Role of IFI16 in Cellular Senescence

描述(申请人提供)：细胞衰老是指细胞已经耗尽其复制潜力，表现出不可逆转的细胞周期停滞。虽然P53、Rb和端粒磨损参与了细胞衰老的调节，但以基因表达明显变化为特征的不可逆细胞周期停滞的分子机制仍未阐明。这些研究的主要目的是了解IFI16蛋白(由位于1q22的IFI16基因编码)，一种可诱导的转录调节因子，参与细胞衰老相关的细胞周期停滞的分子机制。我们用人上皮细胞和人二倍体成纤维细胞(HDFS)进行的实验揭示了IFI16在细胞衰老调节中的一个以前未知的角色。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IFI16通过与转录因子如p53、Rb-E2F和c-Myc相互作用并调节它们的转录活性而导致细胞衰老相关的细胞周期停滞。目的1：确定IFI16和P53之间的相互作用是否有助于p21基因的转录激活和限制HDFS的增殖潜力。我们建议：(I)确定衰老相关的IFI16和P53的翻译后修饰是否影响它们的物理相互作用；(Ii)确定IFI16增加P53介导的p21基因转录激活的分子机制；以及(Iii)比较IFI16对不同P53(P53+/+与P53-/-)或p21CIP1(p21+/+与p21-/-)表达差异的同源HDF对细胞生长的抑制作用。将使用遗传和生化方法，包括染色质免疫沉淀分析(Chlps)。目的#2：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基序依赖的方式与RB Pocket结合，以及Rb的Pocket突变体是否存在与IFI16结合的缺陷；(Ii)IFI16与E2F-家族蛋白的结合抑制了E2F与DP-家族蛋白的二聚化和E2F介导的转录；以及(Iii)IFI16增强了RB介导的异染色质形成和E2F靶基因的沉默，如hTERT。目的#3：阐明IFI16负性调控hTERT基因转录的分子机制。我们将确定：(I)IFI16在HDFS中的表达下调是否增加了hTERT的表达和端粒酶活性；(Ii)IFI16与c-Myc的结合是否抑制了c-Myc与其伴侣Max的二聚化和c-Myc介导的转录；(Iii)IFI16表达的增加或减少影响了c-Myc与hTERT启动子的结合和c-Myc介导的hTERT基因的转录。我们提出的实验的意义在于，他们将识别IFI16调控衰老相关基因转录并有助于衰老相关细胞周期停滞的分子机制。

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.