Cell Type Differences in Human Cytomegalovirus Transcription and Epigenetic Regulation with Insights into Major Immediate-Early Enhancer-Promoter Control [DFF-ChIP]

Cell type differences in the human cytomegalovirus (HCMV) transcriptome may result from variations in transcription and/or post-transcription. Here we report unexpected differences in transcription and epigenetic control in late-stage HCMV infection of human differentiated NTera2 cells (D-NT2) compared to fibroblasts, using integrated functional genomic approaches (PRO-Seq, RNA-Seq, DFF ChIP-Seq, rapid viral protein degradation, and promoter mutation and function assays). In D-NT2, but not fibroblasts, RNA polymerase II initiation and elongation at several viral promoters requires viral DNA synthesis and are independent of host P-TEFb, viral IE2, or viral UL87 late transcription factor (LTF). This includes transcription from the enhancer for the major immediate early (MIE) promoter where GC-box sequence mutations increase transcription and mutations in CREB and NF-kB response elements decrease transcription. The GC-box sequence mutations also alter infected cell morphology and gene expression program, whereas mutations in CREB and NF-kB response elements do not. In D-NT2, LTF-driven promoters constitute a smaller proportion of the viral late promoter population and are generally less active. Additionally, viral genomes have more nucleosomes, potentially restricting LTF access. A TBP-IE2-nucleosome complex, with more nucleosome than in fibroblasts, covers the MIE promoter transcription start site, potentially contributing to epigenetically silence of the promoter. Overall design: Nuclei from D-NT2 or HFF cells were rapidly isolated, either without crosslinking or with crosslinking using 1% paraformaldehyde for 10 minutes, followed by immediate quenching with 1.33 M Tris (pH 7.6). The isolated nuclei were then digested with DNA Fragmentation Factor (DFF) to primarily generate mono-nucleosomes. Chromatin was released from the nuclei through light sonication, and immunoprecipitations were performed on the released chromatin. Finally, the nuclei were prepared for DFF ChIP-Seq as detailed in the methods section.