J. Cosmet. Sci., 68, 25–33 ( January/February 2017) 25 Effect of SIRT6 knockdown on NF- B induction and on residual DNA damage in cultured human skin fi broblasts E. C. GOYARTS, K. DONG, E. PELLE, and N. PERNODET, Estee Lauder Research Laboratories, Melville, NY (E.C.G., K. D., E. P., N. P.) and Environmental Medicine, New York University School of Medicine, New York, NY (E.P.). Accepted for publication October 24, 2016. Synopsis SIRT6 is a member of the sirtuin family, which is involved in multiple cellular pathways related to aging, infl ammation, epigenetics, and a variety of other cellular functions, including DNA repair (1). Multiple pathways involving different cellular functions are impacted by the deacetylase activity of SIRT6. Genomic integrity is maintained by the capacity of SIRT6 to modulate the accessibility of DNA repair proteins. Glucose metabolism is suppressed by SIRT6 via the deacetylation of histones located at the promoter regions of multiple glycolytic genes and the corepression of hypoxia-inducible factor-1α. SIRT6 is also a corepressor of nuclear factor (NF)-κB, silencing NF-κB target genes through the deacetylation of histones at their promoters’ regions. We used SIRT6 small-interfering RNA as a tool to modulate residual DNA damage and NF-κB expression in human dermal fi broblasts. We measured NF-κB levels in the presence or the absence of ultraviolet B (UVB). The impact of SIRT6 knockdown as shown by a decrease in SIRT6 messenger RNA levels resulted in residual DNA damage as evaluated by the comet assay. Our results show that NF-κB was increased signifi cantly (up to 400%) due to SIRT6 silencing in the absence of UVB, illustrating the master regulatory function of SIRT6 in infl ammation. We also found a signifi cant increase in DNA damage without UV exposure as a result of SIRT6 silencing, indicating the importance of SIRT6 in DNA repair pathways in cultured human dermal fi broblasts. INTRODUCTION Sirtuins are a family of NAD+-dependent histone deacetylases impacting a broad range of cellular functions (1,2). Mammals have seven sirtuins (SIRT1–7), which modulate diverse cellular functions including metabolism, cellular stress response, genomic stability, infl am- mation, and aging (3,4). SIRT6, which is localized in the nucleus, catalyzes the removal of an acetyl group at lysine 9 (H3K9) and/or lysine 56 (H3K56) of histone 3. Deacety- lation of histone 3 reduces the accessibility of DNA-binding proteins because the nucleosomal structure becomes compressed. Thus, SIRT6 modulates many genes through posttransla- tional modifi cation of histone 3 resulting in chromatin silencing (5). SIRT6 is considered Address all correspondence to Earl C. Goyarts at egoyarts@estee.com.

JOURNAL OF COSMETIC SCIENCE 26 to have strong antiaging benefi ts as SIRT6-defi cient mice develop a progeroid-like syn- drome resembling premature aging and die after 4 weeks (6). Mammalian cells defi cient in SIRT6 have defects in DNA repair resulting in genomic instability. These include base excision repair (BER) (6,7,8), double strand break (DSB) repair (7,9–12), and telomere maintenance (7,13,14). Current evidence indicates that SIRT6 regulates BER either by modulating BER factors or by regulating the chromatin density permitting the accessibility of BER factors to DNA damage sites (7,13). Success- ful DSB repair requires that SIRT6 recruit a chromatin remodeler, SNF2H, to the DSB (11) and the deacetylation of a C-terminal binding protein, CtIP, to promote DNA- end resectioning (10). Proper telomere maintenance requires the binding of SIRT6 to telomeric chromatin resulting in the deacetylation of H3K9 histones and preventing chromosome end fusion. Thus, SIRT6 functions both as an enzyme and as a scaffold for establishing a unique microenvironment for recruiting other proteins needed for DNA repair. In addition to DNA repair, SIRT6 is involved in infl ammation control (3). Pyrimidine dimers are the primary trigger of post-ultraviolet (UV) erythema and their removal requires DNA nicking in order to mediate excision repair (15). SIRT6 promotes tumor necrosis factor (TNF)-α secretion by deacylating two myristoyl groups from lysine residues at peptide positions 19 and 20 of TNF-α (16). Conversely, SIRT6 is recruited to promoter regions of nuclear factor (NF)-κB target genes and physically docks with the NF-κB subunit, RelA (5,17). SIRT6 silences these NF-κB target genes by modifying the chromatin via deacetylation of H3K9 on nearby histones (5,17). The altered chromatin structure destabilizes RelA binding to the target gene promoter, terminating NF-κB stimulation of the target genes. A SIRT6-defi cient mouse survives only 30 days because both NF-κB genes are unregulated leading to hyperactive NF-kB signaling (18). A SIRT6-defi cient mouse crossed with a RelA +/− mouse rescues early lethality of SIRT6-defi cient mice, giving rise to progeny which survive for more than 100 days, indicating that a reduction of the NF-κB copy number results in longer survival (17,18). Further, recall that transgenic male mice overexpressing SIRT6 have increased longevity, about 15% (19), which is associated with the insulin-like growth factor 1 signaling pathway (3). As mentioned earlier, a reduced capacity to maintain genomic integrity and an increased sensitivity to oxidative stress and infl ammation are causes of premature aging in the SIRT6 knockout mouse. SIRT6-defi cient mice, as well as several mouse models of nucle- otide excision repair defi ciency, exhibit a premature aging phenotype, which is associated with insulin signaling (16). This premature aging phenotype in mice is associated with dysregulation of insulin signaling as a result of glucose metabolism dysfunction (16,20), hyperactive NF-κB signaling (17), and genomic instability (7). SIRT6 is involved in all these pathways. With regard to humans, mesenchymal stem cells defi cient in SIRT6 have dysregulated redox metabolism because SIRT6 functions as a transactivator for nuclear factor erythroid 2-related factor 2, establishing a connection between SIRT6 and oxidative stress (21). The imbalanced relationship between DNA repair and metabolism in SIRT6- defi cient mice is an important theme in premature aging. The upregulation of NF-κB as a consequence of SIRT6 defi ciency further illustrates the multiple connections of SIRT6 with premature aging. We sought to investigate the importance of DNA repair and NF-κB in a tissue culture model. We explored the effects of SIRT6 knockdown in human dermal fi broblasts under normal conditions and after environmental stress from ultraviolet B (UVB) and ultraviolet A (UVA) irradiation. In this report, we evaluated the effectiveness of