Regulation of nucleosome landscape and transcription factor binding at enhancers by BRG1
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE26501 | GEO | 2011/07/01
SECONDARY ACCESSION(S): PRJNA136673
REPOSITORIES: GEO
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Project description:Enhancers of transcription activate transcription via binding of sequence-specific transcription factors to their target sites in chromatin. In this report, we identify GATA1-bound enhancers genome-wide and find a global reorganization of the nucleosomes at these enhancers during differentiation of hematopoietic stem cells (HSCs) to erythrocytes. We show that the catalytic subunit BRG1 of BAF complexes localizes to these enhancers during differentiation and generates a longer nucleosome repeat length surrounding the GATA1 sites by shifting the flanking nucleosomes away. Intriguing, we find that the nucleosome shifting specifically facilitates binding of TAL1 but not GATA1, which subsequently activates transcription of target genes. Human hematopoietic stem cells (referred as CD34+ cells) were differentiated into erythroid lineage expressing the cell surface marker CD36 (referred as CD36+ cells). To study the function of ATP-dependent chromatin remodeling BAF complexes in the establishment of erythroid specific enhancers during differentiation, we mapped the genome-wide binding sites of GATA1 and TAL1, the key transcription regulators of erythroid differentiation, in CD36+ cells, then determined the genomic positions of the catalytic subunit BRG1 of the BAF complexes and the nucleosome landscapes, by ChIP-Seq and Mnase-Seq, respectively, for both HSCs and the differentiated cells. We compared changes in nucleosome landscapes with BRG1 binding at GATA1 and TAL1 binding sites and found that BRG1 binding is correlated with nucleosome shifting away from the binding sites, which is critical for TAL1 binding. To confirm that BRG1 is responsible for the nucleosome shift, we knocked down BRG1 in CD36+ cells and analyzed the nucleosome changes in the knockdown cells. The data indicated that inhibition of BRG1 caused a nucleosome shift towards the GATA1 or TAL1 sites, indicating that BRG1 is indeed responsible for the observed nucleosome shift.
2011-07-01 | E-GEOD-26501 | biostudies-arrayexpress
Project description:Genome-wide mapping of nucleosomes has revealed a great deal about the relationships between chromatin structure and control of gene expression, and has led to mechanistic hypotheses regarding the rules by which chromatin structure is established. High-throughput sequencing has recently become the technology of choice for chromatin mapping studies, yet analysis of these experiments is still in its infancy. Here, we introduce a pipeline for analyzing deep sequencing maps of chromatin structure and apply it to data from S. cerevisiae. We analyze digestion series where nucleosomes are isolated from under- and over-digested chromatin. We find that certain classes of nucleosomes are unusually susceptible or resistant to overdigestion, with promoter nucleosomes easily digested and mid-coding region nucleosomes being quite stable. We find evidence for highly sensitive nucleosomes located within “nucleosome-free regions,” suggesting that these regions are not always completely naked but instead are likely associated with easily-digested nucleosomes. Finally, since RNA polymerase is the dominant energy-consuming machine that operates on the chromatin template, we analyze changes in chromatin structure when RNA polymerase is inactivated via a temperature-sensitive mutation. We find evidence that RNA polymerase plays a role in nucleosome eviction at promoters, and is also responsible for retrograde shifts in nucleosomes during transcription. Loss of RNA polymerase results in a relaxation of chromatin structure to more closely match in vitro nucleosome positioning preferences. Together, these results provide analytical tools and experimental guidance for nucleosome mapping experiments, and help disentangle the interlinked processes of transcription and chromatin packaging. Keywords: Genomic Solexa/Illumina sequencing of mononucleosomes. Titration series: gel-purified mononucleosomal DNA from three different titration levels – underdigested, typical digestion, and overdigested. RP021 series: MNase-seq at 0, 20, and 120 minutes after shifting these cells from 25 C to 37 C.
2009-10-15 | E-GEOD-18530 | biostudies-arrayexpress
Project description:Erythropoiesis is among the best understood examples of cell differentiation. In a required step, GATA1 replaces GATA2 at genomic loci, termed GATA switch enhancers. Little is known about how this switch affects chromatin architecture. Here, we show that cohesin binds GATA1 and occupies most GATA switch enhancers. Cohesin-based chromatin interaction analysis demonstrated that the most dynamic and interactive enhancers are enriched for GATA switch sites/TAL1 occupancy and frequently lack super-enhancer features. A c-kit locus CRISPR/Cas9 screen identified GATA motifs within one such element as strongly impacting expression. These data highlight the role of developmentally exchanged transcription factors in modulating genomic folding.
