Role of IFI16 in Cellular Senescence

IFI16 在细胞衰老中的作用

• 批准号: 7438836
• 负责人: DIVAKER CHOUBEY
• 金额: $ 23.65万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7438836
• 关键词: 1q22; Affect; Binding; Biochemical Genetics; Biological Assay; CDKN1A gene; Cell Aging; Cell Cycle Arrest; Cells; Dimerization; Diploidy; Down-Regulation; E2F1 gene; Ectopic Expression; Epithelial Cells; Fibroblasts; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Growth; Heterochromatin; Human; Human Cell Line; Hypoxia; Ionizing radiation; Length; Mediating; Molecular; Oncogenes; Pathway interactions; Post-Translational Protein Processing; Protein Binding; Protein Family; Proteins; Radiation Induced DNA Damage; Regulation; Role; Serum; Stress; TERT gene; TP53 gene; Telomerase; Telomerase inhibition; Testing; Transcriptional Activation; Up-Regulation; base; c-myc Genes; cell growth; cell growth regulation; chromatin immunoprecipitation; cobaltous chloride; design; exhaust; in vivo; keratinocyte; mutant; novel; oncoprotein p21; promoter; research study; senescence; telomere; transcription factor

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.

细胞衰老是指细胞在耗尽其细胞周期后所显示的不可逆的细胞周期停滞。 复制潜力尽管p53、Rb和端粒的磨损与细胞凋亡的调控有关， 衰老，分子机制，有助于不可逆的细胞周期停滞，其特征在于不同的 基因表达的变化仍有待阐明。这些拟议研究的主要目标是了解 IFI 16蛋白（由位于1 q22的/F/76基因编码）是一种可诱导的蛋白， 转录调节因子，有助于细胞衰老相关的细胞周期停滞。 我们使用人上皮细胞和人二倍体成纤维细胞（HDFs）的实验揭示了以前的 IFI 16在细胞衰老调节中的作用未知。然而，其分子机制仍不清楚。 基于我们的初步和其他观察，我们假设IFI 16有助于细胞衰老- 通过与转录因子如p53、Rb-E2 F和c-Myc相互作用，以及通过调节细胞周期， 它们的转录活性。以下三个具体目标旨在验证我们的假设：目标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p21基因的表达。 HDFs的增殖潜力。我们建议：（i）确定是否衰老相关的翻译后 IFI 16和p53的修饰影响它们的物理相互作用;（ii）确定IFI 16 增加p53介导的p21基因的转录激活;和（iii）比较IFI 16对细胞生长的抑制 在p53（p53+/+对p53 +/-）或p21 CIR 1（p21+/+对p21+/-）表达不同的同基因HDF之间。遗传 和生物化学方法，包括染色质免疫沉淀试验（ChlPs），将被使用。目标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。我们建议 确定：（i）IFI 16是否以LxCxE基序依赖性方式与Rb口袋结合，以及口袋突变体是否 （ii）IFI 16与E2 F家族蛋白的结合抑制了E2 F的二聚化 与DP家族蛋白和E2 F介导的转录;和（iii）IFI 16增强Rb介导的异染色质 E2 F靶基因如hTERT的形成和沉默。目标#3：通过以下方法阐明分子机制： 其中IFI 16负调控hTERT基因的转录。我们将确定是否：（i）IFI 16的敲除 HDFs中的表达增加hTERT表达和端粒酶活性;（ii）IFI 16与c-Myc的结合抑制hTERT表达和端粒酶活性。 c-Myc与其配偶体Max的二聚化和c-Myc介导的转录;（iii）c-Myc与其配偶体Max的二聚化和c-Myc介导的转录的增加或减少。 IFI 16表达对c-Myc与hTERT启动子结合及c-Myc介导的hTERT转录的影响 基因我们提出的实验的意义在于，它们将确定IFI 16 调节衰老相关基因的转录，并促进衰老相关细胞周期 逮捕了