THE ROLE OF p/CIP ONCOGENE IN MOUSE EMBRYONIC STEM CELL PLURIPOTENCY

p/CIP is a transcriptional coactivator that binds liganded nuclear hormone receptors, as well as other transcription factors, and facilitates transcription through direct recruitment of accessory factors. p/CIP is highly expressed in undifferentiated mouse embryonic stem (mES) cells and is downregulated during differentiation. Using siRNA- mediated knockdown of p/CIP in mES cells in combination with microarray analysis I have identified a contingent of essential pluripotency genes which are significantly downregulated including Klf4, Tbx3 and Dax-1. Subsequent chromatin immuno­ précipitation (ChIP) analysis demonstrated that Tbx3 and Dax-1 are direct transcriptional targets of p/CIP. Using the piggyBac transposase system, a mouse ES cell line that expresses Flag-p/CIP in a doxycycline-dependent manner was generated. p/CIP overexpression increased pluripotency gene expression and promoted the formation of undifferentiated ES cell colonies. Collectively, these results indicate that p/CIP contributes to the maintenance of ES cell pluripotency through direct, as well as indirect, regulation o f essential pluripotency genes

The human papillomavirus E7 proteins associate with p190RhoGAP and alter its function

Using mass spectrometry, we identified p190RhoGAP (p190) as a binding partner of human papillomavirus 16 (HPV16) E7. p190 belongs to the GTPase activating protein (GAP) family and is one of the primary GAPs for RhoA. GAPs stimulate the intrinsic GTPase activity of the Rho proteins, leading to Rho inactivation and influencing numerous biological processes. RhoA is one of the best-characterized Rho proteins and is specifically involved in formation of focal adhesions and stress fibers, thereby regulating cell migration and cell spreading. Since this is the first report that E7 associates with p190, we carried out detailed interaction studies. We show that E7 proteins from other HPV types also bind p190. Furthermore, we found that conserved region 3 (CR3) of E7 and the middle domain of p190 are important for this interaction. More specifically, we identified two residues in CR3 of E7 that are necessary for p190 binding and used mutants of E7 with mutations of these residues to determine the biological consequences of the E7-p190 interaction. Our data suggest that the interaction of E7 with p190 dysregulates this GAP and alters the actin cytoskeleton. We also found that this interaction negatively regulates cell spreading on a fibronectin substrate and therefore likely contributes to important aspects of the HPV life cycle or HPV-induced tumorigenesis. © 2014, American Society for Microbiology