Differences in RNA polymerase II complexes and their interactions with surrounding chromatin on human and cytomegalovirus genomes [ChIP-seq]

To investigate the chromatin structure surrounding human RNA polymerase II (Pol II) transcription on host and viral genomes, chromatin immunoprecipitation (ChIP) was performed following digestion of HCMV infected primary human foreskin fibroblasts (HFFs) with DNA Fragmentation Factor (DFF). DFF is a human endonuclease responsible for cleaving between nucleosomes during apoptosis. We found that utilizing DFF as the front end for ChIP-Seq in place of sonication or Micrococcal nuclease (MNase) offered new insights into the connections between active transcription and the local chromatin on the host and that these connections were completely absent on the HCMV genome. Nuclei from multiple different cell types that were either infected with HCMV strains TB40e or Towne or uninfected were rapidly isolated following no treatment, treatment with P-TEFb inhibitor flavopiridol, or treatment with XPB inhibitor triptolide. Resulting nuclei were then digested with DNA fragmentation Factor (DFF), an endonuclease with reduced intra-nucleosomal cutting as compared to Micrococcal nuclease (MNase), to generate primarily mono-nucleosomes. After release of chromatin from the nucleus following light sonication, transcription complex factors and histone modifications were then immunoprecipitated from the released chromatin.