The polycomb group protein Bmi1 binds to the herpes simplex virus 1 latent genome and maintains repressive histone marks during latency

Abstract

The mechanism by which herpes simplex virus 1 (HSV-1) establishes latency in sensory neurons is largely unknown. Recent studies indicate that epigenetic modifications of the chromatin associated with the latent genome may play a key role in the transcriptional control of lytic genes during latency. In this study, we found both constitutive and facultative types of heterochromatin to be present on the latent HSV-1 genome. Deposition of the facultative marks trimethyl H3K27 and histone variant macroH2A varied at different sites on the genome, whereas the constitutive marker trimethyl H3K9 did not. In addition, we show that in the absence of the latency-associated transcript (LAT), the latent genome shows a dramatic increase in trimethyl H3K27, suggesting that expression of the LAT during latency may act to promote an appropriate heterochromatic state that represses lytic genes but is still poised for reactivation. Due to the presence of the mark trimethyl H3K27, we examined whether Polycomb group proteins, which methylate H3K27, were present on the HSV-1 genome during latency. Our data indicate that Bmi1, a member of the Polycomb repressive complex 1 (PRC1) maintenance complex, associates with specific sites in the genome, with the highest level of enrichment at the LAT enhancer. To our knowledge, these are the first data demonstrating that a virus can repress its gene transcription to enter latency by exploiting the mechanism of Polycomb-mediated repression.

FIG. 1.

triMe H3K27 is enriched on the 17syn+ latent genome. (A) Validation of the ChIPs using anti-triMe H3K27 was performed by analyzing bound and unbound fractions of ChIP by real-time PCR with positive control PCR primers/probe to upHoxa5 compared to a negative control PCR primer/probe, APRT. Data are graphed as the B/(U+B) ratio. Average fold enrichment between upHoxa5 and APRT is denoted by the bracket. (B) ChIPs using anti-triMe H3K27 were subjected to real-time PCR using primers specific for the HSV-1 target genes indicated, and the results were graphed as B/(U+B) ratio normalized to the APRT B/(U+B). Mean values are displayed for each gene; data from six independent ChIPs are graphed (n = 6). (C) ChIPs of latently infected mouse DRG using control IgG were performed and analyzed as described for panel B. Mean values for each gene are shown (n = 3). L. pro, LAT promoter; L. enh, LAT enhancer.

FIG. 2.

Histone variant macroH2A is incorporated into the viral chromatin on the 17syn+ latent genome. ChIP of histone variant macroH2A on 17syn+ latently infected mouse DRG. (A) Validation of the ChIPs with anti-macroH2A were performed by analyzing bound and unbound fractions of ChIP by real-time PCR with positive control PCR primers/probe to midline compared to negative control PCR primers/probe, upHoxa5. Data were graphed as the B/(U+B) ratio. Average fold enrichment between midline and upHoxa5 is denoted by the bracket. (B) ChIPs using anti-macroH2A were subjected to real-time PCR using primers specific for the HSV-1 target genes indicated, and the results were graphed as B/(U+B) normalized to APRT B/(U+B). Mean values for each gene are shown; data from six independent ChIPs are graphed (n = 6). L. pro, LAT promoter; L. enh, LAT enhancer.

FIG. 3.

triMe H3K9 is enriched on the 17syn+ latent genome. (A) Validation of the ChIPs using anti-triMe H3K9 were performed by analyzing bound and unbound fractions of ChIP by real-time PCRs with positive control PCR primers/probe to mouse centromere compared to negative control PCR primers/probe, APRT. Data were graphed as the B/(U+B) ratio. Average fold enrichment between mouse centromere and APRT is denoted by the bracket. (B) ChIPs using anti-triMe H3K9 were subjected to real-time PCR using primers specific for the HSV-1 target genes indicated, and the results were graphed as B/(U+B) normalized to APRT B/(U+B). Mean values for each gene are shown; data from five independent ChIPs are graphed (n = 5). L. pro, LAT promoter; L. enh, LAT enhancer.

FIG. 4.

Viral transcript abundance in 17syn+ latently infected mouse DRG. RNA was isolated from latently infected DRG, reverse transcribed into cDNA, and analyzed by real-time PCR as described in Materials and Methods. Data are shown as RNA molecules per genome. (A) Results of PCR analysis for the LAT (5′ exon) and lytic genes. (B) Results of PCR analysis for the lytic genes (note different scale).

FIG. 5.

triMe H3K27 enrichment is increased on the 17ΔPst latent genome. (A) Validation of the ChIPs using anti-triMe H3K27 was performed by analyzing bound and unbound fractions of ChIP by real-time PCR with positive control PCR primers/probe to upHoxa5 compared to negative control PCR primers/probe, APRT. Data were analyzed as the B/(U+B) ratio. Average fold enrichment between upHoxa5 and APRT is denoted by the bracket. (B) ChIPs using anti-triMe H3K27 were subjected to real-time PCR using primers specific for the HSV-1 target genes indicated, and the results were graphed as B/(U+B) normalized to APRT B/(U+B). Mean values for each gene are shown; data from five independent ChIPs are graphed (n = 5). (C) ChIPs of latently infected mouse DRG using control IgG were performed and analyzed as described for panel B. Mean values for each gene are shown (n = 2). L. enh, LAT enhancer.

FIG. 6.

macroH2A replacement in the viral chromatin is increased on the 17ΔPst latent genome. (A) Validation of the macroH2A ChIPs was performed by analyzing bound and unbound fractions of ChIPs by real-time PCR with positive control PCR primers/probe to midline compared to negative control PCR primers/probe, APRT. Data were analyzed as the B/(U+B) ratio. Average fold enrichment between midline and APRT is denoted by the bracket. (B) ChIPs using anti-macroH2A were subjected to real-time PCR using primers specific for the HSV-1 target genes indicated, and the results were graphed as B/(U+B) normalized to APRT B/(U+B). Mean values for each gene are shown; data from five independent ChIPs are graphed (n = 5). L. enh, LAT enhancer.

FIG. 7.

Bmi1 is enriched on the 17syn+ latent genome. (A) Validation of the ChIPs using anti-Bmi1 was performed by analyzing bound and unbound fractions of ChIPs by real-time PCR with positive control PCR primers/probe to HOTAIR compared to negative control PCR primers/probe, myoD1. Data were graphed as the B/(U+B) ratio. Average fold enrichment between HOTAIR and myoD1 is denoted by the bracket. (B) ChIPs using anti-Bmi1 were subjected to real-time PCR using primers specific for the HSV-1 target genes indicated, and the results were graphed as B/(U+B) normalized to APRT B/(U+B). Mean values for each gene are shown; data from eight independent ChIPs are graphed (n = 8). L. pro, LAT promoter; L. enh, LAT enhancer.