Figure 3

PTOV1 interacts with RBP-Jκ and the Notch repressor complex at the HEY1 promoter and its repressive function requires HDACs activity and it is restrained by CBP. (A) PTOV1 interacts with RBP-Jκ. PC-3 cells, transfected with FLAG-RBP-Jκ and HA-PTOV1, were co-transfected with ICN or treated with DAPT for 4 days. Cell lysates were immunoprecipitated with FLAG antibody or control IgG and blots were revealed with anti-HA antibody. (B) Endogenous PTOV1 occupies the endogenous HEY1 promoter under conditions of Notch signaling inhibition. PC-3 cells, transfected with FLAG-RBP-Jκ, were treated with DAPT (left) or co-transfected with ICN (right). Chromatin was immunoprecipitated with antibodies to PTOV1, FLAG, Notch1, or non-specific IgGs. Associated DNA fragments were analyzed by PCR with primers specific for HEY1 promoter regions. Primers from intragenic regions of the HES1 gene were used as a specificity control. (C) PTOV1 interacts with the Notch repressor complex. FLAG-HDAC1, FLAG-HDAC4, FLAG-NCoR and FLAG-RBP-Jκ were separately transfected into PC-3 cells and tested for interaction with GST-PTOV1 (left panel) or GST-A domain, GST-B domain (right panel) or control GST beads. Bound proteins were analyzed by Western blotting with anti-FLAG. (D) PTOV1 repressor activity requires HDACs activity. Cells, transfected with HES1-luciferase, Renilla and the plasmids indicated, were treated with the HDAC inhibitor trichostatin A (TSA) for 48 h. Lysates were used in transactivation assays for firefly luciferase activity (E) Overexpression of the CBP acetyl-transferase, but not p300, overcomes the repressor activity of PTOV1 on the HES1 promoter. Cells, transfected with p300 or CBP plus the indicated plasmids, were used in HES1-driven luciferase transactivation assays. ( F ) PTOV1 interacts with CBP but not with p300. Lysates from cells transfected with FLAG -PTOV1 and HA-CBP (top), or HA-p300 (bottom), were immunoprecipitated with antibody to HA, or control IgG, and analyzed by Western blotting.