show Abstracthide AbstractCTCF and the cohesin complex modify chromatin by binding to DNA and interacting with each other and with other cellular proteins. Both proteins regulate transcription by a variety of local effects on transcription and by long range topological effects. CTCF and cohesin also bind to herpesvirus genomes at specific sites and regulate viral transcription during latent and lytic cycles of replication. Kaposi's sarcoma-associated herpesvirus (KSHV) transcription is regulated by CTCF and cohesin, with both proteins previously reported to act as restrictive factors for lytic cycle transcription and virion production. In this study, we examined the interdependence of CTCF and cohesin binding to the KSHV genome. ChIP-seq analyses revealed that cohesin binding to the KSHV genome is highly CTCF dependent whereas CTCF binding does not require cohesin. Further, depletion of CTCF leads to almost complete dissociation of cohesin from sites at which they colocalize. Thus, previous studies which examined the effects of CTCF depletion actually represent concomitant depletion of both CTCF and cohesin components. Analysis of the effects of single and combined depletion indicate that CTCF primarily activates KSHV lytic transcription whereas cohesin has primarily inhibitory effects. Further, CTCF or cohesin depletion was found to have regulatory effects on cellular gene expression relevant for control of viral infection, with both proteins potentially facilitating expression of multiple genes important in the innate immune response to viruses. Thus, CTCF and cohesin have both positive and negative effects on KSHV lytic replication as well as effects on the host cell that enhance antiviral defenses. Overall design: Three different treatments were applied to cells: control KD, CTCF KD and Rad21 KD. Each treatment has three types of analyses: Input DNA, CTCF Ab IP or Rad21 IP.