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, 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 plays a central role in SG transcription for its ability to sequentially recruit various elongation factors. These studies are extended to analysis of Brd4f/f mice expressing cell type specific Cre. We also studied H3.3 incorporation into IFN stimulated genes using NIH3T3 cells expressing H3.3-fused to the yellow fluorescent protein (YFP). H3.3-YFP expressing cells make it possible to detect H3.3 deposition with high sensitivity. 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. The pattern of H3.3-YFP deposition was highly unusual and did not correlate with patterns of other histone tail modifications, except histoen 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 with 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 H3.3 specific chromatin assembly factor HIRA, causing H3.3 deposition. Thus, in the absence of WHSC1, both transcriptional elongation and H3.3 were impaired while 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 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. Based on these results ChIP-seq analysis is being performed using mouse embryonic fibroblasts (MEFs)

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$937,804
Indirect Cost
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Mochizuki, Kazuki; Nishiyama, Akira; Jang, Moon Kyoo et al. (2008) The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase. J Biol Chem 283:9040-8
Nishiyama, Akira; Mochizuki, Kazuki; Mueller, Florian et al. (2008) Intracellular delivery of acetyl-histone peptides inhibits native bromodomain-chromatin interactions and impairs mitotic progression. FEBS Lett 582:1501-7