Mechanism of Human Papillomavirus E6-Mediated Repression of P53-Target Gene *Transcription

255 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006.The mechanism employed by DNA tumor viruses to inhibit p53-dependent transcription is poorly understood. Here we describe a new paradigm for human papillomavirus (HPV) E6 oncoprotein-mediated repression of human p21 WAF1 cyclin-dependent kinase inhibitor gene transcription, independent of its well known role to promote the ubiquitination and degradation of p53. Using both high-risk and low-risk HPV E6 oncoproteins, each representing a class of oncoproteins that is capable of inducing p53 degradation or unable to degrade p53, respectively, we investigated whether these DNA tumor viruses shared a general repression mechanism to inhibit p53-target gene transcription. We used UV-irradiated human fibrosarcoma HT1080 cells treated with proteasome inhibitors (MG132) and in vitro-assembled chromatin templates reconstituted with purified chromatin assembly factors (Acf1 and ISWI), core histones (H2A, H2B, H3, and H4), and a histone chaperone (NAP-1) in order to define the mechanism used by high-risk and low-risk HPV E6 to inhibit p53-dependent transcription. We demonstrate that E6 does not prevent p53 or p300 recruitment to the chromatin, but inhibits p300-mediated acetylation on p53 and nucleosomal core histones. This suppression of protein acetylation requires the E6-interacting regions of p300. Moreover, high-risk HPV E6 mutants unable to interact with p53 or p300, but not deficient in inducing p53 degradation, fail to inhibit p53-mediated activation in vitro and in vivo, indicating that a p53-E6-p300-containing protein complex is critical for repressing p53-target gene activation. It is likely that this mechanism for repressing p53 target gene transcription is common among DNA tumor viruses based on the finding that SV40 large T-antigen, polyomavirus large T-antigen, adenovirus E1B 19K, and human papillomavirus E6 encoded-oncoproteins inhibited p53-dependent chromatin transcription at a step prior to the entry of p300, indicating that oncoprotein incorporation into p53-p300 complex constitutes a major switch for virus-mediated gene regulation through modulation of p300-targeted acetylation on p53 and nucleosomal core histones.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD