CTCF Occupation of the Herpes Simplex Virus 1 Genome Is Disrupted at Early Times Postreactivation in a Transcription-Dependent Manner

Abstract

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...

Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity

“…Recent findings have shown that, during latency, the cellular CTCF protein is enriched on the two HSV CTRLs and that acetylated histone H3, an epigenetic beacon of active transcription, accumulates in the LAT region between these elements (19,(43)(44)(45). These findings suggested that CTRLs may act as boundary elements to shield the LAT locus against the spreading of repressive epigenetic modifications (29), and it was shown in short-term plasmid transfection assays that a fragment encompassing CTRL2 combines the functions of a typical insulator (19).…”

Occupancy of RNA Polymerase II Phosphorylated on Serine 5 (RNAP S5 P ) and RNAP S2 P on Varicella-Zoster Virus Genes 9, 51, and 66 Is Independent of Transcript Abundance and Polymerase Location within the Gene

“…ChIP has shown that transcriptionally distinct regions of the virus genome are maintained by chromosomal insulators, most notably, the CCCTCbinding factor, CTCF (31). During HSV-1 reactivation, these chromatin boundary elements are released from the virus DNA (32,33). Since the release of CTCF from the virus genome is an attractive mechanism connecting external stress to HSV-1 reactivation (34,35) and the critical CCCTC-binding sites are separated by at least 3,300 nt (31), the HSV-1 DNA fragment size is not a critical aspect of these ChIP analyses.…”

Strength in diversity: Understanding the pathways to herpes simplex virus reactivation

“…This apparent demarcation in the nature of the chromatin likely arises due to binding sites for the cellular insulator protein CCCTC-binding factor (CTCF) on the viral genome. Interestingly, CTCF eviction coincides with reactivation (Ertel et al, 2012; Washington et al, 2018b), and depletion of CTCF in vivo promotes reactivation (Washington et al, 2018a). Furthermore, one important binding site of CTCF, known as CTRL2, lies downstream of the LAT enhancer and separates it from the nearby lytic ICP0 gene (Amelio et al, 2006b).…”

“…It remains unclear if JNK plays a similar mechanistic role following explant of latently infected ganglia, and, as a whole, very little is known about the mechanisms linking cell signaling pathways and changes in viral chromatin following axotomy. However, a number of important observations about the viral chromatin have been made using the explant model, including changes in histone post-translational modification and changes in CTCF binding (Ertel et al, 2012; Washington et al, 2018b). Most notably, it has been shown that, following axotomy, there is a transient increase in the enrichment of euchromatic histone markers (acetylated H3K9/K14) at IE gene promoters (Amelio et al, 2006a).…”

“…This is in contrast to reactivation in intact ganglia, in which VP16 is required (Thompson et al, 2009), again indicating that the mechanisms of reactivation likely differ depending on the nature of the trigger. Lastly, it has been shown that CTCF domains positioned around IE (ICP0 and ICP4) and LAT gene regions lose CTCF occupancy following explant-induced reactivation (Ertel et al, 2012; Washington et al, 2018b), suggesting that disruption of CTCF-mediated chromatin domains may contribute to reactivation.…”