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 leukemia and solid cancers. These inhibitors are also shown to effectively inhibit certain types of inflammatory responses in immune cells. We have studied mechanisms by which BRD4 controls transcription. We have previously shown that BRD4 interacts with the pause-release factor P-TEFb, releasing RNA polymerase II (Pol II) from promoter-proximal pausing. Further BRD4 has been shown to be present in the enhancer and superenhancer regions that influence the intensity of tissue and signal dependent transcription. Interestingly, enhancer regions are also transcribed by Pol II. By ChIP-seq analysis we show that BRD4 occupy widespread genomic regions in MEFs, and stimulated elongation of both protein-coding transcripts and non-coding enhancer RNAs (eRNAs). This was dependent on the ability of the bromodomains to interact with acetylated histones. During transcription BRD4 interacted with the elongating Pol II complexes, and facilitated Pol II progression. These processes were antagonized by the BET inhibitor, JQ1 resulting in reduced synthesis of both coding and eRNA. Thus, BRD4 plays multiple roles in the transcription hierarchy. We also studied H3.3 incorporation into IFN stimulated genes (ISG) using embryonic fibroblasts (MEFs) expressing H3.3-fused to the yellow fluorescent protein (YFP). Following IFN stimulation, H3.3-YFP was rapidly incorporated into at IFN activated genes, with the highest enrichment seen near and at the transcription end site. Surprisingly, H3.3 enrichment in the coding region continued for an extended period of time, long after transcription ceased. The promoter region, although constitutively enriched with H3.3-YFP, did not show an increase in its deposition in response to IFN stimulation. The pattern of H3.3-YFP deposition was did not correlate with histone tail modifications associated with gene expression, except histone H3K36 trimethylation (H3K36me3). Methylation of H3K36 is catalyzed by several histone methytransferases, WHSC1, NSD3 and SETD2. These methyltranserases carry the catalytic domain conserved from yeast to humans. WHSC1 among them is associated with various cancers. We show that disruption of the Whsc1 gene abolishes IFN stimulated H3.3-YFP deposition, by ChIP analysis using Whsc1-/-MEFs. Subsequent studies revealed that WHSC1 itself was recruited to ISG TSS upon IFN stimulation by binding to BRD4, and that it travels across the coding region along with mRNA elongation. Importantly during this step WHSC1 interacted with the H3.3 specific chromatin assembly factor HIRA, enabling H3.3 deposition. Thus, IFN stimulated H3.3 deposition was strongly impaired in Whsc1-/- MEFs, although BRD4 recruitment was unaffected. These studies demonstrate that WHSC1 links transcriptional elongation and H3.3 deposition and provide a new scope in chromatin exchange and epigenetic control. Our long-standing effort has produced new mouse strains in which two of the H3,3 loci are replaced by a HA-tagged H3.3. These mice enable us to study the role of H3.3 in mammalian development and distribution of H3.3 in in vivo cells in the whole animal model. We have confirmed the expression of H3.3-HA in various cells and tissues in these mice, including cells of innate immunity. We also showed that H3.3 is deposited in IFN response genes upon stimulation with IFN-gamma in a transcription-coupled manner. ChIP-seq analysis is underway to describe genome-wide redistribution of H3.3 upon cytokine stimulation.

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U.S. National Inst/Child Hlth/Human Dev
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Kamada, Rui; Yang, Wenjing; Zhang, Yubo et al. (2018) Interferon stimulation creates chromatin marks and establishes transcriptional memory. Proc Natl Acad Sci U S A 115:E9162-E9171
Yu, Xiaoli; Chen, Hui; Zuo, Chen et al. (2018) Chromatin remodeling: demethylating H3K4me3 of type I IFNs gene by Rbp2 through interacting with Piasy for transcriptional attenuation. FASEB J 32:552-567
Ouda, Ryota; Sarai, Naoyuki; Nehru, Vishal et al. (2018) SPT6 interacts with NSD2 and facilitates interferon-induced transcription. FEBS Lett 592:1681-1692
Gegonne, Anne; Chen, Qing-Rong; Dey, Anup et al. (2018) Immature CD8 Single-Positive Thymocytes Are a Molecularly Distinct Subpopulation, Selectively Dependent on BRD4 for Their Differentiation. Cell Rep 24:117-129
Lee, Ji-Eun; Park, Young-Kwon; Park, Sarah et al. (2017) Brd4 binds to active enhancers to control cell identity gene induction in adipogenesis and myogenesis. Nat Commun 8:2217
Morita, Mayu; Yoshida, Shigeyuki; Iwasaki, Ryotaro et al. (2016) Smad4 is required to inhibit osteoclastogenesis and maintain bone mass. Sci Rep 6:35221
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Ozato, Keiko (2016) Introduction to a Special Issue on IFN Regulatory Factors in Innate Immune Responses. J Interferon Cytokine Res 36:413
Bachu, Mahesh; Dey, Anup; Ozato, Keiko (2016) Chromatin Landscape of the IRF Genes and Role of the Epigenetic Reader BRD4. J Interferon Cytokine Res 36:470-5
Huang, X F; Nandakumar, V; Tumurkhuu, G et al. (2016) Mysm1 is required for interferon regulatory factor expression in maintaining HSC quiescence and thymocyte development. Cell Death Dis 7:e2260

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