Project description:Inhibition of Brd4 with Jq1 in neurons with or without BDNF stimulation Examination of the effects of Jq1 treatment on primary mouse cortical neurons
Project description:The estrogen receptor-α (ERα) is a transcription factor which plays a critical role in controlling cell proliferation and tumorigenesis by recruiting various cofactors to estrogen response elements (EREs) to induce or repress gene transcription. A deeper understanding of these transcriptional mechanisms may uncover novel therapeutic targets for ERα-dependent cancers. Here we show for the first time that BRD4 regulates ERα−induced gene expression by affecting elongation-associated phosphorylation of RNA Polymerase II (RNAPII P-Ser2) and histone H2B monoubiquitination (H2Bub1). Consistently, BRD4 activity is required for estrogen-induced proliferation of ER+ breast and endometrial cancer cells and uterine growth in mice. Genome-wide occupancy studies revealed an enrichment of BRD4 on transcriptional start sites as well as EREs enriched for H3K27ac and demonstrate a requirement for BRD4 for H2B monoubiquitination in the transcribed region of estrogen-responsive genes. Importantly, we further demonstrate that BRD4 occupancy correlates with active mRNA transcription and is required for the production of ERα-dependent enhancer RNAs (eRNAs). These results uncover BRD4 as a central regulator of ERα function and potential therapeutic target. ChIP-sequencing of BRD4, ERα and H2Bub1 in MCF7 cells treated with +/- estrogen treatment and or +/- JQ1 treatment in triplicates.
Project description:The BET (bromodomain and extra terminal) protein family members including BRD4 bind to acetylated lysines on histones and regulate the expression of important oncogenes, e.g., MYC and BCL2. Here we demonstrate the sensitizing effects of the histone hyperacetylation inducing pan-histone deacetylase inhibitor (HDI) panobinostat (PS) on human AML blast progenitor cells (BPCs) to the BET protein inhibitor JQ1. Treatment with JQ1 but not its inactive enantiomer (R-JQ1) was highly lethal against AML BPCs expressing mutant NPM1c+ with or without co-expression of FLT3-ITD, or AML expressing MLL fusion oncoprotein. JQ1 treatment reduced binding of BRD4 and RNA polymerase II to the DNA of MYC and BCL2, and reduced their levels in the AML cells. Co-treatment with JQ1 and the HDAC inhibitor panobinostat (PS) synergistically induced apoptosis of the AML BPCs, but not of normal CD34+ hematopoietic progenitor cells. This was associated with greater attenuation of MYC and BCL2, while increasing p21, BIM and cleaved PARP levels in the AML BPCs. Co-treatment with JQ1 and PS significantly improved the survival of the NOD/SCID mice engrafted with OCI-AML3 or MOLM13 cells (p < 0.01). These findings highlight co-treatment with a BRD4 antagonist and an HDI as a potentially efficacious therapy of AML. Two samples were analyzed (untreated cells, cells treated with JQ1)
Project description:Class Switch Recombination (CSR) is a B cell specific genomic alteration induced by activation induced cytidine deaminase (AID)-dependent DNA break, followed by repair and recombination at the immunoglobulin heavy-chain locus. The involvement of several chromatin-associated factors in promoting AID-induced DNA break formation has been reported. However, the involvement of chromatin adaptors at the repair phase of CSR remains unknown. Here, we provide evidence that acetylated histone reader Brd4 is critical to the repair and recombination step of CSR. Brd4 was recruited to the AID-induced DNA break region, and depletion of Brd4 from the S region chromatin by knockdown or a chemical inhibitor JQ1 causes CSR impairment without affecting AID-induced DNA break generation. Such inhibition of Brd4 suppressed the accumulation of 53BP1 and UNG at the cleaved S regions, perturbed switch donor-switch acceptor microhomology length and reduced Igh/c-myc translocation. We conclude that Brd4 serves as a histone-reader platform required for the recruitment of CSR repair components. Brd4 were depleted from the chromatin by either siRNA treatment or JQ1 (40nM) addition in CH12F3-2A cells in the presence of CIT stimulation. RNA from each samples were extracted and relative difference in transcript level were compared with control RNAi- and DMSO-treated, CIT-stimulated samples.
