Biphasic Recruitment of Transcriptional Repressors to the Murine Cytomegalovirus Major Immediate-Early Promoter during the Course of Infection In Vivo^▿

Mice and virus.

Three-week-old, female, specific-pathogen-free BALB/c mice were purchased from Jackson Laboratory, Bar Harbor, ME. Mice were maintained in isolation cages and fed and watered ad libitum. MCMV (Smith strain, ATCC 194-VR) was purchased from the American Type Culture Collection (Rockville, MD). Virus stocks were generated by propagation in salivary glands as previously described (31). Mice were infected by intraperitoneal injection with 10⁵ PFU of MCMV and sacrificed at various times postinfection. This study protocol was reviewed and approved by the Northwestern University Institutional Animal Care and Use Committee.

Nucleic acid extraction.

Kidney DNA was prepared by using a Puregene tissue kit (Gentra, Minneapolis, MN) according to the manufacturer's instruction. Total RNA was isolated and purified with a TRIzol Plus RNA purification kit (Invitrogen, Carlsbad, CA). On-column DNase Ι digestion was performed during RNA purification. DNA and RNA were quantified by A₂₆₀ with a DU640B spectrophotometer (Beckman Coulter, Fullerton, CA).

Reverse transcription (RT).

Oligo(dT)-primed cDNA was generated from 5 μg of total RNA by using an AffinityScript multiple temperature cDNA synthesis kit (Stratagene, La Jolla, CA) in a 20-μl reaction mixture. Reverse transcriptase was inactivated by heating at 70°C for 15 min. Samples were cooled to 37°C and treated for 30 min with 0.15 U/μl RNaseH (Invitrogen) and 1 μl RNase cocktail (Ambion, Austin, TX). Subsequently, the cDNA was purified with a QIAquick PCR purification kit (Qiagen, Valencia, CA) and quantified using a Quant-iT Oligreen single-stranded DNA (ssDNA) assay kit (Invitrogen).

Real-time PCR analysis.

All real-time PCRs were performed using TaqMan gene expression master mix in an ABI 7500 Fast system using the standard curve (absolute quantitation) assay and regular 7500 mode. Primers and TaqMan minor groove binder (MGB) probes described in Table 1 were designed by ABI or by using Primer Express 3.0 software. Reaction mixtures contained 900 nM primers and 250 nM TaqMan MGB probes. Each sample was analyzed in triplicate. The thermal cycling conditions were 50°C for 2 min and 95°C for 10 min followed by 50 cycles of 95°C for 15 s and 60°C for 1 min.

Quantification of MCMV DNA and RNA by real-time PCR.

For absolute quantification of MCMV DNA copy number in kidney tissue, a standard curve was generated by serial dilution of plasmid pIE111 (45), which contains the MIEP and IE genes, in genomic DNA isolated from kidneys of uninfected mice, such that 13.5 μl of the template contained 3 × 10⁶, 3 × 10⁵, 3 × 10⁴, 3 × 10³, 3 × 10², or 3 × 10¹ copies of pIE111 in 800 ng cellular DNA. A standard curve for mouse Eef1α DNA was generated by serial dilution of plasmid pDRIVE-mEF1α (InvivoGene, San Diego, CA), which contains the promoter region of the mouse eukaryotic translation elongation factor 1 alpha (Eef1α) gene, such that 13.5 μl of the template contained 1 × 10⁶, 1 × 10⁵, 1 × 10⁴, 1 × 10³, 1 × 10², or 10 copies of pDRIVE-mEF1α in 800 ng sheared salmon sperm DNA. Sample genomic DNA for each reaction (800 ng) was used as a template for PCR in a 30-μl reaction mixture. Genomic DNA from uninfected mouse kidneys (800 ng) was used as a control for detection of nonspecific amplification. The primers and probes used to detect MCMV MIEP and Eef1α promoter DNA are listed in Table 1. The target DNA copy number in each sample was calculated from a standard curve generated in each individual assay. The viral DNA copy number per million cells was determined by dividing the average copy number of MCMV DNA by the Eef1α copy number in the same sample and multiplying by 2 × 10⁶.

