Chromatin occupancy of key transcriptional regulators in the presence of the BET bromodomain inhibitor JQ1
ABSTRACT: MM1.S cells are an aggressive dexamethasone sensitive multiple myeloma cell line whose transcritional program is driven by deregulated c-Myc activity. We present ChIP-seq analysis of key transcritional regulators that are implicated the c-Myc transcriptional network in MM1.S cells treated with vehicle or 500nM JQ1. Brd4, Cdk9, cMyc, Max, Med1, RNA Pol II, and the chromatin modifications H3K4me3 and H3K27Ac were profiled in MM1.S cells treated with 500nM JQ1 for 24hr
Project description:Chromatin regulators have become highly attractive targets for cancer therapy, yet many of these regulators are expressed in a broad range of healthy cells and contribute generally to gene expression. An important conundrum has thus emerged: how can inhibition of a general regulator of gene expression produce selective effects at specific oncogenes? Here we investigate how inhibition of the transcriptional coactivator BRD4 (Bromodomain containing 4) leads to selective inhibition of disease-critical oncogenes in a highly malignant blood cancer, multiple myeloma (MM). We found that BRD4 generally occupies the promoter elements of active genes together with the Mediator coactivator, but remarkably high levels of these two coactivator proteins were associated with a small set of exceptionally large enhancers. These super-enhancers are associated with genes that feature prominently in MM biology, including the MYC oncogene. Treatment of MM tumor cells with the BET-bromodomain inhibitor JQ1 led to preferential loss of BRD4 at super-enhancers and consequent transcription elongation defects that preferentially impact genes with super-enhancers, including the c-MYC oncogene. Super-enhancers were found at key oncogenic drivers in many other tumor cells. Thus, super-enhancers can regulate oncogenic drivers in tumor cells, which in some cells can be preferentially disrupted by BRD4 inhibition, which in turn contributes to the selective transcriptional effects observed at these oncogenes. These observations have implications for the discovery of novel cancer therapeutics directed at components of super-enhancers in diverse tumor types. ChIP-Seq for chromatin regulators and RNA Polymerase II in multiple myeloma, glioblastoma multiforme, and small cell lung cancer
Project description:Critical drivers of cancer progression are likely controlled through the actions of transcription factors and cofactors that bind to the genome and form enhancers that stimulate gene expression. We present ChIP-seq analysis of key transcriptional regulators, cofactors and histone modifications that indicate transcriptional activity across a range of different cancer cells. ChIP-seq was performed against the cofactors Med1, Brd4 and CDK7 in MM1.S multiple myeloma cells; Brd4 in Sk-MEL-5 melanoma cells; and H3K9/K14 acetylation in MV4;11 leukemia cells,
Project description:ChIP-Seq of RNA Polymerase II, and transcriptional regulators in multiple myeloma (MM.1S), glioblastoma (U87-MG), and small cell lung carcinoma (H2171) treated with the BET bromodomain inhibitor JQ1. Cell lines (MM.1S, U87-MG, and H2171) representing multiple myeloma, glioblastoma, and small cell lung carcinoma, were treated with varying concentrations (5nM to 5µM) of the BET bromodomain inhibitor JQ1 followed by ChIP-Seq for RNA Polymerase II and transcriptional regulators. Other datasets from this series of experiments have been release as a part of GSE42355.
Project description:P493-6 cells are immortalized human peripheral B cells that carry a conditional, tetracycline-regulated myc gene. We present ChIP-seq analysis of key transcritional regulators in P493-6 cells expressing various levels of c-Myc: 0hr (low c-Myc levels), 1hr (intermediate c-Myc levels), 24hr (very high c-Myc levels) and No Tet (steady-state c-Myc levels). Brd4, c-Myc, Max, Med1, RNAPII, and the chromatin modification H3K27Ac were profiled in P493-6 cells
Project description:cMyc and Max ChIP-seq analysis in a high MYC expressing small cell lung carcinoma cell line (H2171) and a low MYC expressing small cell lung carcinoma cell line (H128). H2171 cells (expressing high levels of cMyc protein) and H128 cells (expressing low levels of cMyc protein) were used to analyze how cMyc overexpression influences its genome-wide occupancy.