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Project description:We performed ChIP-Seq for Ets1 and histone modifications in P5424 thymic cells, without and with Ets1 knockdown via shRNA. Overall, we find that loss of Ets1 results in specific, higher occupancy of H3K4me1-marked nucleosomes at the Ets1 binding site, but not H3K4me3 nucleosomes. We verified the specificity of this mechanism as Ets1-dependent by also computing H3K4me1 and 3 nucleosome occupancy in hypersensitive, Ets1-depleted sites. This effect was also found in primary WT DN+DP Ets1 sites. Overall, this suggests that Ets1 induces chromatin remodeling and that its loss increases H3K4me1 nucleosome occupancy in bound sites, with levels of H3K4me3 comparatively unchanged, thus reducing the activation status of these enhancers Genome-wide analysis via ChIP-Seq for Ets1, H3K4me1 and H3K4me3 in WT, sh-scramble and sh-Ets1 the P5424 thymic cell line
2015-12-19 | E-GEOD-74042 | biostudies-arrayexpress
Project description:Nucleosome organization influences gene activity by controlling DNA accessibility to transcription machinery. Here, we develop a chemical biology approach to determine mammalian nucleosome positions genome-wide. Using this strategy, we uncover surprising new features of nucleosome organization in mouse embryonic stem cells. In contrast to the prevailing model, we observe that for nearly all mouse genes a class of fragile nucleosomes occupies previously designated nucleosome-depleted regions around transcription start sites and transcription termination sites. We show that a subset of DNA-binding proteins including insulator CTCF and pluripotency factors co-occupy DNA targets with nucleosomes. Furthermore, we provide in vivo evidence that promoter-proximal nucleosomes, with the +1 nucleosome in particular, contribute to the pausing of RNA Polymerase II. Lastly, we find a characteristic preference for nucleosomes at exon-intron junctions. Altogether, we establish an accurate method for defining the nucleosome landscape, and provide a valuable resource for studying nucleosome-mediated gene regulation in mammalian cells.
2016-11-23 | GSE82127 | GEO
Project description:Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses and candidate enhancers identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the TSS of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or erythroid cell phenotypes. Candidate enhancers exhibited only moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a data set of erythroid-associated, biologically relevant, SNPs from the GWAS catalog of the NHGRI. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function, and provide insights into inherited and acquired hematologic disease. CD34+-selected stem and progenitor cells were expanded for three days in the absence of EPO. The cells were further cultured in the presence of EPO, and formaldehyde crosslinked chromatin was isolated after cells differentiated into R3/R4 nucleated erythroid cells. Chromatin Immunoprecipitation followed by sequencing (chIP-seq) was performed using antibodies against GATA1, KLF1, NFE2, TAL1, p300, H3K4me2 and H3K4me3, along with a total input control. Raw data (fastq, SRA) is missing for the TAL1 chIP-seq dataset
2013-01-28 | E-GEOD-43625 | biostudies-arrayexpress
Project description:The ability to measure epigenetic features, such as histone modifications and occupancy by transcription factors and co-activators, on a genome-wide scale is advancing the accuracy of CRM predictions. While integration of signals from multiple features is expected to improve predictions, the contribution of each feature to prediction accuracy is not known. We began with predictions of 4,915 erythroid enhancers based on genomic occupancy by TAL1, a key hematopoietic transcription factor that is strongly associated with gene induction in erythroid cells. Seventy of these DNA segments occupied by TAL1 (TAL1 OSs) were tested by transient transfections of cultured hematopoietic cells, and 56% of these were active as enhancers. Sixty-six TAL1 OSs were evaluated in transgenic mouse embryos, and 65% of these were active enhancers in various tissues. Inclusion of additional epigenetic features improved the prediction accuracy, with combinations of TAL1, GATA1, EP300, H3K4me1, and H3K27ac giving high accuracy of enhancer prediction (70%-75% success depending on method of clustering) while maintaining good sensitivity and specificity. Motifs that distinguish active from inactive TAL1 OSs implicate IRFs, STATs, and FOX protein families as candidate positive co-factors with TAL1, while REST (NRSF) and HOX family proteins are implicated in inactivity. While signals for evolutionary constraint were weak over the entire TAL1-bound DNA segments regardless of activity in either assay, phylogenetic preservation of a TF-binding site motif was associated with enhancer activity. The contribution of 8 epigenetic features including H3K27ac to identification of enhancers in 24h-induced G1E-ER4 cells.
