Cohesins, which mediate sister chromatin cohesion, and CTCF, which functions at 
chromatin boundaries, play key roles in the structural and functional 
organization of chromosomes. We examined the binding of these two factors on the 
Kaposi's sarcoma-associated herpesvirus (KSHV) episome during latent infection 
and found a striking colocalization within the control region of the major 
latency transcript responsible for expressing LANA (ORF73), vCyclin (ORF72), 
vFLIP (ORF71), and vmiRNAs. Deletion of the CTCF-binding site from the viral 
genome disrupted cohesin binding, and crippled colony formation in 293 cells. 
Clonal instability correlated with elevated expression of lytic cycle gene 
products, notably the neighbouring promoter for K14 and vGPCR (ORF74). siRNA 
depletion of RAD21 from latently infected cells caused an increase in K14 and 
ORF74, and lytic inducers caused a rapid dissociation of RAD21 from the viral 
genome. RAD21 and SMC1 also associate with the cellular CTCF sites at mammalian 
c-myc promoter and H19/Igf2 imprinting control region. We conclude that cohesin 
subunits associate with viral and cellular CTCF sites involved in complex gene 
regulation and chromatin organization.