Project description:The estrogen receptor-M-NM-1 (ERM-NM-1) is a transcription factor which plays a critical role in controlling cell proliferation and tumorigenesis by recruiting various cofactors to estrogen response elements (EREs) to induce or repress gene transcription. A deeper understanding of these transcriptional mechanisms may uncover novel therapeutic targets for ERM-NM-1-dependent cancers. Here we show for the first time that BRD4 regulates ERM-NM-1M-bM-^HM-^Rinduced gene expression by affecting elongation-associated phosphorylation of RNA Polymerase II (RNAPII P-Ser2) and histone H2B monoubiquitination (H2Bub1). Consistently, BRD4 activity is required for estrogen-induced proliferation of ER+ breast and endometrial cancer cells and uterine growth in mice. Genome-wide occupancy studies revealed an enrichment of BRD4 on transcriptional start sites as well as EREs enriched for H3K27ac and demonstrate a requirement for BRD4 for H2B monoubiquitination in the transcribed region of estrogen-responsive genes. Importantly, we further demonstrate that BRD4 occupancy correlates with active mRNA transcription and is required for the production of ERM-NM-1-dependent enhancer RNAs (eRNAs). These results uncover BRD4 as a central regulator of ERM-NM-1 function and potential therapeutic target. mRNA expression profiles of MCF7 cells treated with +/- estrogen treatment under negative control siRNA, BRD4 siRNA or JQ1 treatment, in duplicates.
Project description:Transcriptional elongation by RNA polymerase II (Pol II) is regulated by positive transcription elongation factor b (P-TEFb) in association with Bromodomain-containing protein 4 (BRD4). We used genome-wide chromatin immunoprecipitation sequencing in primary human CD4+ T cells to reveal that BRD4 co-localizes with Ser2-phosphorylated Pol II (Pol II Ser2) at both enhancers and promoters of active genes. Disruption of bromodomain:histone acetylation interactions by JQ1, a small-molecule bromodomain inhibitor, resulted in decreased BRD4 binding, reduced Pol II Ser2, and reduced expression of lineage-specific genes in primary human CD4+ T cells. A large number of JQ1-disrupted BRD4 binding regions exhibited di-acetylated H4 (lysine-5 and -8) and H3K27 acetylation (H3K27ac), which correlated with the presence of histone acetyltransferases and deacetylases. Genes associated with BRD4/H3K27ac co-occupancy exhibited significantly higher activity than those associated with H3K27ac or BRD4 binding alone. Comparison of BRD4 binding in T cells and in human embryonic stem cells revealed that enhancer BRD4 binding sites were predominantly lineage-specific. Our findings suggest that BRD4-driven Pol II phosphorylation at serine 2 plays an important role in regulating lineage-specific gene transcription in human CD4+ T cells. Examination of BRD4, total Pol II, serine 2 phosphorylated Pol II and serine 5 phosphorylated Pol II binding in CD4+ T cells (with and without JQ1 treatment) and BRD4 binding in human embryonic stems cell; PolyA RNA expression in CD4+ T cells( with and without JQ1 treatment) using RNA-seq
Project description:We analyzed anti-proliferative dominant-negative Brd4 mutants that compete with the function of distinct Brd4 domains. We used these Brd4 mutants to compare the Brd4-specific transcriptome with the transcriptome of JQ1 treated cells.
Project description:BRD4 is amplified and/or up-regiulated in a subset of ovarian cancer which correlates with a poor survival siRNA sensitization screens in the presence of CHK1 inhibitor, LY2606368, identify BRD4 as a therapeutic target in ovarian cancer. BRD4 suppression by either siRNA or using JQ1 increases CBX5 expression
Project description:Epigenetic pathways regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. While chromatin alterations are, in principle, reversible and often amendable to drug intervention, the promise of targeting such pathways therapeutically has been hampered by our limited understanding of cancer-specific epigenetic dependencies. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukemia (AML) – an aggressive hematopoietic malignancy often associated with aberrant chromatin states. By screening a custom shRNA library targeting known chromatin regulators in a genetically defined AML mouse model, we identify the bromodomain-containing protein Brd4 as a critical requirement for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust anti-leukemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation. Extensive evaluation of JQ1-sensitivity in primary human leukemia samples and in established cell lines revealed a broad activity of this compound against diverse AML subtypes. These effects are, at least in part, due to a requirement for Brd4 in maintaining Myc expression and promoting aberrant self-renewal. Together, our results indicate that Brd4 is a promising therapeutic target in AML and identify a small molecule that efficiently targets Myc. These findings also highlight the utility of RNAi screening as a discovery platform for revealing epigenetic vulnerabilities for direct pharmacologic intervention in cancer. In order to understand downstream targets of Brd4, we performed array in murine or human MLL-AF9/NrasG12D cell line under the condition that Brd4 was suppressed by using shRNAs or the small molecule inhibitor JQ1. To test the hypothesis that Myc might be an important target of Brd4, we performed arrary on murine ectopic Myc overexpression MLL-AF9/NrasG12D cell under JQ1 treatment.