Role of IFI16 in Cellular Senescence

描述（由申请人提供）：细胞衰老是指已耗尽其复制潜力的细胞所显示的不可逆细胞周期停滞。虽然p53，Rb和端粒磨损已被牵连在细胞衰老的调节，分子机制有助于不可逆的细胞周期停滞，其特征在于在基因表达的明显变化仍有待阐明。这些拟议的研究的主要目的是了解IFI 16蛋白（由位于1 q22的IFI 16基因编码），一种诱导型转录调节因子，有助于细胞衰老相关的细胞周期阻滞的分子机制。我们使用人上皮细胞和人二倍体成纤维细胞（HDF）的实验揭示了IFI 16在细胞衰老调节中的先前未知的作用。然而，其分子机制仍不清楚。基于我们的初步观察和其他观察，我们假设IFI 16通过与转录因子如p53、Rb-E2 F和c-Myc相互作用并通过调节它们的转录活性来促进细胞衰老相关的细胞周期停滞。以下三个具体目的旨在验证我们的假设：目的#1：确定IFI 16和p53之间的相互作用是否有助于p21基因的转录激活并限制HDF的增殖潜力。我们建议：（i）确定IFI 16和p53的衰老相关的翻译后修饰是否影响它们的物理相互作用;（ii）鉴定IFI 16增加p53介导的p21基因转录激活的分子机制;和（iii）比较p53表达不同的同基因HDF之间IFI 16对细胞生长的抑制（p53+/+对比p53-/-）或p21 CIP 1（p21+/+对比p21-/-）。将使用遗传和生物化学方法，包括染色质免疫沉淀试验（ChlPs）。目的#2：确定IFI 16与Rb和E2 F的相互作用是否增强E2 F靶基因的沉默。我们拟确定：（i）IFI 16是否以LxCxE基序依赖的方式与Rb口袋结合，以及Rb口袋突变体是否与IFI 16结合缺陷;（ii）IFI 16与E2 F家族蛋白的结合是否抑制E2 F与DP家族蛋白的二聚化和E2 F介导的转录;和（iii）IFI 16增强Rb介导的异染色质形成和E2 F靶基因如hTERT的沉默。目的3：阐明IFI 16负调控hTERT基因转录的分子机制。我们将确定：（i）在HDF中敲低IFI 16表达增加hTERT表达和端粒酶活性;（ii）IFI 16与c-Myc的结合抑制c-Myc与其配偶体Max的二聚化和c-Myc介导的转录;（iii）IFI 16表达的增加或减少影响c-Myc与hTERT启动子的体内结合和c-Myc介导的hTERT基因的转录。我们提出的实验的意义在于，它们将确定IFI 16调节衰老相关基因转录的分子机制，并有助于衰老相关的细胞周期停滞。

DESCRIPTION (provided by applicant): Cellular senescence refers to the irreversible cell cycle arrest displayed by cells that have exhausted their replicative potential. Although the p53, Rb, and telomere attrition have been implicated in the regulation of cellular senescence, molecular mechanisms contributing to irreversible cell cycle arrest that is characterized by distinct changes in gene expression remain to be elucidated. The main objective of these proposed studies is to understand the molecular mechanisms by which IFI16 protein (encoded by the IFI16 gene located at 1q22), an inducible transcriptional regulator, contributes to cellular senescence-associated cell cycle arrest. Our experiments using human epithelial cells and human diploid fibroblasts (HDFs) have revealed a previously unknown role for IFI16 in the regulation of cellular senescence. However, the molecular mechanisms remain unknown. Based on our preliminary and other observations, we hypothesize that IFI16 contributes to cellular senescence- associated cell cycle arrest by interacting with transcription factors, such as p53, Rb-E2F, and c-Myc, and by regulating their transcriptional activities. The following three specific aims are designed to test our hypothesis: Aim #1: To determine whether interactions between IFI16 and p53 contribute to transcriptional activation of p21 gene and limit the proliferative potential of HDFs. We propose to: (i) determine whether senescence-associated posttranslational modifications of IFI16 and p53 affect their physical interactions; (ii) identify the molecular mechanisms by which IFI16 increases p53-mediated transcriptional activation of p21 gene; and (iii) compare inhibition of cell growth by IFI16 between isogenic HDFs differing in the expression of p53 (p53+/+ versus p53-/-) or p21CIP1 (p21+/+ versus p21-/-). Genetic and biochemical approaches, including chromatin immunoprecipitation assays (ChlPs), will be used. Aim# 2: To determine whether interactions of IFI16 with Rb and E2F potentiate silencing of the E2F target genes. We propose to determine whether: (i) IFI16 binds to Rb pocket in an LxCxE-motif-dependent manner and whether the pocket mutants of Rb are defective in binding to IFI16; (ii) binding of IFI16 to the E2F-family of proteins inhibits dimerization of E2Fs with DP-family of proteins and E2F-mediated transcription; and (iii) IFI16 potentiates the Rb-mediated heterochromatin formation and silencing of the E2F target genes, such as hTERT. Aim# 3: To elucidate molecular mechanisms by which IFI16 negatively regulates the transcription of hTERT gene. We will determine whether: (i) knockdown of IFI16 expression in HDFs increases hTERT expression and the telomerase activity; (ii) binding of IFI16 to c-Myc inhibits dimerization of c-Myc with its partner Max and the c-Myc-mediated transcription; (iii) increases or decreases in the expression of IFI16 affect binding of c-Myc to hTERT promoter in vivo and c-Myc-mediated transcription of hTERT gene. The significance of our proposed experiments is that they will identify molecular mechanisms by which IFI16 regulates the transcription of senescence-associated genes and contributes to the senescence-associated cell cycle arrest.