Project description:An ability to map the global interactions of a chemical entity with chromatin genome-wide could provide new insights into the mechanisms by which a small molecule perturbs cellular functions. we developed a method that uses chemical derivatives and massively parallel DNA sequencing (Chem-Seq) to identify the sites bound by small chemical molecules throughout the human genome. We developed in vivo and in vitro Chem-Seq protocols with a biotinylated derivative of small molecules. In the in vivo protocol, Cells were first treated with biotinylated ligand and cross-linked with formaldehyde at the same time. Cells were then lysed, sonicated to shear the DNA, and streptavidin beads were used to isolate biotinylated ligand and associated chromatin fragments. We then used massively parallel sequencing to identify the enriched DNA fragments, and mapped these sequences to the genome. In in vitrol protocol, MM1.S cells were fixed and the derived sonicated lysate incubated with biotinylated drug to enrich for bound chromatin regions in vitro. We then used massively parallel sequencing to identify the enriched DNA fragments, and mapped these sequences to the genome.
Project description:Excessive expression of c-Myc occurs frequently in human cancers, where high levels are associated with tumor aggression and poor clinical outcome, but the effect of high levels of c-Myc on global gene regulation is poorly understood. We report here that in tumor cells expressing high levels of c-Myc, the transcription factor binds to E-box sequences in the core promoters of most actively transcribed genes and, unexpectedly, the enhancers of these active genes. The predominant effect of increasing c-Myc levels at both proximal and distal promoter elements is to produce higher levels of transcription at existing active genes by promoting RNA polymerase II elongation, as opposed to stimulating transcription of novel target genes. Our results argue that c-Myc overexpression drives increased transcription of growth-promoting genes, and does so by amplifying the levels of transcripts associated with the entire gene expression program of the cancer cell. Thus, excess c-Myc functions to elicit the transcriptional amplification of existing active genes through the invasion of enhancers across the cancer cell genome, thereby reducing the rate-limiting constraints required for continuous tumor growth and proliferation. ChIP-Seq of multiple factors and histone modifications in a variety of human tumor cell lines
Project description:H3K27me3 is a chromatin modification depositied by Suz12, a component of the Polycomb Group 2 complex, and is associated with transcriptional repression. In contrast, H3K79me2 is a chromatin modification associated with active gene transcription. It is deposited by the histone methyltransferase Dot1L and generally is localized just downstream of the transcriptional start site and extends down the body of the gene. To gain insight into the transcriptional state of genes in hES cells, chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) was performed to determine the genome-wide occupancy of the H3K27me3 and H3K79me2 chromatin modifications and genome-wide occupancy of the Suz12. DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing A sample of whole cell extract was sequenced and used as the background to determine enrichment. ChIP was performed using an antibody against H3K27me3, H3K79me2, and Suz12 (Abcam).
Project description:Condensin molecules are loaded onto the genome to mediate essential changes in chromosome condensation during mitosis, but it is not clear why there are two forms of vertebrate condensin that become differentially distributed on chromosomes. We report here that condensin II, the form of condensin present in the nucleus throughout the cell cycle, functions specifically at active genes. Condensin II is loaded at transcriptionally active promoters in embryonic stem cells (ESCs), migrates through these genes in a transcription-dependent fashion and accumulates in transcription termination regions. Unlike cohesin, which is also loaded at active promoters, condensin II has little influence on transcription. We conclude that condensin II is loaded and distributed across actively transcribed chromatin and thus serves to specifically condense this euchromatic portion of chromosomes during the cell division cycle. ChIP-Seq data for Condensin II and Cohesin in v6.5 ESCs treated or not with the RNA polymerase II elongation inhibitor flavopiridol.
Project description:Multiple protein complexes and histone marks have been implicated and/or associated with gene repression in ES cells. To gain insights into repressive complexes present at repressed genes and their associated chromatin state, we profiled REST, MCAF1, Ring1b and H4K20me3 in mouse ES cells. DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. ChIP was performed using an antibody against REST, MCAF1, Ring1b and H4K20me3.