Genome wide analysis of p53 inhibition by high glucose

描述（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突出的作用。许多功能都归因于p53，包括修复和重组，与基因组稳定性和染色质修饰相关的蛋白质的作用。然而，其最广泛的细胞效应是转录因子（TF）。作为TF， p53通过多种细胞应激（包括DNA损伤）介导多种翻译后修饰。我们的实验室最近发现，TAF1磷酸化p53在p21启动子上的Thr55位点，这种磷酸化导致p53与启动子分离，并在DNA损伤后终止p21的转录。TAF1是转录因子TFIID的最大亚基，也是一种细胞周期调节蛋白，对G1期的进展很重要。有趣的是，我们还发现TAF1以细胞ATP水平依赖的方式磷酸化p53。由于高糖条件（HG）可以潜在地增加细胞ATP水平，我们假设它可能会增强Thr55磷酸化，导致DNA损伤时蛋白质失活。为了验证这一假设，我们证明在HG条件下p21的转录确实在DNA损伤时减少。我们的发现提出了一个根本性的问题，即在DNA损伤的反应中，HG在多大程度上抑制了p53的全基因组激活。因此，本研究将侧重于鉴定和探索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个分子机制，通过高血糖，常见于糖尿病患者，可以间接促进恶性转化。我们拟通过以下三个目的开展本研究项目：(1)通过rna测序，在全基因组范围内确定受HG影响的p53靶基因，以响应DNA损伤。(2)通过ChIP-sequencing在全球范围内鉴定出受HG影响的p53结合启动子对DNA损伤的响应。(3)测试atp依赖性TAF1磷酸化作为抑制p53活性机制的普遍性。

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.