Factors involved in the epigenetic regulation of the HSV-1 reactivation critical region (rcr) during latency and reactivation

During herpes simplex virus type 1 (HSV-1) latency, only the latency associated transcript (LAT) is transcribed, while the lytic genes are repressed. Evidence suggests that latent gene expression is regulated by post-translational modification to histones associated with the latent HSV-1 genome. Previous studies show that the LAT region, (which contains a known enhancer) is hyperacetylated, whereas all lytic genes analyzed (ICP0, UL54, ICP4, and DNA polymerase) are hypoacetylated. Those studies suggest that the HSV-1 genome is organized into transcriptionally permissive and non-permissive chromatin domains, possibly regulated by the LAT enhancer. While the LAT enhancer has been implicated in various functions (including reactivation), the extent of its effects on chromatin structure, and on the regulatory elements that establish the transcriptionally permissive domain around the LAT enhancer, remains unknown.; Using an HSV-1 deletion mutant (17DeltaSty, which deletes 371 bp of the LAT enhancer), we showed by chromatin immunoprecipitation (ChIP) that a genetic element encoded within the StyI-StyI region of the LAT enhancer is involved in regulating transcriptional permissivity in the LAT region during latency. Deleting this element resulted in a dramatic increase in transcriptional permissiveness but was not accompanied by an increase in LAT transcription.; Using sequence analysis coupled with ChIP and luciferase reporter assays, we identified the following: (1) clustered CCCTC and CTCCC motifs located in the HSV-1 genome; (2) each immediate-early gene, as well as the LAT enhancer, is flanked by two clusters; (3) clusters are conserved in other alpha herpesviruses; (4) the cellular insulator protein CTCF binds to these clusters in vivo; (5) mutational analysis of a 135 by cluster located in the LAT region revealed LAT enhancer/rcr -blocking insulator function.; These data support the identification of an insulator capable of regulating the LAT enhancer during the viral life cycle.; Using an explant co-cult model of HSV-1 reactivation, we showed that a decrease in both LAT enhancer acetylation and LAT RNA abundance occurs before an increase in acetylation (or transcriptional permissiveness) at the ICP0 promoter. Taken together, these studies begin to characterize the epigenetic mechanisms involved in regulating the transcriptional complexity of HSV-1 latency and reactivation.