Quantification of MCMV IE-1, IE-3, M112, and M100 mRNA was performed using the relative standard curve method as described in the Applied Biosystems support material “Chemistry Guide: Applied Biosystems Real-Time PCR Systems” (http://www.appliedbiosystems.com/support/apptech/, part no. 4348358) with the primers and probes specific to each gene (Table 1). Serially diluted cDNA derived from RNA isolated from kidneys at 5 days postinfection was used to generate the standard curve. Sample cDNA (10 ng) was added to a 20-μl reaction mixture for amplification of virus cDNA; the same amount of cDNA from uninfected mouse kidneys was used as a control for nonspecific amplification. The abundance of the target cDNA in each sample was determined on the basis of a standard curve produced in each run. Mouse eukaryotic Eef2 RNA was employed as an internal control. The relative quantity of MCMV RNA in the sample was determined by dividing the average quantity of MCMV mRNA by that of Eef2, as determined from the standard curve. As a control to detect contamination of the cDNA by genomic DNA, we analyzed cDNAs for the presence of the intronic region between exons 1 and 2 in the immediate-early gene 1/3.

Chromatin immunoprecipitation (ChIP).

Chromatin immunoprecipitation assays were performed essentially as described previously (40). Antibodies against histone H4 (pan), acetyl histone H4 (Lys5, Lys8, Lys12, Lys16), and Daxx were obtained from Millipore Corporation, Billerica, MA. Antibodies for histone deacetylase type 2 (HDAC2) and HDAC3 were purchased from Sigma-Aldrich Inc., St. Louis, MO. Antibodies specifc for RNA polymerase ΙΙ (N-20) and CBF-1/RBP-Jk(H-50) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. Anti-YY1 and anti-CIR were purchased from Abcam, Cambridge, MA, and Novus Biologicals, Littleton, CO, respectively.

Quantitative analysis of ChIP DNA samples by real-time PCR.

The relative amount of MCMV MIEP DNA in each ChIP sample was determined using the relative standard curve method. Serial dilutions of sheared genomic DNA purified from acutely infected mouse kidneys were used to generate the standard curve. The same set of standards was used for all analyses. For analysis of total histone H4, HDACs, RNA Pol II, YY1, CBF-1/RBP-Jk, Daxx, and CIR, the results are expressed as the percentage of immunoprecipitated chromatin relative to the total input of chromatin in the immunoprecipitation mixture. For analysis of acetylated histone H4, the results are expressed as the ratio of the modified histone to corresponding total histone after normalization to input DNA. ChIP assays were analyzed in triplicate, and the results presented are the averages of results of two to four independent assays plus standard error.

Statistical analysis.

A two-tailed Student's t test was used to determine statistical significance. P values of ≤0.05 were considered significant.

Repression of MCMV ie gene expression in the kidney occurs between day 7 and day 10 postinfection.

In order to understand the mechanism of establishment of latency, it is important to know when repression of viral gene expression occurs during the course of infection. We therefore examined the kinetics of expression of IE-1 and IE-3 RNAs and accumulation of viral DNA during acute infection in the kidney. IE-1 and IE-3 RNAs were analyzed by real-time PCR analysis of purified cDNA using the relative standard curve method as described in Materials and Methods. The intron region between exons 1 and 2 was analyzed as a control for genomic DNA contamination of the cDNA. DNA and RNA from uninfected mice were used as controls for nonspecific amplification. These controls were negative (data not shown). Our results show that expression of IE-1 RNA was detectable at day 2 postinfection (p.i.), peaked at day 7, and dropped significantly at day 10 relative to the peak at day 7 (Fig. 1A). Expression of IE-3, which is transcribed from the same promoter as IE-1, followed the same trend as expression of IE-1.

MCMV DNA was detectable in the kidney within 1 day p.i. but did not accumulate significantly above this level until day 4. In contrast to IE RNA expression, which peaked at day 7, MCMV DNA copy number peaked at day 10 and fell gradually until day 28, where it plateaued to levels seen in latently infected mice (Fig. 1B). The decrease in DNA copy number likely reflects clearance of infected cells by the host immune response. However, our results show that loss of ie gene expression precedes the decrease in DNA copy number and is therefore not due simply to clearance of infected cells. These data demonstrate that repression of IE RNA expression in the kidneys of infected mice occurs between day 7 and day 10.

