Mechanisms of gene activation in the Epstein -Barr virus lytic cycle

Expression of the Epstein-Barr virus (EBV) lytic genes occurs in an ordered cascade that is coordinately controlled by the viral transactivators Rta and ZEBRA. Understanding how these activators orchestrate an elaborate program of expression could serve as a paradigm for how transcriptional regulatory circuits function during the development and differentiation of multi-cellular organisms. The combinatorial effect of Rta and ZEBRA was dissected in vitro, using the highly expressed BHLF-1 gene as a model system. Recombinant Rta and ZEBRA recapitulate the endogenous and synergistic regulation of BHLF-1 in crude nuclear extracts and in a coactivator enriched transcription system. Using DNase I footprinting assays, I showed that synergistic activation is in part due to cooperative activator binding on the BHLF-1 enhancer. This reaction can be further stimulated by the architectural factor HMG-1, which facilitates binding of Rta to its sites. Immobilized template assays revealed that synergy can also be attributed to the differential recruitment of distinct coactivator complexes by Rta and ZEBRA during preinitiation complex assembly. ZEBRA bound to the BHLF-1 proximal promoter recruits the general factor TFIID to the core promoter of the gene, whereas Rta bound to the enhancer primarily targets the human mediator complex. In addition, the activators combinatorially recruit the p300 coactivator.;Next, I investigated the mechanism of the stimulatory effect of HMG-1 on Rta binding to the BHLF-1 enhancer. HMG-1 had been previously shown to facilitate cooperative interactions between pairs of closely apposed ZEBRA dimers bound to the BHLF-1 proximal promoter. In contrast, HMG-1 promotes binding of a single Rta dimer to its site. This effect requires the DNA binding domain of Rta, and can be elicited by different and unrelated DNA bending proteins. Furthermore, using hydroxyl radical footprinting, antibody supershift and immobilized template assays, I demonstrate that HMG-1 does not form a stable ternary complex with Rta. Taken together, these results argue that HMG-1 stimulates binding by transiently associating with the Rta sites and inducing a prebent and favorable DNA conformation. Consistent with this hypothesis, Rta bends its site upon binding.