Establishment of murine cytomegalovirus latency in vivo is associated with changes in histone modifications and recruitment of transcriptional repressors to the major immediate-early promoter

Abstract

Human cytomegalovirus (CMV) is a ubiquitous herpesvirus with the ability to establish a lifelong latent infection. The mechanism by which this occurs is not well understood. Regulation of, for example, immediate-early (IE) gene expression is thought to be a critical control point in transcriptional control of the switch between latency and reactivation. Here, we present evidence that supports previous studies showing that the majority of genomes are quiescent with respect to gene expression. To study the possible role of epigenetic factors that may be involved in repression of ie gene expression in latency, we have analyzed changes in the patterns of modifications of histones bound to the major IE promoter (MIEP) in the kidneys of acutely and latently infected mice. Our studies show that, like herpes simplex virus, murine CMV genomes become relatively enriched in histones in latent infection. There are dramatic changes in modifications of histones associated with the MIEP when latency is established: H3 and H4 become hypoacetylated and H3 is hypomethylated at lysine 4, while H3 lysine 9 is hypermethylated in latently infected mice. These changes are accompanied by a relative loss of RNA polymerase and gain of heterochromatin protein 1gamma and Yin-Yang 1 bound to the MIEP. Our studies suggest that, in the majority of cells, CMV establishes a true latent infection, defined as the lack of expression of genes associated with productive infection, and that this occurs through changes in histone modifications and recruitment of transcriptional silencing factors to the MIEP.

FIG. 1.

Validation of ChIP assays. Chromatin from kidneys of acutely (A) and latently (L) infected mice was subject to ChIP analysis with antibodies against histone H3, RNA Pol II, HP-1, or normal IgG as described in Materials and Methods. PCR was performed with primers specific to the β-actin gene promoter region (β-actinP) or the Ant4 gene promoter region (Ant4P).

FIG. 2.

ChIP analysis of RNA polymerase II binding to the MCMV MIEP. (A) Anti-Pol II- or IgG-precipitated DNA samples subjected to semiquantitative PCR amplification with primers specific to the MIEP region. N, uninfected mice; A, acutely infected mice; L, latently infected mice. (B) ChIP/real-time PCR analysis of the association of RNA polymerase II with the MIEP, β-actin, and Ant4 promoters in chromatin from acutely infected and latently infected mice. The results (means of three experiments + standard errors of the means) are shown as percentages of input chromatin immunoprecipitated with anti-RNA polymerase II antibody. *, P < 0.001. Gray bars, acute infection; black bars, latent infection.

FIG. 3.

ChIP analysis of the interaction of the MIEP with histones in acutely and latently infected mice. Chromatin was immunoprecipitated with antibodies recognizing all forms of histone H3 or histone H4. ChIP products were analyzed by real-time PCR using primers and probes specific for the cellular genes β-actin or Ant4 and for the MCMV MIEP. The results (means of three experiments + standard errors of the means) are shown as percentages of input chromatin immunoprecipitated with anti-histone H3 (top) or anti-histone H4 (bottom). *, P < 0.05. Gray bars, acute infection; black bars, latent infection.

FIG. 4.

Association of acetylated histones with the MIEP region. Chromatin from acutely and latently infected mice was subjected to ChIP with antibodies against histone H3, histone H4, acetylated histone H3 (Ac-H3), or acetylated histone H4 (Ac-H4), followed by quantitative real-time PCR with primers/probe specific to β-actin (top panels), Ant4 (middle panels), or the MCMV MIEP region (bottom panels). The data are presented as the percent input (A) or as the ratio of DNA bound to acetylated histones versus that bound to the corresponding total histone after normalization to input DNA (B). Results shown are the means + standard errors of the means of three independent experiments. *, P < 0.01. Gray bars, acute infection; black bars, latent infection.

FIG. 5.

Recruitment of histone deacetylases to the MIEP in latent infection. Chromatin from acutely or latently infected mice was immunoprecipitated with antibodies specific to histone deacetylase 2 (top) or histone deacetylase 3 (bottom) followed by real-time PCR analysis with primers/probe specific to the MCMV MIEP, β-actin, or Ant4. The results (means of three experiments + standard errors of the means) are shown as percentages of input chromatin immunoprecipitated with anti-HDAC antibody. *, P < 0.01. Gray bars, acute infection; black bars, latent infection.

FIG. 6.

Analysis of methylation of histone H3 bound to the MIEP in acute and latent infection. Chromatin from kidneys of acutely or latently infected mice was analyzed by ChIP with antibodies against histone H3, H3K4me3, H3K9me1, H3K9me2, or H3K9me3. Real-time PCR was performed on the immunoprecipitates with primers/probe specific to β-actin (top panels), Ant4 (middle panels), or the MIEP (bottom panels). The data are presented as the percent input (A) or as the ratio of DNA bound to methylated histones versus that bound to the corresponding total H3 after normalization to input DNA (B). Results shown are the means + standard errors of the means of three independent experiments. *, P < 0.02. Gray bars, acute infection; black bars, latent infection.

FIG. 7.

Heterochromatin protein 1γ binds to the MIEP in latent infection. ChIP/quantitative real-time PCR was performed on chromatin from acutely and latently infected mice with antibody specific to HP-1γ. Immunoprecipitated DNA was analyzed by real-time PCR with primers/probe specific to the MIEP, β-actin, or Ant4 promoters. The results (means of three experiments + standard errors of the means) are shown as percentages of input chromatin immunoprecipitated with anti-HP-1γ antibody. *, P < 0.001. Gray bars, acute infection; black bars, latent infection.

FIG. 8.

Recruitment of YY1 to the MIEP in latent infection. ChIP/quantitative real-time PCR was performed on chromatin from acutely and latently infected mice with antibody specific to YY1. Immunoprecipitated DNA was analyzed by real-time PCR with primers/probe specific to the MIEP, β-actin, or rpL30 promoters. The results (means of three experiments + standard errors of the means) are shown as percentages of input chromatin immunoprecipitated with anti-YY1. *, P < 0.001. Gray bars, acute infection; black bars, latent infection.