CMV gene expression during productive infection occurs in a cascade fashion typical of other herpesviruses, in which the immediate-early genes induce expression of the early genes, followed by DNA replication and expression of the late genes. We therefore analyzed expression of M112 (e1) and M100 (gM), which are representative of the early and late genes, respectively. The kinetics of expression of M112 RNA was indistinguishable from that of the immediate-early genes during acute infection in vivo (Fig. 1A). In contrast, relatively high levels of M100 RNA were still observed at day 10 p.i., when expression of IE RNA had fallen, but DNA copy number was still high. Interestingly, expression of this RNA was rapidly lost after day 10, despite continued presence of the DNA (Fig. 1A and B). With the exception of results for one mouse at day 60, expression of M112 and M100 RNA was undetectable after day 28. Taken together, our results suggest that latency is established in the kidney by day 28 p.i.

Transcriptional activation of ie gene expression correlates with recruitment of RNA polymerase and acetylated H4 to the MIEP.

We then examined binding of RNA polymerase II to the MIEP during acute infection (Fig. 2A). As expected, binding of RNA polymerase paralleled ie gene expression, with binding detectable at day 2, peaking at day 7, and falling significantly at day 10. In addition, we analyzed binding of RNA polymerase II to the β-actin and Ant4 cellular promoters as controls for transcriptionally active and inactive genes, respectively (Fig. 2B). β-Actin is widely expressed in all organs; The Ant4 gene is a developmentally regulated gene which is silenced through DNA methylation in adult mouse tissues (61). Binding of RNA polymerase to the β-actin promoter was observed at all time points at comparable levels. Thus, the changes in binding of RNA polymerase to the MIEP during the course of infection are not due to differences in the chromatin preparations. At the peak of infection, the percentage of MIEP DNA bound to RNA polymerase was similar to that of β-actin, indicating that the proportion of viral genomes being actively transcribed was similar to that of a highly active cellular gene and, thus, is biologically relevant. In contrast, very little binding of RNA polymerase to the transcriptionally inactive Ant4 promoter was observed, indicating the specificity of the binding to the MIEP that we observed.

We previously demonstrated that MCMV DNA is bound to histones during infection and that the percentage of genomes associated with histones rises dramatically in latent infection (40). Here, we have examined the kinetics of histone deposition to the MIEP (Fig. 2C). Our previous studies showed that binding of histone H4 to the MIEP was detectable during acute infection; here, we show that binding is detectable as early as day 2. Statistically significant accumulation of histones above this level was observed starting at day 14, when repression of ie gene expression was apparent (Fig. 1). At the peak of ie gene expression at day 7, binding of H4 to the MIEP was comparable to that of cellular genes (Fig. 2C and D). In contrast to cellular promoters, whose association with H4 did not change during the course of infection, binding of H4 to the MIEP increased more than 14-fold in latently infected mice compared to that in mice at 7 days postinfection.

Transcriptional activity of cellular genes is controlled by posttranslational modifications of histones bound to promoter regions (32). Acetylation of histone H4 is associated with active transcription, while deacetylation is associated with transcriptional repression. Because the percentage of genomes associated with histones changes during the course of infection, we analyzed the ratio of acetylated histones to total histones bound to the MIEP. Our results show that binding of acetylated histone H4 to the MIEP paralleled that of RNA polymerase, with a peak at day 7 and a significant drop at day 10 (Fig. 2E). In contrast, binding of acetylated H4 to the β-actin promoter, which was analyzed as a positive control, remained high throughout the course of infection (Fig. 2F). Very little binding of acetylated H4 to the Ant4 promoter, which was analyzed as a negative control, was observed at any time point.

Biphasic recruitment of repressors to the MIEP.

