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. 2013 Aug 6;110(32):13126-31.
doi: 10.1073/pnas.1305548110. Epub 2013 Jul 22.

Nucleosome maps of the human cytomegalovirus genome reveal a temporal switch in chromatin organization linked to a major IE protein

Einat Zalckvar  1 , Christina Paulus, Desiree Tillo, Alexandra Asbach-Nitzsche, Yaniv Lubling, Carla Winterling, Nicholas Strieder, Katrin Mücke, Felicia Goodrum, Eran Segal, Michael Nevels
Affiliations

Nucleosome maps of the human cytomegalovirus genome reveal a temporal switch in chromatin organization linked to a major IE protein

Einat Zalckvar et al. Proc Natl Acad Sci U S A. .

Abstract

Human CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes deficient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-deficient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function.

Keywords: ChIP-chip; epigenetic regulation; functional genomics; herpesvirus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Massive changes in nucleosome organization across the hCMV genome during productive infection. (A) Nucleosome occupancy profiles of the hCMV (TB40E) WT strain at 8, 48, and 96 h postinfection. At the top, the hCMV genome organization is schematically depicted [terminal repeat long (TRL), UL1–150, unique short ORFs 7–34 (US7–34), and terminal repeat short (TRS)]. The location of BAC sequences replacing viral ORFs IRS1, US1, US2 (partial), and US3–US6 in the TB40-BAC4 is indicated. (B) Summary of correlations between hCMV genome-wide nucleosome maps from this study and between these maps and the model by Kaplan et al. (6) (n = 204,747). Red squares mark the correlation between 8 and 96 h postinfection for the WT or dlIE1 maps. Correlations were grouped using 2D hierarchical clustering. (C) Autocorrelation plots (44) of nucleosome occupancy data from WT and dlIE1 virus genomes. The y axis on each plot shows Pearson correlation values obtained from correlating each MNase-derived nucleosome occupancy map set with shifted versions of itself from 0 to 1,000 bp (offset; x axis). Dashed vertical lines represent the typical length of one nucleosome including linker DNA.
Fig. 2.
Fig. 2.
IE1-dependent temporal changes in nucleosome organization across hCMV genomes. (A) Global views of nucleosome occupancy on WT and dlIE1 viral genomes across time postinfection. The model is by Kaplan et al. (6), and it predicts intrinsic nucleosome occupancy. Data were grouped based on WT nucleosome occupancy profiles (MNase data) of all time points postinfection from −1 to +1 kb relative to the manually annotated transcription start sites (k = 2, correlation distance metric), resulting in clusters #1 (higher occupancy of transcribed elements of the WT strain at 8 h postinfection) and #2 (higher occupancy of transcribed elements of the WT strain at 96 h postinfection). (B) Examples of nucleosome occupancy profiles from WT and dlIE1 at 8 and 96 h postinfection in viral genomic regions UL80–UL83 (cluster #1) and UL1–UL6 (cluster #2).
Fig. 3.
Fig. 3.
IE1-dependent effects on global nucleosome occupancy across hCMV genomes. (A) MRC-5 cells were infected with WT or dlIE1 strains of hCMV TB40E (3 pfu/cell) for 8 h. (Upper) Nuclei were reacted with the indicated amounts of MNase, and purified DNA was separated in a 1.2% (wt/vol) agarose gel stained with ethidium bromide. (Lower) The same DNA samples were subjected to Southern blotting using a whole-genome probe derived from TB40-BAC4. (B) MRC-5 cells were infected with WT or dlIE1 strains of hCMV Towne (0.5 pfu/cell) for 8 h. (Upper) Nuclei were reacted with the indicated amounts of MNase, and DNA was separated in a 1.2% (wt/vol) agarose gel stained with ethidium bromide. The same DNA samples were subjected to chromatin accessibility real-time PCR at the indicated viral genomic sites. Columns represent dlIE1-to-WT DNA ratios from two measurements or ratios averaged across all tested sites (Ø), with SDs shown as error bars. (C) MRC-5 cells were infected with WT or dlIE1 strains of hCMV Towne (3 pfu/cell) for 0.5 h. ChIP was performed using antibodies for the indicated core histones, and DNA was quantified by qPCR at the indicated viral genomic sites. Columns represent mean output-to-input DNA ratios from two measurements normalized to the respective ratios at the cellular tubulin beta (TUBB) locus.
Fig. 4.
Fig. 4.
Negative correlation between IE1-dependent changes in nucleosome organization and expression across hCMV genomes. (A) Summary of correlations between IE1-dependent changes in transcription and nucleosome occupancy across viral promoters and transcribed regions for the indicated times postinfection and gene sets [set 1: annotations from the work by Zhang et al. (32), n = 135; set 2: manual annotations, n = 86]. An average transcription (from total and nascent cDNA data) and nucleosome occupancy score (from MNase data) was calculated across promoters (defined as −200 to 0 bp relative to the transcription start site) or transcribed regions (defined as ORFs or sequences from transcription start to end), and differences between dlIE1 and WT were determined. Spearman correlations between differences in nucleosome occupancy and differences in transcript levels are shown. Asterisks indicate correlations with P < 0.05. (B) Scatterplots illustrating the negative relationship (ρ = Spearman correlation, P = P value) between IE1-dependent changes in nucleosome occupancy (from MNase data) and gene expression (from total cDNA data, set 2) across viral promoters and transcribed regions at 96 h postinfection (Figs. S5, S6, and S7).
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References

    1. Davey CA, Sargent DF, Luger K, Maeder AW, Richmond TJ. Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 Å resolution. J Mol Biol. 2002;319(5):1097–1113. - PubMed
    1. Luger K, Mäder AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature. 1997;389(6648):251–260. - PubMed
    1. Segal E, Widom J. What controls nucleosome positions? Trends Genet. 2009;25(8):335–343. - PMC - PubMed
    1. Kaplan N, Hughes TR, Lieb JD, Widom J, Segal E. Contribution of histone sequence preferences to nucleosome organization: Proposed definitions and methodology. Genome Biol. 2010;11(11):140. - PMC - PubMed
    1. Tillo D, et al. High nucleosome occupancy is encoded at human regulatory sequences. PLoS One. 2010;5(2):e9129. - PMC - PubMed

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