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Regulation of Global Genome Nucleotide Excision Repair by SIRT1 Through Xeroderma Pigmentosum C

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Regulation of Global Genome Nucleotide Excision Repair by SIRT1 Through Xeroderma Pigmentosum C Regulation of global genome nucleotide excision repair by SIRT1 through xeroderma pigmentosum C Mei Minga, Christopher R. Sheaa, Xiumei Guob, Xiaoling Lib, Keyoumars Soltania, Weinong Hana, and Yu-Ying Hea,1 aSection of Dermatology, Department of Medicine, University of Chicago, Chicago, IL 60637; and bLaboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 Edited by C. Thomas Caskey, University of Texas-Houston Health Science Center, Houston, TX, and approved November 22, 2010 (received for review July 16, 2010) Disruption of the nucleotide excision repair (NER) pathway by found reduced levels of SIRT1 mRNA in ovarian, prostatic, and mutations can cause xeroderma pigmentosum, a syndrome predis- bladder carcinomas, and glioblastoma, as compared with normal posing affected individuals to development of skin cancer. The tissues. But they did not find any changes in SIRT1 protein levels xeroderma pigmentosum C (XPC) protein is essential for initiating in skin cancer compared with normal skin. global genome NER by recognizing the DNA lesion and recruiting In this study, we used an in vitro cell culture model and human downstream factors. Here we show that inhibition of the deacety- skin tumor analysis to study the role of SIRT1 in GG-NER in cells lase and longevity factor SIRT1 impairs global genome NER through exposed to UVB radiation and in human skin tumors. Our data suppressing the transcription of XPC in a SIRT1 deacetylase-de- provide strong evidence that mammalian SIRT1 plays a critical pendent manner. SIRT1 enhances XPC expression by reducing AKT- role in tumor-suppressing GG-NER, and that down-regulation of dependent nuclear localization of the transcription repressor of XPC. SIRT1 is associated with human skin tumorigenesis. Finally, we show that SIRT1 levels are significantly reduced in human skin tumors from Caucasian patients, a population at highest risk. Results These findings suggest that SIRT1 acts as a tumor suppressor through SIRT1 Is Required for Efficient GG-NER of UVB-Induced DNA Damage. its role in DNA repair. To determine whether SIRT1 plays a role in GG-NER to remove CPD and 6-4PP lesions, we measured the percentage of UVB | PTEN | AKT CPDs or 6-4PPs remaining at different intervals post-UVB, and thereby determined the percentage of repair, in parental and MEDICAL SCIENCES ucleotide excision repair (NER) is a versatile DNA repair SIRT1-inhibited cells. To genetically delete SIRT1, SIRT1 WT Npathway that eliminates a wide variety of helix-distorting mouse embryonic fibroblasts (MEFs) were used for comparison base lesions, including UV radiation-induced cyclobutane py- with SIRT1 KO cells. To inhibit SIRT1 in keratinocytes, which rimidine dimers (CPD) and (6-4) photoproducts (6-4PPs) (1, 2), are major targets for human UVB exposure, we transfected as well as bulky adducts induced by numerous chemical com- human HaCaT cells with siRNA targeting SIRT1. In both MEFs pounds. Defects in NER by mutations cause the autosomal re- and HaCaT cells, inhibition of SIRT1 significantly inhibited re- cessive xeroderma pigmentosum (XP) and Cockayne syndromes pair of CPDs (Fig. 1 A and B; P < 0.05, two-way ANOVA), as (3–5). XP patients are clinically characterized by cutaneous sen- well as 6-4PPs (Fig. 1 C and D; P < 0.05, two-way ANOVA). The sitivity to sunlight exposure and a predisposition to skin cancer. inhibition of CPD repair by SIRT1 loss was more profound than 2 Seven NER-deficient genetic complementation groups of XP that of 6-4PP repair. At these UVB doses (MEFs, 10 mJ/cm ; 2 (XP-A to -G) have been identified, and all of the corresponding HaCaT, 20 mJ/cm ) and time points (up to 48 h), there was no genes have now been cloned (3–5). significant difference in initial DNA damage, growth, or UVB- Mammalian NER consists of two distinct subpathways: global induced apoptosis between WT and KO MEFs or between NC- genome NER (GG-NER), which operates throughout the ge- and siSIRT1-transfected HaCaT cells (SI Appendix, Table S1, nome, and transcription-coupled NER (TC-NER), which spe- Figs. S1 and S2). Our findings demonstrated that loss of SIRT1 cifically removes lesions on the transcribed DNA strand of active results in the inhibition of DNA repair in UVB-induced CPD genes. A major difference between these two pathways appears to and 6-4PPs, strongly indicating that SIRT1 is required for lie in the strategies for detecting damaged bases. Accumulating efficient GG-NER. evidence indicates that the XP group C (XPC) protein plays an essential role in GG-NER-specific damage