Human cytomegalovirus IE2 both activates and represses initiation and modulates elongation in a context-dependent manner

Using PRO-Seq, which profiles nascent transcripts, and a recently developed DFF-ChIP approach that informs on local chromatin environment, we show that IE2 controls viral gene transcription in three distinct capacities during late HCMV infection and reveal mechanisms involving direct binding of IE2 to viral DNA. IE2 represses a subset of viral promoters by binding within the target core promoter region, blocking the assembly preinitiation complexes (PICs). Remarkably, IE2 forms a repressive complex at the major immediate-early promoter region involving direct association of IE2 with nucleosomes and TBP. IE2 activates transcription by binding proximal to, but not within, core promoter regions. In addition, evidence is provided that IE2 functions as a direct roadblock to transcription elongation. At one locus, this function of IE2 is demonstrated to be important for the synthesis of a spliced RNA. Consistent with the minimal observed effects of IE2 depletion on host gene transcription, IE2 only sparingly engages the host genome. Overall design: HCMV IE2-FKBP TB40/E and Towne strain viruses were developed as described in our previous study. Both raw and processed PRO-Seq data generated from that study are available under GEO accession number GSE139114. For RNA-Seq data generation, HFF were infected for 96 h with Towne IE2-FKBP virus and treated for the last 6 h of infection with DMSO or the last 6 or 12 h of infection with dTAG. Total RNA was isolated, processed for library generation, and sequenced as described in the manuscript. For this study, a TB40/E IE2-GFP virus was developed using GalK recombination, as described in the methods. HFF were infected for 48 h with TB40/E IE2-GFP virus. Infected cells were harvested for utilized for standard ChIP-Seq or DFF-ChIP using GFP nanobody conjugated agarose as described in the manuscript. Sequencing data were processed as described. Raw and processed data files are provided here.