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. ? ? ? ? ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
1R01AG025036-01A2
Application #
7091879
Study Section
Cellular Mechanisms in Aging and Development Study Section (CMAD)
Program Officer
Sierra, Felipe
Project Start
2006-05-01
Project End
2010-04-30
Budget Start
2006-05-01
Budget End
2007-04-30
Support Year
1
Fiscal Year
2006
Total Cost
$243,540
Indirect Cost
Name
Loyola University Chicago
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
791277940
City
Maywood
State
IL
Country
United States
Zip Code
60153
Veeranki, Sudhakar; Choubey, Divaker (2012) Interferon-inducible p200-family protein IFI16, an innate immune sensor for cytosolic and nuclear double-stranded DNA: regulation of subcellular localization. Mol Immunol 49:567-71
Choubey, Divaker (2012) DNA-responsive inflammasomes and their regulators in autoimmunity. Clin Immunol 142:223-31
Veeranki, Sudhakar; Duan, Xin; Panchanathan, Ravichandran et al. (2011) IFI16 protein mediates the anti-inflammatory actions of the type-I interferons through suppression of activation of caspase-1 by inflammasomes. PLoS One 6:e27040
Duan, Xin; Ponomareva, Larissa; Veeranki, Sudhakar et al. (2011) Differential roles for the interferon-inducible IFI16 and AIM2 innate immune sensors for cytosolic DNA in cellular senescence of human fibroblasts. Mol Cancer Res 9:589-602
Duan, Xin; Ponomareva, Larissa; Veeranki, Sudhakar et al. (2011) IFI16 induction by glucose restriction in human fibroblasts contributes to autophagy through activation of the ATM/AMPK/p53 pathway. PLoS One 6:e19532
Choubey, Divaker; Panchanathan, Ravichandran; Duan, Xin et al. (2011) Emerging roles for the interferon-inducible p200-family proteins in sex bias in systemic lupus erythematosus. J Interferon Cytokine Res 31:893-906
Song, Lynda Li; Ponomareva, Larissa; Shen, Hui et al. (2010) Interferon-inducible IFI16, a negative regulator of cell growth, down-regulates expression of human telomerase reverse transcriptase (hTERT) gene. PLoS One 5:e8569
Choubey, Divaker; Duan, Xin; Dickerson, Eric et al. (2010) Interferon-inducible p200-family proteins as novel sensors of cytoplasmic DNA: role in inflammation and autoimmunity. J Interferon Cytokine Res 30:371-80
Veeranki, Sudhakar; Choubey, Divaker (2010) Systemic lupus erythematosus and increased risk to develop B cell malignancies: role of the p200-family proteins. Immunol Lett 133:1-5
Choubey, Divaker; Deka, Ranjan; Ho, Shuk-mei (2008) Interferon-inducible IFI16 protein in human cancers and autoimmune diseases. Front Biosci 13:598-608

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