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. 2010 Apr;84(7):3631-43.
doi: 10.1128/JVI.02380-09. Epub 2010 Jan 27.

Biphasic recruitment of transcriptional repressors to the murine cytomegalovirus major immediate-early promoter during the course of infection in vivo

Xue-Feng Liu  1 , Shixian Yan, Michael Abecassis, Mary Hummel
Affiliations

Biphasic recruitment of transcriptional repressors to the murine cytomegalovirus major immediate-early promoter during the course of infection in vivo

Xue-Feng Liu et al. J Virol. 2010 Apr.

Abstract

Our previous studies showed that establishment of murine cytomegalovirus (MCMV) latency in vivo is associated with repression of immediate-early gene expression, deacetylation of histones bound to the major immediate-early promoter (MIEP), changes in patterns of methylation of histones, and recruitment of cellular repressors of transcription to the MIEP. Here, we have quantitatively analyzed the kinetics of changes in viral RNA expression, DNA copy number, and recruitment of repressors and activators of transcription to viral promoters during the course of infection. Our results show that changes in viral gene expression correlate with changes in recruitment of RNA polymerase and acetylated histones to viral promoters. Binding of the transcriptional repressors histone deacetylase type 2 (HDAC2), HDAC3, YY1, CBF-1/RBP-Jk, Daxx, and CIR to the MIEP and HDACs to other promoters showed a biphasic pattern: some binding was detectable prior to activation of viral gene expression, then decreased with the onset of transcription and increased again as repression of viral gene expression occurred. Potential binding sites for CBF-1/RBP-Jk and YY1 in the MIEP and for YY1 in the M100 promoter (M100P) were identified by in silico analysis. While recruitment of HDACs was not promoter specific, binding of CBF-1/RBP-Jk and YY1 was restricted to promoters with their cognate sites. Our results suggest that sequences within viral promoters may contribute to establishment of latency through recruitment of transcriptional repressors to these genes. The observation that repressors are bound to the MIEP and other promoters immediately upon infection suggests that latency may be established in some cells very early in infection.

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Figures

FIG. 1.
FIG. 1.
Kinetics of viral gene expression (A) and DNA copy number (B) in kidneys of MCMV-infected mice. RNA and DNA were isolated from each mouse at various times postinfection (n = 6 to 10/time point). (A) Relative quantification of MCMV RNA was performed as described in Materials and Methods. Results are shown as the normalized average quantity plus standard error. *, P ≤ 0.05 relative to the peak of expression (day 7 for IE-1, IE-3, and M112; day 10 for M100). (B) Absolute quantification of MCMV DNA copy number, determined as described in Materials and Methods. The data are expressed as the average number of copies per million cells plus standard error. *, P ≤ 0.05 relative to the result on day 10.
FIG. 2.
FIG. 2.
Kinetics of recruitment of histones, HDACs, and RNA polymerase to the MIEP in kidneys of mice infected with MCMV. Chromatin from kidneys of infected mice was isolated at various times after infection and immunoprecipitated with the indicated antibodies. Immunoprecipitated DNA was amplified with MIEP-specific primers and probe (A, C, E, G, and I) or with primers and probe specific for the β-actin or Ant 4 promoters (B, D, F, H, and J), and the percentage of input DNA bound to antibody was calculated as described in Materials in Methods. n = 3 to 4/time point, except for day 21, where n = 2. Each sample was analyzed in triplicate. Results for panels A to D and G to H are shown are the mean percentage of input DNA bound to antibody plus standard error. Results for panels E and F are shown as the ratio of acetylated histone H4 to total H4, calculated from the percent input determined with antibodies specific to acetylated and total H4. The ratio is greater than 1 due to differences in the efficiencies of the antibodies. The horizontal axis indicates the days postinfection. *, P < 0.05 relative to the result on day 7.
FIG. 3.
FIG. 3.
Biphasic recruitment of cellular repressors to the MIEP in kidneys of mice infected with MCMV. Chromatin from kidneys of infected mice was isolated at various times after infection and immunoprecipitated with the indicated antibodies. Immunoprecipitated DNA was amplified with primers and probe specific for the MIEP (A, C, E, and F), the RpL30 or Ant 4 promoters (B), or the Hes1 or β-actin promoters (D) and the percentage of input DNA bound to antibody was calculated as described in Materials and Methods. n = 2 to 4/time point. Each sample was analyzed in triplicate. Results shown are the mean percentage of input DNA bound to antibody plus standard error. The horizontal axis indicates the days postinfection. *, P < 0.05 relative to the result on day 7.
FIG. 4.
FIG. 4.
Kinetics of recruitment of transcriptional regulatory factors to viral promoters. Chromatin from kidneys of infected mice was isolated at various times after infection and immunoprecipitated with the indicated antibodies. Immunoprecipitated DNA was amplified with primers and probe specific for the M112P or M100P as described in Materials and Methods, and the percentage of input DNA bound to antibody was calculated as described in Materials and Methods. The data for ChIP analysis of the MIEP in Fig. 2 and 3 are shown with analysis of M112P and M100P for comparison. n = 2 to 4/time point. Each sample was analyzed in triplicate. Results for panels A, C, D, E, and F shown are the mean percentage of input DNA bound to antibody plus standard error. Results for panel B are shown as the ratio of acetylated H4 to total H4. The horizontal axis indicates the days postinfection. *, P < 0.05. For the sake of clarity, not all statistically significant differences are indicated: significant differences in binding to the MIEP are shown in Fig. 2 and 3; significant differences in binding of RNA polymerase and acetylated H4 to the M100P and M112P and in binding of H4, HDAC2, and HDAC3 to M112P at different times are not shown.
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