Development of small molecule inhibitors such as JQ1 and I-BET which specifically block binding of BET proteins to acetylated histone showed that BRD4 has a major role in cells growth regulation, affecting transcription of c-Myc and Fos. Thus these inhibitors can restrict growth of various blood cancers and solid tumors. These inhibitors are also shown to effectively inhibit certain types of inflammatory responses in immune cells. Although these inhibitors facilitated our understanding of BRD4 action, it has remained unclear what BRD4 does and how it does it during development and in adult cells.
Our aim for the project has been to elucidate the role of BRD4 and its mode of action by studying Brd4 conditional knockout (cKO) mice. We found that BRD4 is critically required for the development of hematopoietic cells, in that deletion of Brd4 in embryonic hematopoietic stem cells (HSC) led to almost complete blockade of the subsequent progenitor differentiation required for generation of erythrocytes, lymphocytes and myeloid cells. As a results, Brd4 cKO were found embryonic lethal. We found that the requirement of BRD4 for inflammatory responses in macrophages is less striking, in that Brd4 deletion caused only a partial reduction in the production of proinflammatory factors (cytokines, chemokines and reactive oxygen species). Nevertheless, BRD4 was broadly present in super-enhancers in both quiescent and inflammatory macrophages. Our long-standing effort has produced new mouse strains in which two of the H3,3 loci (H3f3a, H3.3A, H3f3b, H3.3B) are replaced by a HA-tagged H3.3 cDNA and free GFP. H3f3a and H3f3b heterozygous mice are found viable and fertile. The advantage of these mice has been verified when we found that the H3.3 expression and the chromatin deposition can be studied simply by anti-HA antibody. These mice overcome the long standing difficulty in studying H3.3 that stems from the fact that high quality antibodies specific for H3.3 are not easily available. We investigated H3.3-HA expression in adult tissues by immunoblot, and in different immune cells by flowcytometry and showed that both H3.3A and H3.3B are expressed on most of adult cells. Likewise, H3.3 chromatin distribution can be clarified by anti-HA antibody. By ChIP -seq analysis of bone marrow derived macrophages and fibroblasts, we have shown that H3.3 (both A and B) are rapidly incorporated into transcriptionally activated genes in both cell types in response to interferon gamma or interferon beta.
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