Herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection within sensory neurons. During this time, the latent genomes persist as circular episomes associated with histones (9
). Although HSV-1 lytic gene expression is repressed during latency, a latency-associated transcript (LAT) is abundantly expressed in a subset of neurons (20
). Previous chromatin immunoprecipitation (ChIP) analyses of latent genomes have shown that several HSV-1 lytic genes are hypoacetylated in acetyl histone H3 (K9, K14), whereas the LAT region is heavily enriched in this transcriptionally permissive histone (15
). The region of hyperacetylation within the LAT locus encompasses an enhancer that maps to the LAT 5′ exon, and transcription does not seem to be required to maintain this hyperacetylated state (15
). Further, enrichment of acetyl histone H3 (K9, K14) does not include the ICP0 promoter. This suggests that the HSV-1 latent genome is organized into chromatin domains by boundary or insulator elements, similar to those found on cellular chromosomes, which would separate these distinct transcriptional domains (repressed lytic gene regions versus a transcriptionally active LAT region).
CTCF, or “CCCTC-binding factor,” is a DNA-binding protein containing 11 zinc fingers that is highly conserved among vertebrates. CTCF is ubiquitously expressed in most cell types and possesses transcriptional activator activity that is regulated by phosphorylation. In addition to the sequence “CCCTC,” CTCF binds to several other pentanucleotide motifs (21
). While a single DNA-binding motif is sufficient for binding, the binding motifs are often present as clusters of these consensus sequences, which affords higher CTCF-binding affinity (6
). CTCF binding results in a number of distinct activities, including gene activation and repression, though it is most often associated with the formation of chromatin insulators (31
Chromatin insulators are a class of DNA elements found on cellular chromosomes that protect genes in one region of a chromosome from the regulatory influence of another region (for a review, see reference 28
). In the simplest example, an insulator separates a region of transcriptionally active euchromatin from a region of transcriptionally repressed heterochromatin (5
). Insulator elements are believed to act via protein-protein interactions spanning a chromatin domain, as well as through the recruitment of specific histone-modifying enzymes. For example, several chromatin-modifying proteins have been shown to bind to CTCF at insulator elements, including sin3 and YB-1 (18
). There are two main classes of insulators that have been characterized: enhancer-blocking and boundary/barrier elements. Enhancer-blocking insulators have the specific ability to block an enhancer from enhancing gene expression on the distal side of the insulator (32
). Boundary elements, on the other hand, act primarily to separate transcriptionally polar regions of DNA and, in many cases, block the “spread” of transcriptionally repressive heterochromatin into regions that are transcriptionally active or permissive (13
). In addition, a number of cellular insulators are associated with silencer activity (4
). In these cases, CTCF acts as a corepressor with other proteins to recruit transcriptionally repressive histones (30
). While there is a wide range of transcriptional properties associated with specific insulators characterized to date, all vertebrate elements bind CTCF, which plays an essential role in insulator function (31
In the present study, we sought to identify the locations of putative insulators that might separate the transcriptionally permissive LAT region from the nearby transcriptionally repressed, hypoacetylated ICP0 region. A previous study suggested that this boundary would be located approximately 5 kb 3′ to the region of LAT that is hyperacetylated during latency (15
). We report here the identification of a sequence cluster 3′ of this hyperacetylated LAT region, composed of a repeated motif known to bind the cellular protein CTCF and to have a role in the formation of chromosomal boundaries. This cluster of CTCF motifs encompasses approximately 145 bp in the region encoding the LAT intron. ChIP analysis using an antibody specific for CTCF demonstrated that during a latent infection of murine dorsal root ganglia (DRG), this site is enriched in CTCF. In order to determine if this cluster of CTCF motifs marks the location of a functional insulator element, we analyzed a 1.5-kb fragment that contains this CTCF cluster for insulator functions. Insulator functions were assessed by in vitro analysis using luciferase reporter constructs similar to those used to define cellular insulators (24
). These analyses revealed that this CTCF cluster is not only capable of blocking the LAT enhancer from acting on an adjacent promoter, but it also possesses silencing activity. This suggests that the 1.5-kb fragment containing a 135-bp cluster of repeated CTCF motifs possesses cellular-insulator-like properties. This element may therefore contribute to the formation of nucleation sites for the assembly of a functional chromatin boundary, which could play an essential role in insulating the LAT enhancer. Such an arrangement would allow the LAT enhancer to act solely on the LAT promoter during latency and not on surrounding lytic promoters, such as ICP0.
Further analysis of the HSV-1 genome revealed the existence of five other clusters of CTCF motifs. ChIP analysis revealed that during a latent infection of murine DRG, these sites are also enriched in CTCF binding. Interestingly, if all these motifs were to form functional boundaries, the LAT enhancer/rcr, as well as each of the five HSV-1 immediate-early (IE) genes, would exist in a separate chromatin domain. Finally, analysis of other alphaherpesvirus genomes reveals that CTCF motifs (flanking IE genes) are conserved, raising the possibility that definitive chromatin domains may be an important regulatory component of alphaherpesviral latent-gene expression and may contribute in a mechanistic way to the control of latency and reactivation.