Genome wide analysis of p53 inhibition by high glucose

描述(申请人提供)：肿瘤抑制基因P53在癌症和许多人类生物学中扮演着重要的角色。P53具有许多功能，包括在修复和重组中的作用，与参与基因组稳定的蛋白质的联系，以及染色质修饰。然而，它最广泛的细胞效应是转录因子(Tf)。作为一种转铁蛋白，P53受到包括DNA损伤在内的多种细胞胁迫的翻译后修饰的调控。我们的实验室最近发现TAF1在p21启动子上的Thr55处磷酸化P53，这种磷酸化导致P53与启动子的解离，并在DNA损伤后终止p21转录。TAF1是转录因子TFIID的最大亚基，是细胞周期调节蛋白，对细胞周期通过G1期起重要作用。有趣的是，我们还发现TAF1以细胞内ATP水平依赖的方式磷酸化P53。由于高糖条件(HG)可能会增加细胞内的ATP水平，我们推测它可能会增强Thr55的磷酸化，导致DNA损伤时蛋白质的失活。为了验证这一假设，我们确实证明了在HG条件下DNA损伤时p21转录减少。我们的发现提出了基本的问题，即HG在多大程度上有助于抑制基因组范围内对DNA损伤的P53激活。因此，这项研究将集中于鉴定和探索HG对DNA损伤调控的p53靶基因的全基因组影响，并探索这种调控发生的分子途径。我们的长期目标是建立一种分子机制，通过这种机制，糖尿病患者中常见的高血糖可以间接促进恶性转化。我们建议通过以下三个目标开展这一研究计划：(1)通过RNA测序确定HG对全基因组DNA损伤的影响。(2)通过芯片测序，在全球范围内识别受HG影响的P53结合启动子。(3)检测依赖于ATP的TAF1磷酸化作为抑制P53活性的机制的普遍性。

DESCRIPTION (provided by applicant): The tumor suppressor p53 plays a prominent role in cancer and much of human biology. Many functions have been attributed to p53, including roles in repair and recombination, association with proteins involved in genome stability, and chromatin modification. However, its broadest cellular effect is that of a transcription factor (TF. As a TF, p53 is regulated through multiple post translational modifications by a variety of cellula stresses including DNA damage. Our laboratory recently uncovered that TAF1 phosphorylates p53 at Thr55 on the p21 promoter and this phosphorylation leads to dissociation of p53 from the promoter and termination of p21 transcription after DNA damage. TAF1 is the largest subunit of transcription factor TFIID and a cell cycle regulatory protein important for progression through the G1 phase. Interestingly, we also uncovered that TAF1 phosphorylates p53 in a cellular ATP level-dependent manner. Because high glucose conditions (HG) can potentially increase cellular ATP levels, we hypothesize that it may enhance Thr55 phosphorylation, leading to inactivation of the protein upon DNA damage. To test this hypothesis, we show indeed that p21 transcription is reduced upon DNA damage under HG conditions. Our findings raised fundamental questions as to what extent HG contributes to inhibition of p53 activation genome wide in response to DNA damage. This research will thus focus on identifying and exploring the genome wide effect of HG on p53-target genes regulated by DNA damage and exploring the molecular pathway through which this regulation occurs. Our long term goal is to establish a molecular mechanism by which hyperglycemia, commonly found in diabetics, can indirectly contribute towards malignant transformation. We propose to carry out this research project through the following three aims: (1) Identify p53 target genes that are affected by HG in response to DNA damage genome wide by RNA-sequencing. (2) Globally identify p53 bound promoters that are affected by HG in response to DNA damage by ChIP-sequencing. (3) Test the generality of ATP-dependent TAF1 phosphorylation as a mechanism for inhibition of p53 activity.