Werner syndrome is an autosomal recessive disorder associated with pre-mature aging and cancer predisposition caused by mutations of the WRN gene. Sir2, an NAD-dependent histone deacetylase has been proved to extend life span in yeast and C. elegans. Mammalian Sir2 (SIRT1) has also been found regulating premature cellular senescence induced by the tumor suppressors PML and p53. Recent studies also indicate that SIRT1 plays an important role in calorie restriction promoted mammalian cell survival. The specific hypothesis behind the proposed research is that SIRT1 regulates WRN function through deacetylation. This hypothesis is based on the following observations. First, our preliminary studies demonstrated that WRN can be acetylated both in vitro and in vivo. Acetylated WRN translocates from nucleolar to nucleoplasm. Second, treatment of cells with histone deacetylase inhibitors can strongly increase WRN acetylation level in vivo. Third, WRN can interact with SIRT1 both in vitro and in vivo and SIRT1 can deacetylate WRN in cells indicating WRN is a novel target for SIRT1 deacetylation. The interaction of these two aging-related protein, SIRT1 and WRN, suggests they may work together to regulate cellular senescence and aging. Based on these observations, the overall objective of this application is to demonstrate the precise role of SIRT1 in regulation of cellular senescence through WRN deacetylation, and elucidate the molecular mechanism of this WRN acetylation and deacetylation pathway in response to DNA damage. To achieve this goal, the following specific aims are proposed: 1. Demonstrate WRN acetylation in vivo and its effect on the activities of WRN. We will (i) Study the cellular localization, steady-state levels of acetylated WRN protein before and after DNA damage. (ii). Investigate the WRN acetylation status in response to various DNA damage agents. (iii). Test the effect of WRN acetylation on its helicase activity and exonuclease activity.2. Elucidate the role of SIRT1 in the regulation of WRN function. We will (i) Map the physical interactions between SIRT1 and WRN both in vitro and in vivo, and identify binding-defective mutants. (ii) Investigate the regulation of WRN helicase activity by SIRT1 deacetylation in presence and absence of DNA damage. (iii) Test the role of SIRT1 and WRN interaction in response to DNA damage. 3. Investigate the role of WRN-SIRT1 pathway in telomere function and cellular senescence. We will (i) Examine the role of WRN-SIRT1 interaction in telomere function. (ii)Test the role of WRN acetylation and deacetylation pathway in cellular senescence. (iii). Test the role of SIRT1 and WRN interaction in response to DNA damage. ? ? ? ?
