Differences in RNA polymerase II complexes and their interactions with surrounding chromatin on human and cytomegalovirus genomes

Abstract

Interactions of the RNA polymerase II (Pol II) preinitiation complex (PIC) and paused early elongation complexes with the first downstream (+1) nucleosome are thought to be functionally important. However, current methods are limited for investigating these relationships, both for cellular chromatin and the human cytomegalovirus (HCMV) genome. Digestion with human DNA fragmentation factor (DFF) before immunoprecipitation (DFF-ChIP) precisely revealed both similarities and major differences in PICs driven by TBP on the host genome in comparison with PICs driven by TBP or the viral-specific, late initiation factor UL87 on the viral genome. Host PICs and paused Pol II complexes are frequently found in contact with the +1 nucleosome and paused Pol II can also be found in a complex involved in the initial invasion of the +1 nucleosome. In contrast, viral transcription complexes have very limited nucleosomal interactions, reflecting a relative lack of chromatinization of transcriptionally active regions of HCMV genomes.

Human Cytomegalovirus IE2 Both Activates and Represses Initiation and Modulates Elongation in a Context-Dependent Manner

“…This enrichment generally did not correlate with the abundance of H3K4me3 nucleosomes, although there is a notable congruency in nucleosome position between the IE2-GFP and H3K4me3 DFF-ChIP data sets. In addition, IE2 occupancy did not correlate well with DFF-ChIP data for TBP, which defines the position of Pol II PICs ( 35 ), an important observation in light of the fact that TBP and other components of TFIID are documented IE2 interactors ( 16 , 21 ).…”

“…Both standard ChIP-Seq and DFF-ChIP for IE2-GFP were then performed with HFF infected for 48 h with TB40/E mCherry IE2-GFP. In our previous work, DFF-ChIP was carried out only under native conditions ( 35 ). Here, to preserve interactions between IE2 and chromatin, DFF-ChIP was also carried out on cross-linked cells.…”

“…HCMV enters and leaves cells with a genome denuded of chromatin ( 54 ). The HCMV genome is highly permissive to transcription initiation during late infection, and Pol II DFF-ChIP data strongly suggest that the transcriptional machinery does not interface extensively with chromatin ( 35 , 55 ). ATAC-Seq data for HCMV in semipermissive Kasumi-3 cells indicate broad accessibility ( 56 ).…”

“…To further investigate a possible IE2-nucleosome interaction on viral chromatin, a fragMap for cross-linked IE2-GFP DFF-ChIP data was generated. A fragMap is a 2-dimensional heatmap of fragment position versus fragment size, described in Spector et al ( 35 ). The fragMap represents all identified IE2 binding sites except for the crs , which was removed from the analysis due to its high IE2 occupancy ( Fig.…”

“…To gain mechanistic insight into how IE2 differentially operates as an activator, repressor, and elongation barrier during late HCMV infection, standard IE2 chromatin immunoprecipitation sequencing (ChIP-Seq) and a novel method developed by our lab, DFF-ChIP, which employs chromatin digestion by the endonuclease DNA fragmentation factor prior to IP, were performed. Initial studies have demonstrated that DFF-ChIP significantly increases ChIP resolution and has the ability to inform on the local chromatin environment of immunoprecipitated factors through analysis of chromatin digestion patterns ( 35 ). Here, we show that IE2 engages numerous crs -like elements on viral chromatin during late infection and differentially influences transcription in a manner dependent on binding site context.…”