Deacetylation of histones is catalyzed by histone deacetylases (HDACs). Relative to the situation on day 7, binding of HDAC2 to the MIEP began to increase at day 10 and continued to increase as latency was established (Fig. 2G). Thus, binding of HDAC2 to the MIEP correlated inversely with binding of RNA polymerase and acetylated histone H4. Interestingly, binding of HDAC2 to the MIEP was initially observed at day 2 and day 4 and then fell at day 7 before increasing again at day 10. Binding of HDAC3 to the MIEP followed the same trend (Fig. 2I). As controls, we analyzed binding of HDACs to the β-actin and Ant4 promoters (Fig. 2H and J). As expected, little binding of HDACs to the transcriptionally active β-actin promoter was observed. We also observed little binding of HDACs to the transcriptionally silent Ant4 promoter. As we noted previously, this gene is silenced by DNA methylation, and its expression is apparently not regulated by histone deacetylation (40, 61). No changes in the levels of HDACs bound to cellular promoters were observed at different time points, indicating that differences in binding of HDACs to the MIEP were not due to differences in chromatin preparations.

Repression of ie gene expression is likely to occur through interaction of repressive factors in addition to HDACs with the MIEP. Recruitment of these factors may occur directly through recognition of specific DNA sequences in the promoter/enhancer region or indirectly through interaction with other factors. We used MatInspector (release 8.01; Genomatix) (9) to identify potential binding sites for repressive transcription factors in the MCMV enhancer. This analysis identifies sites using position weight matrices, in which every nucleotide in the sequence is assigned a score based on its conservation with other residues at that position in the database and, thus, accounts for the fact that some positions are more highly conserved than others (73). The algorithm defines a short, highly conserved core sequence common to all sequences in the database and calculates a conservation score (Ci value) at each position of the matrix. This is used to identify sequences in the gene of interest with perfect matches for the core sequence and calculates the similarity of the sequence to the matrix. A matrix score of 1 indicates that the test sequence corresponds to the most conserved nucleotide at each position of the matrix. Many of the sites in the MIEP identified by MatInspector are sequences recognized by positively acting transcription factors, such as NF-κB, AP-1, Sp1, and CREB, which may have a role in driving IE gene expression during productive infection or during reactivation from latency.

Of particular interest to this study, we identified 10 sites with high matrix similarity to known CBF-1/RBP-Jk sites and 12 sites with high matrix similarity to known YY1 sites (Table 2). YY1 is a DNA-binding transcription factor (26, 30) that acts as a repressor of some promoters and an activator of others (17, 66). Previous studies have shown that YY1 activates the ribosomal protein L30 (RpL30) promoter and represses the HCMV enhancer (19, 39). We previously demonstrated that YY1 is bound to the MIEP in kidneys of latently infected mice (40). CBF1/RBP-Jk is a component of the Notch signaling pathway (reviewed in references 7 and 28). It is constitutively bound to its cognate sites and, in the absence of Notch ligands (Jagged and Delta-like), acts as a repressor of transcription through recruitment of HDACs and other corepressors, including the CBF-1/RBP-Jk-associated corepressor CIR. CBF-1/RBP-Jk binds constitutively to the promoter of the developmental regulatory gene Hes1 (28). CBF-1/RBP-Jk and CIR have not been previously associated with regulation of CMV IE gene expression. Previous studies have also suggested that Daxx may have a role in silencing ie gene expression (29, 43, 62, 63, 78).

We therefore examined interaction of YY1, CBF-1/RBP-Jk, CIR, and Daxx with the MIEP as a function of time of infection (Fig. 3A, C, E, and F). Our results show that all of these repressors bind to the MIEP in latently infected mice with a pattern similar to that of HDACs. Recruitment of repressors to the MIEP was detectable as early as day 2 postinfection, prior to the activation of ie gene expression (Fig. 1). This was followed by a loss of binding as activation of ie gene expression occurred at days 4 to 7, with a nadir of binding at day 7, when transcription of the IE genes reached its peak. Relative to the findings for day 7, there was a statistically significant increase in the percentage of genomes bound to these factors at day 14, which did not increase further at later times. As positive controls, we analyzed binding of YY1 and CBF-1/RBP-Jk to the promoter regions of RpL30 and Hes1, respectively (Fig. 3B and D). In contrast to the MIEP, binding of these factors did not change during the course of infection, indicating that differences in binding of YY1 and CBF-1/RBP-Jk to the MIEP were not due to variability in the chromatin preparations. Furthermore, the level of binding to the MIEP in latently infected mice was similar to or higher than that observed with cellular promoters, indicating that the binding activity is likely to be biologically relevant. Very little binding to the negative controls (the Ant4 promoter for YY1 and the β-actin promoter for CBF-1/RBP-Jk) was observed, indicating the specificity of the interaction with the MIEP. Changes in the levels of binding of these factors to the MIEP are unlikely to be due to differences in the levels of expression, since real-time PCR analysis of YY1 and CBF-1 RNA levels and Western blot analysis of YY1 protein levels showed no change in expression of these genes over the course of infection (data not shown). Binding of CIR and Daxx to the MIEP followed the same biphasic pattern observed for YY1 and CBF-1/RBP-Jk (Fig. 3E and F). Collectively, our results demonstrate that (i) cellular repressors are recruited to the MIEP during infection and (ii) there are dramatic changes in the proportion of MIEP molecules bound to these repressors as the infection progresses from the very early stages of infection prior to activation of IE gene expression to acute infection and then to latency.

