation, namely that POF specifically targets two loci on the X-chromosome, PoX1 and PoX2 (POF-on-X). PoX1 and PoX2 are located close to the roX1 and roX2 genes, which encode ncRNAs important for the correct targeting and spreading of the MSL-complex. We also found that the targeting of POF to PoX1 and PoX2 is largely dependent on roX expression and identified a high-affinity target region which ectopically recruits POF. The results presented support a model linking the MSL-complex to POF and dosage compensation to regulation of heterochromatin. POF salivary glands ChIP
Project description:Heterochromatin protein 1 (HP1) proteins are important regulators of heterochromatin mediated gene silencing and chromosome structure and it is well known as the reader of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me). In Drosophila three different histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9; Su(var)3-9, Setdb1 and G9a. To gain insights on the dependence of HP1a on the three different HKMTs, the division of labor between these methyl transferases and the dependence of HP1a on H3K9me we have studied HP1a binding in relation to H3K9me in mutants of these HKMTs. We show that Su(var)3-9 is responsible for the HP1a H3K9me-dependent binding in pericentromeric regions while Setdb1 controls the HP1a H3K9me-dependent binding to cytological region 2L:31 and together with POF chromosome 4. HP1a binds to the promoters and within gene bodies of active genes in these three regions. More importantly, HP1a bound at promoters of active genes are independent of H3K9me and POF and is associated to heterochromatin protein 2 (HP2) and open chromatin. Our results supports a model where HP1a nucleates with high affinity independent of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites. In total 44 samples; 2 replicates for each genotype and for each ChIP (HP1a, H3K9me2 and H3K9me3)
Project description:Transcription factors are key components of light signalling as they amplify the signal, which results in massive changes in genome-wide expression during photomorphogenesis. Three bZIP transcription factors (TFs), namely GBF1, HY5, and HYH, form heterodimers with each other and regulate photomorphogenesis in an interdependent manner. GBF1 acts as both a positive and negative regulator of photomorphogenesis, whereas HY5 and HYH mainly act as positive regulators of photomorphogenesis. In this study, the impact of heterodimerization of GBF1 with HY5 and HYH was analyzed for genome-wide binding of GBF1 through ChIP-chip in GBF1. We identified more than 2000 direct targets of GBF1 in the presence of HY5 and HYH. However, in the absence of HY5, very few binding sites were found, and in the absence of functional HYH protein, the number of GBF1 direct targets reduced to only half compared to when functional HYH was present. ChIPed DNA with GBF1 antibody from GBF1OE, hy5 GBF1OE, and hyh GBF1OE lines vs. ChIPed DNA with GBF1 antibody from wild-type.