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.
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