BRD4 regulates genes involved in cell cycle progression from G0, G1 through S, thus impacting on cell growth. Recently, it has been shown that BRD4 is required for induction of inflammatory genes in macrophages, suggesting its role in innate immunity. We asked whether BRD4 plays a role in transcription of interferon (IFN) stimulated genes (ISGs). Type I IFN activates a large number of genes through the JAK/STAT pathway and establishes natural resistance against viruses and other pathogens. We found that IFN stimulation triggers recruitment of BRD4 to the transcription start site (TSS) of a number of ISGs. Recruitment of BRD4 to ISGs coincided with increased histone acetylation at the TSS and induction of ISG mRNAs. Further, BRD4 recruitment coincided with the binding of the elongation factor, P-TEFb, which is known to interact with BRD4. We also show that the pausing complex, NELF and DSIF are recruited to the ISGs after IFN stimulation. IFN-induced NELF/DSIF recruitment was unexpected, since the pausing complex was reported to be present prior to stimulation and dissociate after activation as studed for some inducible genes. Furthermore, another elongation factor, SPT6 was recruited to ISGs after IFN stimulation, a factor known to function as a histone H3 chaperon. These results gave a picture where a number of elongation factors are induced to assemble on the ISGs. To delineate a hierarchical order of the assembly of these factors, we tested the effect of small molecule inhibitor that inhibits binding of acetylated histones to bromodomains. Results showed that this inhibitor not only inhibits BRD4 recruitment, but recruitment of P-TEFb, NELF/DSIF and SPT6. In addition, the inhibitor markedly reduced ISG transcription. Similarly shRNA-based BRD4 knockdown led to reduced recruitment all of above factors. These data support a view that BRD4 recruitment is the primary event that initiates a cascade of factor binding that shapes overall ISG transcription. Additional knockdown studies showed that NELF and SPT6 have opposing activities in elongation, the former repressing ISG transcription, while the latter promoting transcription. Together, our results indicate that BRD4 orchestrates ISG transcription by coordinating positive and negative elongation. We also studied H3.3 incorporation into IFN stimulated genes using NIH3T3 cells expressing H3.3-fused to the yellow fluorescent protein (YFP). It should be noted here that a reliable anti-H3.3 antibody is not available, in that H3.3 is structurally too similar to H3.1/2 to raise specific antibody. This makes it very difficult to study the activity of endogenous H3.3. Following IFN stimulation, H3.3-YFP was rapidly incorporated into all four IFN activated genes tested, with the highest enrichment seen in the distal end of the coding region. 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. Further, while H3.3-YFP deposition stably remained in non-dividing cells for days after IFN stimulation, it was rapidly diminished in dividing cells. Lastly, we examined the role of H3.3 in IFN stimulated transcription by an shRNA approach and found that IFN stimulated transcription was significantly impaired in H3.3-knockdown cells. Results indicate that H3.3 plays a role in IFN mediated-transcription and its deposition leaves a prolonged post-transcriptional mark on these genes. Our four year-long effort has produced new mouse strains in which two of the H3,3 loci are replaced by HA-tagged H3.3. These mouse strains enable us to study the behavior of H3.3 in the whole animal model, which was not possible before. qRT-PCR, Western blot and FACs analyses confirmed the expression of H3.3-HA in various cells and tissues including macrophages and lymphocytes. We also confirmed that transcription-coupled deposition can be detected in macrophages and lymphocytes by ChIP analysis using a commercially available anti-HA antibody. These preliminary results ensure that our newly constructed mice offer a new model to study transcription-coupled histone exchange and epigenetic regulation.

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Miyagawa, Fumi; Tagaya, Yutaka; Ozato, Keiko et al. (2016) Essential Requirement for IFN Regulatory Factor 7 in Autoantibody Production but Not Development of Nephritis in Murine Lupus. J Immunol 197:2167-76
Mancino, Alessandra; Termanini, Alberto; Barozzi, Iros et al. (2015) A dual cis-regulatory code links IRF8 to constitutive and inducible gene expression in macrophages. Genes Dev 29:394-408
Sasaki, Haruka; Kurotaki, Daisuke; Osato, Naoki et al. (2015) Transcription factor IRF8 plays a critical role in the development of murine basophils and mast cells. Blood 125:358-69
Bradfute, Steven B; Anthony, Scott M; Stuthman, Kelly S et al. (2015) Mechanisms of immunity in post-exposure vaccination against Ebola virus infection. PLoS One 10:e0118434
Gupta, Monica; Shin, Dong-Mi; Ramakrishna, Lakshmi et al. (2015) IRF8 directs stress-induced autophagy in macrophages and promotes clearance of Listeria monocytogenes. Nat Commun 6:6379
Ayithan, Natarajan; Bradfute, Steven B; Anthony, Scott M et al. (2015) Virus-like particles activate type I interferon pathways to facilitate post-exposure protection against Ebola virus infection. PLoS One 10:e0118345
Kanno, Tomohiko; Kanno, Yuka; LeRoy, Gary et al. (2014) BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones. Nat Struct Mol Biol 21:1047-57
Sarai, Naoyuki; Nimura, Keisuke; Tamura, Tomohiko et al. (2013) WHSC1 links transcription elongation to HIRA-mediated histone H3.3 deposition. EMBO J 32:2392-406
Patel, Mira C; Debrosse, Maxime; Smith, Matthew et al. (2013) BRD4 coordinates recruitment of pause release factor P-TEFb and the pausing complex NELF/DSIF to regulate transcription elongation of interferon-stimulated genes. Mol Cell Biol 33:2497-507
Devaiah, Ballachanda N; Lewis, Brian A; Cherman, Natasha et al. (2012) BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain. Proc Natl Acad Sci U S A 109:6927-32

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