To address the role of Brd4 in mitotic gene expression, we studied binding of Brd4 to mitotic chromosomes. Chromatin immunoprecipitation analysis found that Brd4 preferentially binds to the transcription start sites of genes that are expressed immediately after mitosis. These regions also showed high levels of acetylation both in histone H3 and H4, indicating that Brd4 marks mitotic chromatin by recognizing acetylated histones and directs post-mitotic gene expression. It has been shown that acetylated histones to which Brd4 binds are enriched with the histone variant H3.3 H3.3 is synthesized throughout cell cycle, and deposited onto chromatin in a replication independent manner. It is enriched in transcriptionally active regions of chromatin and is implicated in epigenetic memory. Dynamics of H3.3 deposition during transcriptional activation, however, has not been fully studied so far. Here we examined H3.3 incorporation into interferon (IFN) stimulated genes in confluent mouse NIH3T3 cells expressing H3.3-fused to the yellow fluorescent protein (YFP). 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 in these genes.
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