The H3.3 histone variant 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. The dynamics of H3.3 deposition during transcriptional activation, however, have 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, although 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 a short hairpin RNA 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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