Regulation of p53 Transcription by Viral Oncoproteins & Covalent Modifications

描述(申请人提供)：P53是一种肿瘤抑制蛋白，其功能是细胞基因组的守护者。在正常生长条件下，由于蛋白质的快速周转，P53保持在较低的水平。当细胞受到压力时，P53变得稳定，并导致细胞周期停滞。这种短暂的阻断使细胞能够克服压力，并在必要时有效地修复DMA损伤。当损伤严重到无法恢复时，P53会诱导细胞凋亡。P53的双重功能在于它能够作为序列特异的转录因子，激活参与细胞周期控制和细胞凋亡的基因产物的转录。毫不奇怪，p53是DNA肿瘤病毒编码的癌蛋白的常见靶标，这些癌蛋白能够在功能上灭活P53，并阻止P53靶基因转录的激活。十多年来，这种对P53反式激活的抑制被归因于这些病毒癌蛋白对P53蛋白稳定性的影响。最近，我们发现了人类乳头瘤病毒(HPV)编码的E6癌蛋白通过抑制P53和核小体核心组蛋白在P53靶向的人p21基因上的乙酰化来灭活染色质结合的P53功能的新途径。为了了解HPVE6和其他DNA肿瘤病毒编码的癌蛋白所使用的抑制机制，我们提出了两个目的。1)鉴定与E6介导的抑制p53靶基因转录有关的细胞蛋白。我们发现P53和组蛋白乙酰转移酶(HAT)p300在E6介导的p21基因转录抑制中都是必不可少的。由于p300的自身乙酰化不受E6的抑制，我们推测，乙酰化的p300可能为募集其他细胞蛋白提供了一个蛋白质编码，进一步改变了p300和p53的功能，导致了p53靶基因上的染色质结构浓缩。这一假设将通过进行体外染色质转录、HAT分析、DNA/染色质结合分析以及体内芯片、RT-PCR、siRNA和报告基因检测来验证，以跟踪转录因子和辅助因子对P53调节基因的招募。此外，我们将通过分离和鉴定E6细胞复合体来识别参与这一抑制途径的其他细胞蛋白。还将进行无偏见的生化分级，以确定与E6抑制物功能有关的细胞因子。2)明确其他DNA肿瘤病毒编码的癌蛋白的抑制机制。我们将研究SV40和多瘤病毒大T抗原和腺病毒E1B-55K和E1B-19K蛋白是否也使用相似但不相同的机制来抑制p53靶基因的转录。总之，这些研究将确立DNA肿瘤病毒编码的癌蛋白所采用的这种新的抑制机制的一般原理，而不依赖于蛋白酶体介导的降解途径，并为开发药物抑制物来阻断这些人类病原体的传播提供了新的方向。

DESCRIPTION (provided by applicant): 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.