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Olga D. González-López
Interdisciplinary Biomedical Sciences Program
B.S. 2009, University of Puerto Rico, Cayey, P.R
Thesis Advisor: David Lukac, Ph.D.
Department of Microbiology, Biochemistry and Molecular Genetics
Friday, May 8, 2015
10:00 A.M., (ICPH) 1st floor auditorium
Interaction of KSHV Rta protein with the cellular Notch signaling effector, RBP-Jk, is essential for KSHV reactivation. Our lab previously showed using transactivation assays and ChIP/q-PCR that Rta stimulates RBP-Jk DNA binding during reactivation in a promoter-specific fashion. This was a mechanism for RBP-Jk-dependent promoter specification that was fundamentally different from that proposed for Notch. The KSHV genome contains at least 203 predicted RBP-Jk binding sites, but only Rta, and not the RBP-Jk-dependent activators NICD-1 nor EBV EBNA2, productively reactivates KSHV from latency. We hypothesized that specific DNA elements would distinguish those promoters to which Rta stimulates RBP-Jk binding, from those to which Rta has no effect on RBP-Jk DNA binding. Using DNAse I footprinting, we showed that Rta formed an extended footprint that covered and flanked the RBP-Jk site in the Mta promoter, and included seven units of a 14-bp sequence that we termed “CANT DNA repeats”. CANT DNA repeats have a consensus sequence of ANTGTAACANTA/TA/TT. Varying the number and relative positions of the CANT DNA repeats influenced the affinity of Rta DNA binding and stimulation of RBP-Jk DNA binding. Based on the results from the single Mta promoter, we concluded that Rta stimulates DNA binding of RBP-Jk to promoters that contain a specific architecture, with a specific combination of CANT DNA repeats and RBP-Jk DNA binding sites. We hypothesized that this architecture would be conserved across the viral genome for additional promoters on which Rta stimulated RBP-Jk DNA binding necessary for viral reactivation. Using chromatin immunoprecipitation-Deep Sequencing (ChIP-Seq), we identified Rta, RBP-Jk and NFkB p50 binding sites on the viral genome during KSHV latency and reactivation. Although previous work suggested that NFkB represses lytic reactivation by inhibiting Rta/RBP-Jk interactions, we found that p50 and Rta/RBP-Jk did not bind to the genome in a mutually-exclusive fashion. We devised a new classification system for KSHV genes using as a reference RBP-Jk DNA binding during latency and reactivation. However, we found incomplete concordance between this classification scheme and results of transient reporter gene transactivation by Rta and RBP-Jk. In fact, these data supported previous reports from our lab and others in which the canonical view of RBP-Jk isoform 1 constitutively bound to DNA is not always sufficient for RBP-JK-dependent promoter specification.
Based on our ChIP/Seq data, we hypothesized that exceptions to this mechanism might be explained by expression of non-canonical members of the Notch pathway in infected cells. The RBP-Jk gene contains fourteen exons that have been annotated to alternatively splice into more than four isoforms. There are no published roles for these isoforms in the Notch signaling pathway or in host-virus interactions that regulate KSHV reactivation. We identified many new splice variants, and cloned two new RBP-Jk cDNAs from PEL infected cells. We determined that these non-canonical RBP-Jk isoforms have differential DNA binding properties. Our qRT-PCR experiments demonstrated that the relative expression of different RBP-Jk cDNAs varies quantitatively and qualitatively in different cell types that KSHV is known to infect, suggesting new mechanisms that govern viral reactivation. Protein-protein interaction studies showed that binding of Rta to RBP-Jk isoforms 2, 3 and 4 is sufficient to rescue Rta transactivation in RBP-Jk null B cells, but in a promoter specific fashion. Ectopic expression of RBP-Jk isoforms 2 and 4 inhibits viral reactivation in a dominant negative fashion. In this thesis, we show a novel mechanism for KSHV gene regulation. Our approaches demonstrate the strength of the KSHV model system for revealing mechanisms that regulate RBP-Jk-dependent promoter specification and their biologic consequences. These mechanisms have the potential to also affect the Notch signaling pathway.