Figure 4. Prox1 regulates the expression of RTA by direct binding to its promoter.

(A) FAIRE assays were performed on K-LECs and K-BECs at 16 hours post-infection as described in Method. The accessibility of the indicated regions of K-LECs was normalized against the corresponding regions of K-BECs. (B) Luciferase assays showing the RTA promoter activity in LECs vs. BECs. Primary LECs and BECs were transfected with a control (CTR) or an RTA promoter-luciferase reporter vector (RTA-3.0K) (31), and their luciferase activity was normalization against total plasmid amount within the cells after 48 hours. (C) Luciferase assays showing Prox1-mediated activation of the RTA promoter. HEK293 cells were co-transfected with an activator vector (empty control vector, Prox1-expressing vectors (Prox1 WT), or DNA-binding defective mutant Prox1 (Prox1 MT (32)) and a RTA-promoter reporter construct (−3 Kb (RTA-3.0k), −1.9 Kb (RTA-1.9k), or −0.9 Kb (RTA-0.9k)) (31). (D) ChIP assay demonstrating Prox1 binding to the RTA promoter in LECs. Primary LECs were infected with KSHV ^GFP for 1 day and subjected to ChIP assays using IgG or anti-Prox1 antibody. The location of the PCR primer sets are listed as −2044, −1264, −310, and 0, counting from the translation start codon of RTA. Consistent results were obtained from three independent experiments for each panel. Statistics, 2-tailed t-test, *, p <0.05; **, p <0.01; ***, p <0.001.