Genome wide analysis of p53 inhibition by high glucose

描述（由申请人提供）：肿瘤抑制因子p53在癌症和许多人类生物学中起着重要作用。p53具有许多功能，包括修复和重组，与基因组稳定性相关的蛋白质以及染色质修饰。然而，其最广泛的细胞效应是转录因子（TF）的效应。作为一种TF，p53通过多种翻译后修饰受到包括DNA损伤在内的多种细胞应激的调节。我们的实验室最近发现，TAF1磷酸化p53在Thr55上的p21启动子和这种磷酸化导致p53从启动子和DNA损伤后终止p21转录的解离。TAF1是转录因子TFIID的最大亚基，也是一种对G1期进展很重要的细胞周期调节蛋白。有趣的是，我们还发现TAF1以细胞ATP水平依赖性方式磷酸化p53。由于高糖条件（HG）可能会增加细胞ATP水平，我们假设它可能会增强Thr55磷酸化，导致DNA损伤后蛋白质失活。为了验证这一假设，我们确实表明，在HG条件下DNA损伤后，p21转录减少。我们的研究结果提出了一个根本性的问题，即在何种程度上HG有助于抑制p53激活基因组范围内的DNA损伤。因此，本研究将集中于鉴定和探索HG对DNA损伤调控的p53靶基因的全基因组效应，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症可以间接导致恶性转化。本研究拟从以下三个方面开展：（1）通过RNA测序技术，在全基因组范围内鉴定HG对DNA损伤反应的p53靶基因。(2)通过ChIP测序，全面鉴定在DNA损伤反应中受HG影响的p53结合启动子。(3)测试ATP依赖性TAF1磷酸化作为抑制p53活性机制的一般性。

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.