YY1 and CBF-1/RBP-Jk are specifically recruited to the MIEP in latent infection.

The observations that the MIEP contains sequences similar to binding sites for YY1 and CBF-1/RBP-Jk and that these factors are bound to the MIEP suggested that these factors might be recruited to the MIEP through direct recognition of sequences in the MIEP. To test this hypothesis further, we examined interaction of YY1 and CBF-1/RBP-Jk with the promoters for the early gene M112 and the late gene M100. In addition, we analyzed binding of RNA polymerase, histone H4, acetylated H4, HDAC2, and HDAC3 to these promoters. These data are shown in Fig. 4, with the results of ChIP analysis of the MIEP in Fig. 2 and 3 included for comparison. Our results show that at day 4, when viral gene expression is increasing, binding of RNA polymerase to the MIEP was significantly higher than binding to the M100 promoter (M100P), while at day 10, the converse was true (Fig. 4A). These changes in recruitment of RNA polymerase to the promoter correlate with changes in RNA expression (Fig. 1). Binding of acetylated histone H4 to these promoters followed the same trend (Fig. 4B). These results are consistent with the temporal regulation of gene expression typical of herpesviruses. Binding of H4 to the MIEP appeared to be higher than binding to other promoters at some time points, but these differences were not significant (Fig. 4C). In latently infected mice, binding of H4 to all viral promoters was approximately 10-fold higher than binding to cellular promoters (2 to 4% of input versus ∼0.3% of input [compare Fig. 2D and Fig. 4C]). These results suggest that, like that of HCMV (11, 18, 49), the entire MCMV genome is likely to be chromatinized.

Recruitment of HDAC2 and HDAC3 to the M100P was also observed, and the percentages of M100 DNA bound to HDACs at all time points followed the same trend observed with the MIEP (Fig. 4D and E). Binding of HDACs to the M112 promoter (M112P) appeared to be lower than binding to the MIEP and M100P at early times, but these differences were not significant. HDACs were bound at similar levels to all promoters analyzed in latently infected mice. Thus, in latently infected mice, deacetylated histones are likely to be distributed throughout the genome.

In contrast to HDACs, which do not bind to specific DNA sequences, we observed promoter-specific binding of YY1 and CBF-1/RBP-Jk. Using Genomatix analysis, we identified a potential YY1 binding site in the M100P, in addition to that in the MIEP. No YY1 sites were identified in the M112P. ChIP analysis of chromatin isolated from infected mice at various times showed that binding of YY1 to the M100P was detectable and that binding to this promoter followed the biphasic pattern observed with binding of YY1 to the MIEP, with initial binding very early in infection, followed by a statistically significant decrease at day 7, and an increased level in latently infected mice (Fig. 4F). However, the percentage of MIEP molecules bound to YY1 was significantly higher than the percentage bound to M100 during latent infection. In contrast, binding to M112P, which lacks potential YY1 sites, was negligible at all time points. We also examined binding of CBF-1/RBP-Jk to the M112 and M100 promoters, which lack CBF-1/RBP-Jk binding sites. In contrast to the results for the MIEP (Fig. 3C), very little interaction with these promoters was observed at any time point (data not shown). These observations support the hypothesis that binding of YY1 and CBF-1/RBP-Jk to the MIEP may be mediated by direct interaction with specific sequences within the promoter, and they suggest that these factors may play a role in recruitment of corepressor molecules and, thus, in repression of IE gene expression during latency.