p53 is a tumor suppressor protein that functions as a cellular genome guardian. Under normal growth conditions, p53 is kept at low levels due to a fast protein turnover rate. When cells are stressed, p53 becomes stabilized and leads to cell cycle arrest. This transient block allows cells to overcome the stress and efficiently repair DMA lesions, if necessary. When the damage is too severe to be restored, p53 induces apoptosis. The dual function of p53 lies in its ability to act as a sequence-specific transcription factor that activates transcription of gene products involved in cell cycle control and apoptosis. Not surprisingly, p53 is a frequent target for DNA tumor virus-encoded oncoproteins that are able to functionally inactivate p53 and block the activation of p53 target gene transcription. For over a decade, this inhibition of p53 transactivation has been attributed to the effect of these viral oncoproteins on p53 protein stability. Recently, we have uncovered a new pathway for human papillomavirus (HPV)-encoded E6 oncoprotein to inactivate the function of chromatin-bound p53 by inhibiting acetylation of p53 and nucleosomal core histones on the p53-targeted human p21 gene. To understand the repression mechanisms employed by HPV E6 and other DNA tumor virus-encoded oncoproteins, we propose two aims. 1) To identify cellular proteins involved in E6-mediated repression of p53 target gene transcription. We found that both p53 and histone acetyltransferase (HAT) p300 are essential for E6-mediated repression of p21 gene transcription. Since p300 autoacetylation is not inhibited by E6, we hypothesize that acetylated p300 may provide a protein code for recruitment of other cellular proteins to further modify the function of p300 and p53, resulting in a condensed chromatin structure on p53 target genes. This hypothesis will be tested by performing in vitro chromatin transcription, HAT assays, DNA/chromatin-binding assays as well as in vivo ChIP, RT-PCR, siRNA and reporter gene assays to follow the recruitment of transcription factors and cofactors to p53-regulated genes. Moreover, we will identify additional cellular proteins involved in this repression pathway by isolating and characterizing E6 cellular complexes. An unbiased biochemical fractionation will also be conducted to identify cellular factors involved in E6 repressor function. 2) To define the repression mechanism used bv other DNA tumor virus-encoded oncoproteins. We will examine whether SV40 and polyomavirus large T-antigens and adenovirus E1B-55K and E1B-19K proteins also employ a similar but non-identical mechanism to repress p53 target gene transcription. Collectively, these studies will establish a general principle of this novel repression mechanism employed by DNA tumor virus- encoded oncoproteins, independently of the proteasome-mediated degradation pathway, and provide a new direction for the development of drug inhibitors to block the propagation of these human pathogens.
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