Transcription factor binding in human cells occurs in dense clusters formed around cohesin anchor sites [Validation Set]
Ontology highlight
ABSTRACT: During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis, and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered, and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M-phase. These results suggest that cohesin binding functions as a cellular memory that promotes re- stablishment of TF clusters after DNA replication and chromatin condensation. Examination of TF binding by ChIP-seq in a CRC cell-line.
Project description:During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis, and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered, and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M-phase. These results suggest that cohesin binding functions as a cellular memory that promotes re- stablishment of TF clusters after DNA replication and chromatin condensation. Examination of TF binding by ChIP-seq in a CRC cell-line treated with non-targeting siRNA, or siRNA specific to the cohesin subunit RAD21.
Project description:During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis, and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered, and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M-phase. These results suggest that cohesin binding functions as a cellular memory that promotes re- stablishment of TF clusters after DNA replication and chromatin condensation. Examination of TF binding by ChIP-seq in a CRC cell-line treated with non-targeting siRNA, or siRNA specific to the cohesin subunit RAD21.
Project description:During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis, and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered, and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M-phase. These results suggest that cohesin binding functions as a cellular memory that promotes re- stablishment of TF clusters after DNA replication and chromatin condensation. Examination of TF binding by ChIP-seq in LoVo CRC cell-lines.
Project description:Examination of EBf1 binding by ChIP-seq in differentiated human adipose stromal cell (hASC) pre-adipocyte Pre-Adipocytes differentiated in-vitro were fixed in 1% formaldehyde for 15 min at room temperature and quenched for 5 min by adding glycine to a final concentration of 0.125 M. ChIP assays were then performed with custom-made EBF1 antibody or rabbit IgG . ChIP-sequencing libraries were prepared using NEBnext chip-seq library Prep master mix set from 5 ng of anti-EBF1 and anti-IgG ChIP DNA, respectively. Sequence data were generated with Illumina HiSeq 2000 single-read sequencing and aligned against the human genome (hg19, NCBI).
Project description:Different mechanisms for CBF-MYH11 function in acute myeloid Leukemia (AML) with inv(16) have been proposed such as tethering of RUNX1 outside the nucleus, interference with transcription factor complex assembly and recruitment of histone deacetylases, all resulting in transcriptional repression of RUNX1 target genes. Here, through genome-wide CBF-MYH11 binding site analysis and quantitative interaction proteomics we found that CBF-MYH11 localizes to RUNX1 occupied promoters where it interacts with TAL1, FLI1 and TBP associated factors (TAFs) in the context of the hematopoietic transcription factors ERG, GATA2 and PU.1/SPI1 and the co regulators EP300 and HDAC1. Transcriptional analysis revealed that upon fusion protein knock down a subset of the CBF-MYH11 target genes show increased expression, confirming a role in transcriptional repression. However, the majority of CBF-MYH11 target genes, including genes implicated in hematopoietic stem cell (HSC) self-renewal such as ID1, LMO1 and JAG1, are actively transcribed and upon fusion protein knock down repressed. Together these results suggest an essential role for CBF-MYH11 in regulating expression of genes involved in maintaining a stem cell phenotype. 17 ChIP-seq samples using antibodies recognizing the indicated proteins and one RNA-seq file from ME-1 cells were analyzed. In addition 2 ChIP-seq profiles were generated using patient AML cells. A CBFβ-MYH11 inducible U937 system (U937CM) was used to examine binding patterns before (1 profile) and after (2 profiles) induction of CBFb-MYH11. In addition, expression was measured through RNA-seq analysis of the two states. The U937CM cells were maintained in the presence of tetracyclin (Tet, 1 uM) and grown in the absence of tetracycline for 3 days to induce expression of CBFβ-MYH11. Finally, a CBFb-MYH11 knock down system was developed in ME-1 cells. Two ME-1 cell lines were created, one with a stably integrated shRNA construct that targets CBFb-MYH11 (ME-1_knockdown) and one with a scrambled shRNA construct (ME-1_SCR). Expression of the shRNA constructs was induced using doxycyclin (dox; 1 mM) treatment for 3 days.
Project description:We carried out a genome-wide investigation of the primary transcriptional targets of 1α,25(OH)2D3 in breast epithelial cancer cells using RNA-Seq technology. We identified early transcriptional targets of 1α,25(OH)2D3 involved in adhesion, growth regulation, angiogenesis, actin cytoskeleton regulation, hexose transport, inflammation and immunomodulation, apoptosis, endocytosis and signaling. Furthermore, we found several transcription factors to be regulated by 1α,25(OH)2D3 that subsequently amplify and diversify the transcriptional output driven by 1α,25(OH)2D3 leading finally to a growth arrest of the cells. Moreover, we could show that 1α,25(OH)2D3 elevates the trimethylation of histone H3 lysine 4 at several target gene promoters. Our present transcriptomic analysis of differential expression after 1α,25(OH)2D3 treatment provides a resource of primary 1α,25(OH)2D3 targets that might drive the antiproliferative action in breast cancer epithelial cells. ChIP-Seq for trimethylated histone H3K4 with SKBr3 cells treated for 2h with 100nM 1α,25(OH)2D3 or vehicle as control.
Project description:We report how CSB affects globally the density of RNA Pol II at TSS and how this effect correlates with the gene expression alterations observed from microarray analysis. We also show globally the distribution of CSB. RNA Pol II and CSB ChIP-Seq in CS1AN cells and CSB reconstituted wild type cells, in duplicate, using Illumina GAIIx
Project description:The androgen receptor (AR) is a ligand-inducible transcription factor that mediates androgen action in target tissues. Upon ligand binding, the AR binds to thousands of genomic loci and activates a cell-type specific gene program. Prostate cancer growth and progression depend on androgen-induced AR signalling. Treatment of advanced prostate cancer through medical or surgical castration leads to initial response and durable remission, but resistance inevitably develops. In castration-resistant prostate cancer (CRPC), AR activity remains critical for tumor growth despite androgen deprivation. While previous studies have focused on ligand-dependent AR signalling, in this study we explore AR function under the androgen-deprived conditions characteristic of CRPC. Our data demonstrate that the AR persistently occupies a distinct set of genomic loci after androgen deprivation in CRPC. These androgen-independent AR occupied regions have constitutively open chromatin structures that lack the canonical androgen response element and are independent of FoxA1, a transcription factor involved in ligand-dependent AR targeting. Many AR binding events occur at proximal promoters, which can act as enhancers to augment transcriptional activities of other promoters through DNA looping. We further show that androgen-independent AR binding directs a distinct gene expression program in CRPC, which is necessary for the growth of CRPC after androgen withdrawal. LNCaP, C4-2B, or 22RV1 cells were cultured in hormone-free media for 3 days and then treated with ethanol vehicle or DHT (10nM) for 4h or 16h prior to ChIP-seq or RNA-seq assays. For siRNA transfection, cells were transfected with AR siRNA or control siRNA for 3 days prior to RNA-seq assays.
Project description:We investigated the RNAPII and γH2AX occupancy genome wide by ChIP-Seq in MLL2 F/F and FC/FC80 MEF cells. We found that a week after MLL2 excision (FC/FC cells), a group of genes present higher levels of γH2AX and RNAPII near the TSS, as compared to the control (F/F cells). H3K4Me1, H3K4M2 and H3K4Me3 levels near the TSS were also studied. There is a total of 52 samples. 3 independent replicates for each experiment were performed. H3, H2AX and IgG ChIPs were used for normalisation or as controls.The experiments were performed using immortalised mouse embryonic fibroblasts (MEF) in which both MLL2 alleles were targeted by the loxp system (F/F cells). Tamoxifen treatment of the F/F cells for 24 hours results in the excision of both MLL2 alleles (FC/FC cells).
Project description:We report that hyperglycemia-mediated induction of genes and pathways associated to endothelial dysfunction occur through modulation of acetylated H3K9/K14 inversely correlated with methyl-CpG content. Examination of H3K9/K14 histone acetylation and DNA methylation in hyperglycemic conditions.