Regulation of WRN protein cellular localization and enzymatic activities by SIRT1-mediated deacetylation

Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations of the WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Sir2, an NAD-dependent histone deacetylase, has been proven to extend life span in yeast and Caenorhabditis elegans. Mammalian Sir2 (SIRT1) has also been found to regulate premature cellular senescence induced by the tumor suppressors PML and p53. SIRT1 plays an important role in cell survival promoted by calorie restriction. Here we show that SIRT1 interacts with WRN both in vitro and in vivo; this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, and SIRT1 can deacetylate WRN both in vitro and in vivo. WRN acetylation decreases its helicase and exonuclease activities, and SIRT1 can reverse this effect. WRN acetylation alters its nuclear distribution. Down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that SIRT1 regulates WRN-mediated cellular responses to DNA damage through deacetylation of WRN.     

SIRT1 regulates the function of the Nijmegen breakage syndrome protein

MRE11-RAD50-NBS1 (MRN) is a conserved nuclease complex that exhibits properties of a DNA damage sensor and is critical in regulating cellular responses to DNA double-strand breaks. NBS1, which is mutated in the human genetic disease Nijmegen breakage syndrome, serves as the regulatory subunit of MRN. Phosphorylation of NBS1 by the ATM kinase is necessary for both activation of the S phase checkpoint and for efficient DNA damage repair response. Here, we report that NBS1 is an acetylated protein and that the acetylation level is tightly regulated by the SIRT1 deacetylase. SIRT1 associates with the MRN complex and, importantly, maintains NBS1 in a hypoacetylated state, which is required for ionizing radiation-induced NBS1 Ser343 phosphorylation. Our results demonstrate the presence of crosstalk between two different posttranslational modifications in NBS1 and strongly suggest that deacetylation of NBS1 by SIRT1 plays a key role in the dynamic regulation of the DNA damage response and in the maintenance of genomic stability.     

Post-translational Modification in Control of SIRT1 Stability during DNA Damage Response

SIRT1 (silent mating type information regulation 2 homolog 1), a class III histone deacetylase, is known to participate in multiple steps of the DNA damage response (DDR) by deacetylating several key DDR proteins. At present, the mechanisms regulating SIRT1 protein stability upon DNA damage have yet to be fully elucidated. In this study, we reveal that, under severe DNA damage, SIRT1 undergoes two forms of post-translational modifications (PTMs): (i) increased polyubiquitination and proteasomal degradation mediated by TRIM28 (tripartite motif-containing protein 28), a RING-domain E3 ligase; and (ii) cleavage at C-terminal mediated by caspases. Importantly, there is reciprocal effects between these forms of PTMs: while suppression of proteasome reduces caspases-mediated cleavage, the cleaved SIRT1 has enhanced interaction with TRIM28, thus facilitating the ubiquitination and proteasomal degradation of SIRT1. Functionally, SIRT1 works as an anti-apoptotic protein in DDR, and the above-mentioned PTMs of SIRT1 subsequently enhances cell death induced by DNA damage agents. Thus, our study has uncovered a pivotal role of SIRT1 post-translational regulation in determining cell fate in DDR.     

A deacetylase-deficient SIRT1 variant opposes full-length SIRT1 in regulating tumor suppressor p53 and governs expression of cancer-related genes

SIRT1 is an NAD-dependent deacetylase and epigenetic regulator essential for normal mammalian development and homeostasis. Here we describe a human SIRT1 splice variant, designated SIRT1-Δ2/9, in which the deacetylase coding sequence is lost due to splicing between exons 2 and 9. This work aimed to determine if SIRT1-Δ2/9 is a novel functional product of the SIRT1 gene. Endogenous SIRT1-Δ2/9 protein was identified in human cell lysate by immunoblotting and splice variant-specific RNA interference (RNAi). SIRT1-Δ2/9 mRNA is bound by CUGBP2, which downregulates its translation. Using pulldown assays, we demonstrate that SIRT1-Δ2/9 binds p53 protein. SIRT1-Δ2/9 maintains basal p53 protein levels and supports p53 function in response to DNA damage, as evidenced by RNAi-mediated depletion of SIRT1-Δ2/9 prior to damage. In turn, basal p53 downregulates SIRT1-Δ2/9 RNA levels, while stress-activated p53 eliminates SIRT1-Δ2/9. Loss of wild-type (wt) p53 has been correlated with overexpression of SIRT1-Δ2/9 in a range of human cancers. Exogenous SIRT1-Δ2/9 protein associates with specific promoters in chromatin and can regulate cancer-related gene expression, as evidenced by chromatin immunoprecipitation analysis and RNAi/genomic array data. SIRT1 is of major therapeutic importance, and potential therapeutic drugs are screened against SIRT1 deacetylase activity. Our discovery of SIRT1-Δ2/9 identifies a new, deacetylase-independent therapeutic target for SIRT1-related diseases, including cancer.     

SIRT1 contributes to telomere maintenance and augments global homologous recombination

Yeast Sir2 deacetylase is a component of the silent information regulator (SIR) complex encompassing Sir2/Sir3/Sir4. Sir2 is recruited to telomeres through Rap1, and this complex spreads into subtelomeric DNA via histone deacetylation. However, potential functions at telomeres for SIRT1, the mammalian orthologue of yeast Sir2, are less clear. We studied both loss of function (SIRT1 deficient) and gain of function (SIRT1(super)) mouse models. Our results indicate that SIRT1 is a positive regulator of telomere length in vivo and attenuates telomere shortening associated with aging, an effect dependent on telomerase activity. Using chromatin immunoprecipitation assays, we find that SIRT1 interacts with telomeric repeats in vivo. In addition, SIRT1 overexpression increases homologous recombination throughout the entire genome, including telomeres, centromeres, and chromosome arms. These findings link SIRT1 to telomere biology and global DNA repair and provide new mechanistic explanations for the known functions of SIRT1 in protection from DNA damage and some age-associated pathologies.     

SIRT1 negatively regulates the activities, functions, and protein levels of hMOF and TIP60

SIRT1 is a NAD(+)-dependent histone H4K16 deacetylase that controls several different normal physiologic and disease processes. Like most histone deacetylases, SIRT1 also deacetylates nonhistone proteins. Here, we show that two members of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, hMOF and TIP60, are SIRT1 substrates. SIRT1 deacetylation of the enzymatic domains of hMOF and TIP60 inhibits their acetyltransferase activity and promotes ubiquitination-dependent degradation of these proteins. Importantly, immediately following DNA damage, the binding of SIRT1 to hMOF and TIP60 is transiently interrupted, with corresponding hMOF/TIP60 hyperacetylation. Lysine-to-arginine mutations in SIRT1-targeted lysines on hMOF and TIP60 repress DNA double-strand break repair and inhibit the ability of hMOF/TIP60 to induce apoptosis in response to DNA double-strand break. Together, these findings uncover novel pathways in which SIRT1 dynamically interacts with and regulates hMOF and TIP60 through deacetylation and provide additional mechanistic insights by which SIRT1 regulates DNA damage response.