recognition (6–8). Inhibition of SIRT1 Down-Regulates XPC Levels. XPC is essential for Although biochemical and genetic analyses have characterized GG-NER of UVB-induced DNA damage (7, 24). To determine the function of XPC in considerable detail, much remains to be the mechanism by which SIRT1 participates in GG-NER, we elucidated with regard to its regulation and interaction with other investigated whether XPC is regulated by SIRT1. When we critical cellular pathways. compared the protein level of XPC from SIRT1 WT MEFs with that from SIRT1 KO MEFs by immunoblotting, we found that Sirtuin 1 (SIRT1), a mammalian counterpart of the yeast silent fi information regulator 2 (Sir2) and a proto member of the sirtuin the protein level of XPC in SIRT1 KO cells was signi cantly lower than in SIRT1 WT cells (Fig. 2 A and B; P < 0.05, Stu- family, is an NAD-dependent longevity-promoting deacetylase. ’ t SIRT1 is crucial for cell survival, metabolism, senescence, and dent s test). Similarly, the XPC protein level in HaCaT cells stress response in several cell types and tissues (9–13). Both his- tone and nonhistone targets of SIRT1 have been identified, in- α κ γ Author contributions: M.M., C.R.S., X.L., and Y.-Y.H. designed research; M.M. performed cluding FOXO, p53, PGC-1 , NF- B, and PPAR (10, 12, 14). research; X.G., X.L., K.S., and W.H. contributed new reagents/analytic tools; M.M., C.R.S., SIRT1 has been implicated as an important player in cancer. K.S., and Y.-Y.H. analyzed data; and Y.-Y.H. wrote the paper. However, it remains unclear whether SIRT1 serves as a tumor The authors declare no conflict of interest. – suppressor or a tumor promoter (13 16). SIRT1 expression is This article is a PNAS Direct Submission. relatively higher in many malignancies, including colon, breast, 1To whom correspondence should be addressed. E-mail: [email protected]. prostate, and skin cancers and leukemia, as compared with their edu. – fi corresponding normal tissues (17 22). But the speci c correlation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. is not clear-cut. Wang et al. (23) analyzed a public database and 1073/pnas.1010377108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1010377108 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 MEFs HaCaT The mRNA level of XPC was significantly lower in SIRT1 KO A B NC E 80 cells than in SIRT1 WT cells (Fig. 3B; P < 0.05, Student’s t test). 60 WT SIRT1 MEF KO 60 40 SIRT1 WT KO Using the promoter reporter assay, we found that the tran- p 40 * SIRT1 scriptional activity of the 1.5-kb WT mouse XPC promoter (SI 20 * * * 20 -actin Appendix, Fig. S4) in SIRT1 KO cells was significantly lower than % CPD Repair % CPD Repair 0 0 0 24 48 0 24 48 in SIRT1 WT cells (Fig. 3C; P < 0.05, Student’s t test). Trans- Hours post-UVB Hours post-UVB fection of KO MEFs with WT SIRT1 significantly increased the transcriptional activity of the XPC promoter (Fig. 3D; P < 0.05, MEFs HaCaT T1 C D F siRNA NC SIR Student’s t test), further demonstrating that SIRT1 is required SIRT1 100 100 SIRT2 for XPC transcription. * * 80 80 * SIRT3 Previous reports indicated that the XPC gene is repressed by 60 * 60 SIRT4 40 40 the E2F4-p130 repressor complex (27), and that disrupting this WT NC SIRT5 20 KO 20 SIRT6 %Repair 6-4pp %Repair 6-4pp SIRT1 complex by the tumor suppressor ARF (alternative reading 0 0 SIRT7 0 24 48 0 24 48 -actin frame) stimulates XPC transcription (28). To investigate whether Hours post-UVB Hours post-UVB SIRT1 regulates the E2F4-p130 complex, we first determined Fig. 1. SIRT1 is essential for efficient DNA GG-NER. ELISA of percent repair whether inhibition of SIRT1 alters the nuclear location of E2F4 of CPDs (A and B) and 6-4PPs (C and D) at intervals post-UVB (MEFs, 10 mJ/cm2; and p130. E2F4 is a nuclear protein, and p130 is mainly localized HaCaT, 20 mJ/cm2). (A and C) WT or SIRT1 KO MEFs. (B and D) HaCaT cells in the cytosol (Fig. 3 E and F). In SIRT1 KO cells, though the transfected with negative control (NC) or siRNA targeting SIRT1. (E) Immu- nuclear E2F4 levels remained unaltered, the nuclear p130 levels noblot analysis of SIRT1 and β-actin (equal loading control) in WT and KO increased as compared with SIRT1 WT cells (Fig. 3 E and F), MEFs. (F) Immunoblot analysis of SIRT1-7 and β-actin in HaCaT cells trans- suggesting that inhibition of SIRT1 increased the p130 levels in fected with siRNA targeting SIRT1 or NC. Error bars show SE. *P < 0.05, the nucleus. To further examine the role of the E2F site in the fi signi cant differences between KO and WT cells, or SIRT1 and NC cells. XPC promoter, sequences between −51 and +7 positions of the mouse XPC promoter, with either an intact (XPC-WT) or mu- SI Appendix transfected with siRNA targeting SIRT1 was significantly re- tated (XPC-Mut) E2F site ( , Fig.
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