# Cell type differences in human cytomegalovirus transcription and epigenetic regulation with insights into major immediate-early enhancer-promoter control

## TL;DR

The study reveals how human cytomegalovirus transcription and epigenetic regulation differ in neural lineage cells compared to fibroblasts, uncovering new regulatory mechanisms.

## Contribution

The paper identifies cell type-specific regulatory mechanisms of HCMV transcription and epigenetic control, independent of known viral factors.

## Key findings

- Transcription at several viral promoters in D-NT2 cells requires viral DNA synthesis and is independent of host P-TEFb and viral IE2.
- GC-box mutations in the MIE enhancer increase enhancer transcription and alter cell morphology and gene expression in D-NT2 cells.
- Viral genomes in D-NT2 cells have more nucleosomes, potentially restricting access to late transcription factors.

## Abstract

Cell type differences in the human cytomegalovirus (HCMV) transcriptome may arise from variations in transcription or post-transcription regulation. Here we report unexpected differences in transcription and epigenetic control in late-stage HCMV infection of human differentiated NTera2 neural lineage cells (D-NT2) compared to fibroblasts, using integrated functional genomic approaches (PRO-Seq, RNA-Seq, DNA fragmentation factor-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 immediate-early protein 2 (IE2), or viral late transcription factor (LTF). This includes transcription from the enhancer for the major immediate early (MIE) promoter where GC-box sequence mutations increase enhancer transcription, while mutations in CREB and NF-kB response elements reduce it. The GC-box mutations also alter infected D-NT2 cell morphology and gene expression program without affecting viral MIE gene expression levels, whereas mutations in CREB and NF-kB response elements do not induce these changes. 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 TATA-binding protein (TBP)-IE2-nucleosome complex, with more nucleosome than in fibroblasts, occupies the MIE promoter transcription start site, potentially contributing to its epigenetic silencing.

Human cytomegalovirus persists silently in most human tissues. Upon reactivation, it infects various cell types, leading to outcomes ranging from asymptomatic shedding to invasive disease, depending on the affected organ and host response. We identify cell type-specific differences in human cytomegalovirus transcription during late-stage infection, revealing novel promoter regulatory mechanisms independent of viral IE2 or late transcription factors. These mechanisms govern multiple viral promoters, including a promoter within the enhancer for the major immediate-early promoter (MIEP). This enhancer-associated promoter is selectively activated in a cell type-dependent manner under cis-regulatory element control, producing a stable enhancer RNA. While histone H3.3, H3 lysine 4 trimethylation, and H3 lysine 27 acetylation are absent from the enhancer, their proportion within the TBP-IE2-nucleosome complex at the MIEP varies by cell type. These structural differences suggest an epigenetic mechanism of MIEP regulation influenced by cell type-specific chromatin architecture.