Regulation of human cytomegalovirus gene expression by changes in histone H3 acetylation and methylation, and through binding of transcription factors to promoter regions during replication in fibroblasts

Human cytomegalovirus DNA is packaged in virions without histones, but associates with histones upon reaching the nucleus of an infected cell. Since transcription is modulated by the interplay of histone modifications, I employed chromatin immunoprecipitation to detect acetylation and methylation of histone H3 at viral promoters at different times during the viral replication cycle. Histone H3 at immediate-early promoters is acetylated at the start of infection, while it is initially methylated at early and late promoters. Acetylation at immediate-early promoters is dynamic, with a high level of activating modifications at 3 and 6 h postinfection (hpi), followed by a marked reduction at 12 hpi. At the start of infection, histone H3 at immediate-early promoters was hypo-methylated at lysine 9. In contrast, early and late promoters were associated with hyper-methylated and hypo-acetylated H3 at the start of infection. As the infection entered the late phase, the amount of methylated H3 at the immediate-early promoter increased, whereas histone H3 methylation at other promoters and non-promoter regions was reduced. All viral promoters, as well as nonpromoter regions, are modified with activating acetylations at 24 to 72 hpi. This global modification was inhibited if viral DNA replication was blocked. The viral UL122-coded IE2 protein was present at the major immediate-early promoter and UL112 early promoter, both of which have binding sites for the factor, beginning at 6 h post infection; and it was at the other early and late promoters beginning at 24 h post infection. It was not detected at the UL37 immediate-early promoter or at non-promoter regions. The transient reduction in histone H3 acetylation at the major immediate-early promoter depends on the cis-repressive sequence to which the IE2 protein binds. A mutant virus lacking this element exhibited increased IE2 expression caused by decreased IE2 binding at the major immediate-early promoter and failed to show reduced acetylation of histone H3 residing at this promoter at 12 hpi. Among other proteins found to bind the major immediate-early promoter were the tegument pUL83 protein in complex with IFI16 and NF-kappaB. My results demonstrate that cytomegalovirus chromatin undergoes dynamic, promoter-specific histone modifications early in the infectious cycle, after which the entire chromosome becomes highly acetylated as the viral chromosome is replicated. I have adapted the chromatin immunoprecipitation technique to study the association of different cellular and viral proteins with the HCMV genome that might play an important role in viral gene regulation.