Role of IFI16 in Cellular Senescence

细胞衰老是指细胞耗尽后表现出的不可逆的细胞周期停滞 复制潜力。尽管P53、Rb和端粒磨损与细胞周期调控有关 衰老，导致不可逆的细胞周期停滞的分子机制，其特征是 基因表达的变化仍有待阐明。这些拟议研究的主要目的是了解 IFI16蛋白(由位于1q22的/F/76基因编码)作为诱导剂的分子机制 转录调节因子，参与细胞衰老相关的细胞周期停滞。 我们用人类上皮细胞和人类二倍体成纤维细胞(HDFS)进行的实验揭示了以前 IFI16在调节细胞衰老中的作用未知。然而，其分子机制仍不清楚。 根据我们的初步观察和其他观察，我们假设IFI16有助于细胞衰老- 通过与转录因子如P53、Rb-E2F和c-Myc相互作用以及通过调节 它们的转录活动。以下三个具体目标旨在检验我们的假设：目标1： 确定IFI16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 高密度脂蛋白纤维的增殖潜能。我们建议：(I)确定衰老与翻译后相关 IFI16和P53的修饰影响它们的物理相互作用；(Ii)确定IFI16的分子机制 增加p53介导的p21基因的转录激活；以及(Iii)比较IFI16对细胞生长的抑制 P5 3+/+与p5 3“‘~)或p2 1CIR1(p2 1+/+与p2 1”A)表达不同的等位基因HDF之间。遗传 并将使用包括染色质免疫沉淀分析(Chlps)在内的生化方法。目标2：达到 确定IFI16与Rb和E2F的相互作用是否增强了E2F靶基因的沉默。我们建议 确定：(I)IFI16是否以LxCxE依赖的方式与RB Pocket结合，以及Pocket突变体是否 与IFI16结合有缺陷；(Ii)IFI16与E2F-家族蛋白的结合抑制E2F的二聚化 与DP蛋白家族和E2F介导的转录有关；以及(Iii)IFI16增强RB介导的异染色质 E2F靶基因的形成和沉默，如hTERT。目的#3：阐明分子机制 其中IFI16负向调控hTERT基因的转录。我们将确定是否：(I)击倒IFI16 HDFS表达增加hTERT表达和端粒酶活性；(Ii)IFI16与c-Myc结合抑制 C-Myc与其伴侣Max的二聚化和c-Myc介导的转录；(Iii)增加或减少 在v/vov和c-Myc介导的hTERT转录中，IFI16的表达影响c-Myc与hTERT启动子结合 吉恩。我们提出的实验的意义在于，它们将确定IFI16 调节衰老相关基因的转录，参与衰老相关的细胞周期 逮捕。

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 /F/76gene 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 p21CIR1 (p21+/+ versus p21"A). 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 v/Voand 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.