Project description:BRD4 is a BET family protein that binds acetylated histones and regulates transcription. BET/BRD4 inhibitors block blood cancer growth and inflammation and serve as a new therapeutic strategy. However, the biological role of BRD4 in normal hematopoiesis and inflammation is not fully understood. Analysis of Brd4 conditional knockout (KO) mice showed that BRD4 is required for hematopoietic stem cell expansion and progenitor development. Nevertheless, BRD4 played limited roles in macrophage development and inflammatory response to LPS ChIP-seq analysis showed that despite its limited importance, BRD4 broadly occupied the macrophage genome and participated in super-enhancer (SE) formation. Although BRD4 is critical for SE formation in cancer, BRD4 was not required for macrophage SEs, as KO macrophages created alternate, BRD4-less SEs that compensated BRD4 loss. This and additional mechanisms led to the retention of inflammatory responses in macrophages. Our results illustrate a context-dependent role of BRD4 and plasticity of epigenetic regulation.
Project description:Macrophages are cells belongs to innate immune system, which response to pathogen by the production of inflammatory proteins those that are effective in both combating pathogen and wound healing. Using small molecule inhibitors it has been shown that many of the inflammatory response genes were under control of BET proteins. We used LysM-Cre to conditionally delete floxed BRD4 in resident peritoneal macrophages and monitored the effect on inflammatory gene expression Overall design: Peritoneal macrophages were treated with either Interferon gamma and IL-4 alone or with LPS and pre treatment of interferon gamma ex-vivo. In some experiment, animals were treated with IL-4 in vivo. RNA was extracted and biotin labeled cDNA was hybridized on Affym3trix microarray. Untreated samples were used as control. To compare differential gene expression samples were prepared from wild type and BRD4 deleted macrophages. Biological duplicates were used for each treatment condition
Project description:We are reporting here genome wide distribution of BRD4 in bone marrow derived macrophages at steady state level and in reponse to LPS stimulation. In both condition, BRD4 distribution was found to be widespread in intragenic as well as intergenic regions with enrichment specifically near TSS. Characteristically BRD4 signal were highly enriched on enhancers of some active genes and hence were classified as BRD4 rich super-enhancers (>12kb long stretches). Super-enhancers are formed, in some instances by redistribution of BRD4 on LPS stimulated genes. BRD4 binding well correlated PolII binding at the chromatin acetylated at H3K27, H3K9 and H4tetraK. Interestingly many LPS induced BRD4 independent genes, despite BRD4 loss in the KO, maintained PolII binding at the chromatin enriched with acetylated lysines at H3K27, H3K9 and H4tetraK. Additionally among some BRD4 independent LPS inducible genes we detected stronger p65 enrichment in the KO. In contrast, P65 binding was not detected in BRD4 dependent genes. These observations provided evidence of plasticity of epigenetic changes in retaining inflammatory reponses in macrophages. Overall design: Bone marrow derived macrophages were obtained from Brd4Fl/Fl;Ert2-cre and Brd4Fl/Fl mice (WT), Brd4 was deleted from the former by treating bone marrow cells with 4-hydroxytamixfen (KO). For inflammatory stimulus differentiated macrophages were treated with LPS (100ng/ml) for 4 hours.
Project description:Macrophages are cells belongs to innate immune system, which response to pathogen by the production of inflammatory proteins those that are effective in both combating pathogen and wound healing. Using small molecule inhibitors it has been shown that many of the inflammatory response genes were under control of BET proteins. We used LysM-Cre to conditionally delete floxed BRD4 in bone marrow derived macrophages and monitored it's effect on inflammatory gene expression Overall design: Bone marrow derived macrophages were treated with either LPS, Interferon gamma and IL-4 alone or with LPS with a pre treatment of interferon gamma for RNA extraction and hybridization on Affymetrix microarray. Samples from untreated cells were used as control. To compare differential gene expression samples were prepared from wild type and BRD4 deleted macrophages. Biological duplicates were used for each treatment condition
Project description:Method: mRNA profiles were generated from pair-end sequencing of duplicate samples using Illumina Hiseq 2000. Results: Genes with an expression change of more than 2 fold were considered to be differentially expresed Overall design: Macrophages are cells belongs to innate immune system, which response to pathogen by the production of inflammatory proteins those that are effective in both combating pathogen and wound healing. Using microarray approach with BET inhibitors it was shown that many of the inflammatory response genes were under control of BET proteins. Purpose of this study was to assess the effect of BRD4 KO in NGS derived transcriptome profiles of both stimulated and unstimulated macrophages.
Project description:The epigenomic reader Brd4 is an important drug target for cancers. However, its role in cell differentiation and animal development remains largely unclear. Using two conditional knockout mouse strains and derived cells, we demonstrate that Brd4 controls cell identity gene induction and is essential for adipogenesis and myogenesis. Brd4 co-localizes with lineage-determining transcription factors (LDTFs) on active enhancers during differentiation. LDTFs coordinate with H3K4 mono-methyltransferases MLL3/MLL4 (KMT2C/KMT2D) and H3K27 acetyltransferases CBP/p300 to recruit Brd4 to enhancers activated during differentiation. Brd4 deletion prevents the enrichment of Mediator and RNA polymerase II transcription machinery, but not that of LDTFs, MLL3/MLL4-mediated H3K4me1, and CBP/p300-mediated H3K27ac, on enhancers. Consequently, Brd4 deletion prevents enhancer RNA production, cell identity gene induction and cell differentiation. Interestingly, Brd4 is dispensable for maintaining cell identity genes in differentiated cells. These findings identify Brd4 as an enhancer epigenomic reader that links active enhancers with cell identity gene induction in differentiation.
Project description:High-grade serous ovarian cancer (HGSOC), with its high recurrence rates, urges for reasonable therapeutic strategies that can prolong overall survival. A tumor microenvironment (TME) discloses prognostic and prospective information on cancer, such as the expression level of PD-1 or PD-L1. However, in HGSOC, the impact of the therapies aiming at these targets remains unsatisfying. Tumor-associated macrophages (TAMs) in HGSOC make up a large part of the TMEs and transform between diverse phenotypes under different treatments. AZD5153 inhibiting BRD4, as a potential therapeutic strategy for HGSOC, was demonstrated to confer controversial plasticity on TAMs, which shows the need to uncover its impact on TAMs in HGSOC. Therefore, we established models for TAMs and TAMs co-culturing with T lymphocytes in vitro. Via RT-PCR and flow cytometry, we find that AZD5153 resets TAMs from M2-type macrophages to M1-like macrophages, consequently promoting pro-inflammatory cytokine secretion and thus activating CD8+ cytotoxic T lymphocytes (CTLs) in vitro. This modification occurs on MAF transcripts in TAMs and modified by BRD4, which is the target of AZD5153. Importantly, the 3-D microfluid model demonstrates that AZD5153 sensitizes ovarian cancer to anti-PD-L1 therapy. Our results clarify that besides eliminating tumor cells directly, AZD5153 works as a regulator of the TAM phenotype, providing a novel strategy combining AZD5153 and PD-1/PD-L1 antibody that could benefit HGSOC patients.