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Project description:Nucleosome structure and positioning play pivotal roles in gene regulation, DNA repair and other essential processes in eukaryotic cells. Nucleosomal DNA is thought to be uniformly inaccessible to DNA binding and processing factors, such as MNase. Here, we show, however, that nucleosome accessibility and sensitivity to MNase varies. Digestion of Drosophila chromatin with two distinct concentrations of MNase revealed two types of nucleosomes: sensitive and resistant. MNase-resistant nucleosome arrays are less accessible to low concentrations of MNase, whereas MNase-sensitive arrays are degraded by high concentrations. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C containing dinucleotides. In contrast, MNase-sensitive nucleosomes form on A/T rich sequences represented by transcription start and termination sites, enhancers and DNase hypersensitive sites. Lowering of cell growth temperature to ~10°C stabilizes MNase-sensitive nucleosomes suggesting that variations in sensitivity to MNase are related to either thermal fluctuations in chromatin fiber or the activity of enzymatic machinery. In the vicinity of active genes and DNase hypersensitive sites nucleosomes are organized into synchronous, periodic arrays. These patterns are likely to be caused by “phasing” nucleosomes off a potential barrier formed by DNA-bound factors and we provide an extensive biophysical framework to explain this effect. RNA-seq S2 cells Drosophila melanogaster
2015-09-17 | E-GEOD-69335 | biostudies-arrayexpress
Project description:Erythroid development and differentiation from multiprogenitor cells to red blood cells requires precise transcriptional regulation. Key erythroid transcription factors, GATA1 and TAL1, co-operate, along with other proteins, to regulate many aspects of this process. How GATA1 and TAL1 are positionally organized with respect to each other and their cognate DNA binding site across the mouse genome remains unclear. We applied high resolution ChIP-exo to GATA1 and TAL1 to study their positional organization across the mouse genome during GATA1-dependent maturation. Two complementary methods, MultiGPS and peak-pairing, were used to determine high confidence binding locations by ChIP-exo. We identified ~10,000 GATA1 and ~15,000 TAL1 locations, which were essentially confirmed by ChIP-seq. Of these, ~4,000 locations were bound by both GATA1 and TAL1. About three-quarters of these were tightly linked (<40 bp away) to a partial E-box located 7-8 bp upstream of a WGATAA motif. Both TAL1 and GATA1 generated distinct characteristic ChIP-exo peaks around WGATAA motifs, that reflect on their positional arrangement within a complex. We show that TAL1 and GATA1 form a precisely organized complex at a compound motif consisting of a TG 7-8 bp upstream of a WGATAA motif across thousands of genomic locations. Genome wide analysis of GATA1 and TAL1 in G1E and G1E-ER4 cells using ChIP-exo experiments
2015-09-17 | E-GEOD-68964 | biostudies-arrayexpress
Project description:The positioning of nucleosomes with respect to DNA plays an important role in regulating transcription. However, nucleosome mapping has been performed for only limited genomic regions in humans. We have generated genome-wide maps of nucleosome positions in both resting and activated human CD4+ T cells by direct sequencing of nucleosome ends using the Solexa high-throughput sequencing technique. Corresponding ChIP-Seq data are available in NCBI's Short Read Archive at ftp://ftp.ncbi.nih.gov/pub/TraceDB/ShortRead/SRA000234/; We find that nucleosome phasing relative to the transcription start sites (TSSs) is directly correlated to RNA polymerase II binding. Furthermore, the first nucleosome downstream of TSSs exhibits differential positioning in active and silent genes. TCR signaling induces extensive nucleosome reorganization in promoters and enhancers to allow transcriptional activation or repression. Our results suggest that H2A.Z-containing and modified nucleosomes are preferentially lost from the -1 nucleosome position. Our data provide a comprehensive view of the nucleosome landscape and its dynamic regulation in the human genome. This microarray dataset is the gene expression data from resting and activated CD4+ T cells. We used this data to look at nucleosome positioning, Pol II and a few chromatin modifications at genes that are silent and activated in both resting and activated T cells as well as genes that are induced or repressed with T cell activation. Experiment Overall Design: Gene expression data from resting (M0 and T0) and activated (M18 and T18) T cells.
2008-04-08 | E-GEOD-10437 | biostudies-arrayexpress