cIn herpes simplex virus 1 (HSV-1), binding clusters enriched in CTCF during latency have been previously identified. We hypothesized that CTCF binding to CTCF clusters in HSV-1 would be disrupted in a reactivation event. To investigate, CTCF occupation of three CTCF binding clusters in HSV-1 was analyzed following sodium butyrate (NaB)-and explant-induced reactivation in the mouse. Our data show that the CTCF domains positioned within the HSV-1 genome, specifically around the latency-associated transcript (LAT) and ICP0 and ICP4 regions of the genome, lose CTCF occupancy following the application of reactivation stimuli in wild-type virus. We also found that CTCF binding clusters upstream of the ICP0 and ICP4 promoters both function as classical insulators capable of acting as enhancer blockers of the LAT enhancer. Finally, our results suggest that CTCF occupation of domains in HSV-1 may be differentially regulated both during latency and at early times following reactivation by the presence of lytic transcripts and further implicate epigenetic regulation of HSV-1 as a critical component of the latency-reactivation transition.T he human alphaherpesvirus herpes simplex virus 1 (HSV-1), infects sensory neurons, where it establishes a lifelong latent infection as a circular episome associated with histones (6,16,38,40,44). During HSV-1 latency, the latency-associated transcript (LAT) is abundantly transcribed, and lytic regions are, in essence, silent (6,38,40). Recent studies have shown that there are epigenetic components involved in HSV-1 latency and in reactivation (1,13,21,31,36,39). For example, during latency, the LAT regions of the LAT reactivation-critical promoter and LAT 5= exon (containing the LAT enhancer) (8) are enriched in euchromatic histone markers compared to the promoters of the immediateearly (IE) ICP0, ICP4, and ICP27 genes (1,13,21,24,31). Further, these permissive marks established on the LAT are independent of LAT transcription (24). Additional reports indicated that the LAT region of HSV-1 also contains facultative heterochromatin (12, 25), a form of repressed chromatin that can convert to euchromatin via posttranslational histone tail modifications. Consequently, the nearby IE regions are also enriched with repressive histone marks that have been characterized predominantly as facultative heterochromatin (12,25,26,45). These key findings provide evidence that HSV-1 latency is established and maintained, at least in part, by complex epigenetic mechanisms that further poise the virus for reactivation. To support this, recent data show that the enrichments of euchromatic histone marks on the HSV-1 LAT and IE promoters change in response to reactivation stimuli in both rabbit and mouse models latently infected with wild-type HSV-1 (1, 13, 26, 31, 36). Specifically, histone marks on ICP0 and ICP4 rapidly and transiently become more euchromatic in nature, while the LAT region loses enrichment of euchromatic histone marks as LAT is degraded at early times in reactivation in the two different in...