A chromatin insulator-like element in the herpes simplex virus type 1 latency-associated transcript region binds CCCTC-binding factor and displays enhancer-blocking and silencing activities

Abstract

A previous study demonstrated that the latency-associated transcript (LAT) promoter and the LAT enhancer/reactivation critical region (rcr) are enriched in acetyl histone H3 (K9, K14) during herpes simplex virus type 1 (HSV-1) latency, whereas all lytic genes analyzed (ICP0, UL54, ICP4, and DNA polymerase) are not (N. J. Kubat, R. K. Tran, P. McAnany, and D. C. Bloom, J. Virol. 78:1139-1149, 2004). This suggests that the HSV-1 latent genome is organized into histone H3 (K9, K14) hyperacetylated and hypoacetylated regions corresponding to transcriptionally permissive and transcriptionally repressed chromatin domains, respectively. Such an organization implies that chromatin insulators, similar to those of cellular chromosomes, may separate distinct transcriptional domains of the HSV-1 latent genome. In the present study, we sought to identify cis elements that could partition the HSV-1 genome into distinct chromatin domains. Sequence analysis coupled with chromatin immunoprecipitation and luciferase reporter assays revealed that (i) the long and short repeats and the unique-short region of the HSV-1 genome contain clustered CTCF (CCCTC-binding factor) motifs, (ii) CTCF motif clusters similar to those in HSV-1 are conserved in other alphaherpesviruses, (iii) CTCF binds to these motifs on latent HSV-1 genomes in vivo, and (iv) a 1.5-kb region containing the CTCF motif cluster in the LAT region possesses insulator activities, specifically, enhancer blocking and silencing. The finding that CTCF, a cellular protein associated with chromatin insulators, binds to motifs on the latent genome and insulates the LAT enhancer suggests that CTCF may facilitate the formation of distinct chromatin boundaries during herpesvirus latency.

FIG. 1.

The HSV-1 genome contains clustered CTCF-binding sites. An algorithm that searched for CCCTC or CTCCC motifs was used (see Materials and Methods) to analyze the HSV-1 strain 17syn+ genome (GenBank accession no. NC_001806) in 1,000-bp segments to determine the frequencies with which these CTCF binding sites occur in the (A) positive (direct) and (C) negative (complement) DNA strands. (B) Diagram of the HSV-1 genome illustrating locations of identified CTCF motifs. The shading (blue/red, interleaved CTCCC/CCCTC motifs; blue, CTCCC; red, CCCTC) indicates the regions of the genome where a high frequency of CTCF motifs cluster.

FIG.2.

Genome segments containing a high frequency of motifs reveal tandem CTCF motif clustering. Shown is an expanded view of a portion of the HSV-1 U_L, internal R_L and R_S (IR_L and IR_S), U_S, and terminal R_S regions illustrating the locations of clustered CTCF motifs that occur in tandem. The corresponding positive (direct)- or negative (complement)-strand DNA sequence is shown with 5′-CTCCC-3′ motifs highlighted in blue and 5′-CCCTC-3′ motifs highlighted in red. The CTCF motifs are shown in capital letters. (A) CTCCC/CCCTC cluster on the positive (direct) DNA strand that maps to the U_L/IR_L junction (nt 117,158 to 117,342). Since both CTCCC and CCCTC motifs cluster at this site, two copies of identical sequences are shown, but one motif type is selectively highlighted for viewing simplicity. (B) CTCCC cluster on the positive (direct) DNA strand that maps to the LAT intron region (nt 120,503 to 120,635). (C) CTCCC cluster on the positive (direct) DNA strand that maps to the a′ sequence (nt 126,057 to 126,274). (D) CCCTC/CTCCC cluster on the positive (direct) DNA strand that maps near the a′ sequence/IR_S junction (nt 126,571 to 127,141). The adjacent blocks of sequence are identical, but one motif is selectively highlighted for viewing simplicity. (E) CCCTC cluster on the negative (complement) strand that maps near the IR_S/U_S junction (nt 132,396 to 132,507). (F) CTCCC cluster on the negative (complement) strand that maps to the U_S region (nt 143,722 to 143,861). (G) Linear diagram of a portion of the genome labeled with relative locations of CTCF motif clusters and immediate-early genes. (H) Circular diagram of the entire genome labeled with relative locations of CTCF motif clusters and immediate-early genes.

FIG.2.

Genome segments containing a high frequency of motifs reveal tandem CTCF motif clustering. Shown is an expanded view of a portion of the HSV-1 U_L, internal R_L and R_S (IR_L and IR_S), U_S, and terminal R_S regions illustrating the locations of clustered CTCF motifs that occur in tandem. The corresponding positive (direct)- or negative (complement)-strand DNA sequence is shown with 5′-CTCCC-3′ motifs highlighted in blue and 5′-CCCTC-3′ motifs highlighted in red. The CTCF motifs are shown in capital letters. (A) CTCCC/CCCTC cluster on the positive (direct) DNA strand that maps to the U_L/IR_L junction (nt 117,158 to 117,342). Since both CTCCC and CCCTC motifs cluster at this site, two copies of identical sequences are shown, but one motif type is selectively highlighted for viewing simplicity. (B) CTCCC cluster on the positive (direct) DNA strand that maps to the LAT intron region (nt 120,503 to 120,635). (C) CTCCC cluster on the positive (direct) DNA strand that maps to the a′ sequence (nt 126,057 to 126,274). (D) CCCTC/CTCCC cluster on the positive (direct) DNA strand that maps near the a′ sequence/IR_S junction (nt 126,571 to 127,141). The adjacent blocks of sequence are identical, but one motif is selectively highlighted for viewing simplicity. (E) CCCTC cluster on the negative (complement) strand that maps near the IR_S/U_S junction (nt 132,396 to 132,507). (F) CTCCC cluster on the negative (complement) strand that maps to the U_S region (nt 143,722 to 143,861). (G) Linear diagram of a portion of the genome labeled with relative locations of CTCF motif clusters and immediate-early genes. (H) Circular diagram of the entire genome labeled with relative locations of CTCF motif clusters and immediate-early genes.

FIG. 3.

ChIP analysis of the CTCF motif clusters within latent HSV-1 DNA using antiserum specific for anti-CTCF. DRG from mice latently infected with HSV-1 strain 17syn+ were processed and subjected to ChIP analysis as described in Materials and Methods. The relative enrichment of CTCF at each motif cluster was determined by PCR analysis of the ChIP (bound) fraction. Lanes 6 to 8 represent three independent ChIP experiments, and lanes 1 to 4 represent dilutions of the input material for comparison. Lanes 5 are the no-input controls. (A) ChIPs were validated by serial dilutions of input and a 1/10 dilution of bound ChIP sample using primers to the cellular target Tsix imprinting/choice center CTCF site A (positive control) and MT498 (negative control) (6). (B) PCRs performed with the same titrated input and bound ChIP sample with primers to the CTRL1, CTRS1 and -2, CTRS3, CTUS1, and gC viral targets. (C) PCRs performed with titrated input and 1/100 dilution of bound ChIP sample with primers to the CTRL2 and gC viral targets. Band intensities of PCR products generated with ChIP-precipitated DNA were quanti- tated with respect to twofold dilutions of input and used to demonstrate enrichments.

FIG. 4.

The 1.5-kb region encompassing CTRL2 functions as an insulator with enhancer-blocking and -silencing activities. (A) Luciferase reporter constructs were generated to test the ability of the LAT insulator to specifically block the LAT enhancer/rcr. The data were collected as relative luciferase units, normalized to an internal Renilla luciferase control, and expressed relative to the pGL3-SV40 promoter control. The P values represent unpaired one-tailed Student's t tests in pairwise comparisons to the pGL3-SV40 promoter control. The error bars represent standard deviations. (B) Mutational analysis of a luciferase reporter construct that specifically deletes the 135-bp core CTRL2 cluster. The single-asterisk P values represent unpaired one-tailed Student's t tests in pairwise comparisons to the pGL3-SV40 promoter control, while the double-asterisk P value represents a comparison to the nonmutated enhancer-blocking insulator construct.

FIG. 5.

Clustered CTCF-binding sites are conserved across the Alphaherpesvirus family and flank the immediate-early genes. (A) The algorithm used to analyze the HSV-1 strain 17syn+ genome was applied in the same manner to analyze 1,000-bp segments of each alphaherpesvirus genome to determine the frequency of CTCF-binding sites on the positive (direct) or negative (complement) DNA strand. Additionally, tandem-repeat analysis was performed to characterize the CTCF motif clustering (2). Analyses were performed using published NCBI GenBank sequences for HSV-2 strain HG52 (NC_001798; D. J. McGeoch), suid herpesvirus 1 (pseudorabies virus) (BK001744; L. W. Enquist), human herpesvirus 3 strain Dumas (varicella-zoster virus) (X04370; J. E. Scott), and cercopithecine herpesvirus 1 (monkey B virus) (NC_004812; J. K. Hilliard). (B) Representative CTCF pentanucleotide motifs found clustered within the Alphaherpesvirus family members. The solid triangles represent consensus CTCF motifs previously reported to bind CTCF (17, 21). The open triangles represent nonconsensus CTCF pentanucleotide motifs. Partially solid/open triangles represent clusters composed of interleaved consensus and nonconsensus motifs. The pointed end of each triangle indicates the DNA strand direction (direct or complement).