SIRT 7 activates quiescent hair follicle stem cells to ensure hair growth in mice

Article21 July 2020Open Access SIRT7 activates quiescent hair follicle stem cells to ensure hair growth in mice Guo Li Guo Li Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Xiaolong Tang Xiaolong Tang orcid.org/0000-0002-4744-5846 Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China Search for more papers by this author Shuping Zhang Shuping Zhang Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Meiling Jin Meiling Jin CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China Search for more papers by this author Ming Wang Ming Wang Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China Search for more papers by this author Zhili Deng Zhili Deng Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Zuojun Liu Zuojun Liu Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China Search for more papers by this author Minxian Qian Minxian Qian orcid.org/0000-0002-1763-2325 Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China Search for more papers by this author Wei Shi Wei Shi Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Zimei Wang Zimei Wang Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China Search for more papers by this author Hongfu Xie Hongfu Xie Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Ji Li Corresponding Author Ji Li [email protected] orcid.org/0000-0003-0931-5562 Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan, China Key Laboratory of Organ Injury, Aging and Regenerative Medicine of Hunan Province, Changsha, Hunan, China Department of Dermatology, The Second Affiliated Hospital of Xinjiang Medical University, Urumqi, China Search for more papers by this author Baohua Liu Corresponding Author Baohua Liu [email protected] orcid.org/0000-0002-1599-8059 Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China Guangdong Provincial Key Laboratory of Regional Immunity and Diseases, School of Basic Medical Sciences, Shenzhen University Health Science Center, Shenzhen, China Search for more papers by this author Guo Li Guo Li Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Xiaolong Tang Xiaolong Tang orcid.org/0000-0002-4744-5846 Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China Search for more papers by this author Shuping Zhang Shuping Zhang Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Meiling Jin Meiling Jin CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China Search for more papers by this author Ming Wang Ming Wang Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China Search for more papers by this author Zhili Deng Zhili Deng Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Zuojun Liu Zuojun Liu Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China Search for more papers by this author Minxian Qian Minxian Qian orcid.org/0000-0002-1763-2325 Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China Search for more papers by this author Wei Shi Wei Shi Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Zimei Wang Zimei Wang Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China Search for more papers by this author Hongfu Xie Hongfu Xie Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China Search for more papers by this author Ji Li Corresponding Author Ji Li [email protected] orcid.org/0000-0003-0931-5562 Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan, China Key Laboratory of Organ Injury, Aging and Regenerative Medicine of Hunan Province, Changsha, Hunan, China Department of Dermatology, The Second Affiliated Hospital of Xinjiang Medical University, Urumqi, China Search for more papers by this author Baohua Liu Corresponding Author Baohua Liu [email protected] orcid.org/0000-0002-1599-8059 Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China Guangdong Provincial Key Laboratory of Regional Immunity and Diseases, School of Basic Medical Sciences, Shenzhen University Health Science Center, Shenzhen, China Search for more papers by this author Author Information Guo Li1,‡, Xiaolong Tang2,3,‡, Shuping Zhang1, Meiling Jin4, Ming Wang2,3, Zhili Deng1, Zuojun Liu2,3, Minxian Qian2,3, Wei Shi1, Zimei Wang2,3, Hongfu Xie1, Ji Li *,1,5,6,7 and Baohua Liu *,2,3,8 1Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China 2Shenzhen Key Laboratory for Systemic Aging and Intervention (SAI), National Engineering Research Center for Biotechnology (Shenzhen), International Cancer Center, Shenzhen University, Shenzhen, China 3Guangdong Key Laboratory of Genome Stability and Human Disease Prevention, Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Shenzhen University, Shenzhen, China 4CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China 5National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan, China 6Key Laboratory of Organ Injury, Aging and Regenerative Medicine of Hunan Province, Changsha, Hunan, China 7Department of Dermatology, The Second Affiliated Hospital of Xinjiang Medical University, Urumqi, China 8Guangdong Provincial Key Laboratory of Regional Immunity and Diseases, School of Basic Medical Sciences, Shenzhen University Health Science Center, Shenzhen, China ‡Those authors contributed equally to this work *Corresponding author. Tel: +86 731 89753799; E-mail: [email protected] *Corresponding author. Tel: +86 755 86674609; E-mail: [email protected] The EMBO Journal (2020)39:e104365https://doi.org/10.15252/embj.2019104365 See also: M Simon et al (September 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Hair follicle stem cells (HFSCs) are maintained in a quiescent state until activated to grow, but the mechanisms that reactivate the quiescent HFSC reservoir are unclear. Here, we find that loss of Sirt7 in mice impedes hair follicle life-cycle transition from telogen to anagen phase, resulting in delay of hair growth. Conversely, Sirt7 overexpression during telogen phase facilitated HSFC anagen entry and accelerated hair growth. Mechanistically, Sirt7 is upregulated in HFSCs during the telogen-to-anagen transition, and HFSC-specific Sirt7 knockout mice (Sirt7f/f;K15-Cre) exhibit a similar hair growth delay. At the molecular level, Sirt7 interacts with and deacetylates the transcriptional regulator Nfatc1 at K612, causing PA28γ-dependent proteasomal degradation to terminate Nfatc1-mediated telogen quiescence and boost anagen entry. Cyclosporin A, a potent calcineurin inhibitor, suppresses nuclear retention of Nfatc1, abrogates hair follicle cycle delay, and promotes hair growth in Sirt7−/− mice. Furthermore, Sirt7 is downregulated in aged HFSCs, and exogenous Sirt7 overexpression promotes hair growth in aged animals. These data reveal that Sirt7 activates HFSCs by destabilizing Nfatc1 to ensure hair follicle cycle initiation. Synopsis The mechanisms underlying hair follicle stem cell (HFSC) exit from dormancy and its impairment during ageing remain unclear. Here, in vivo genetic work identifies protein deacetylase Sirt7 as a critical guardian of HFSC dynamics and the hair cycle. Depletion of Sirt7 in stem cells delays telogen-to-anagen transition and HFSC activation in mice. Sirt7 deacetylates transcriptional regulator Naftc1, promoting its proteasomal degradation. Sirt7-dependent Nfatc1 downregulation activates HFSCs during telogen phase. Depletion of Sirt7 accelerates and forced Sirt7 expression ameliorates age-related hair follicle dysfunction in mice. Introduction Hair follicles (HFs) build up within 2 weeks after birth in mice to produce what is considered a “mini-organ” in the skin (Stenn & Paus, 2001). HFs undergo many growth–rest cycles over time; these cycles comprise anagen (growth), catagen (regression), and telogen (rest) phases that are driven by HF stem cells (HFSCs) (Millar, 2002; Schmidt-Ullrich & Paus, 2005; Rishikaysh et al, 2014). These stem cells are found within the HF outer root sheath (ORS), or “bulge” (Cotsarelis et al, 1990). A complex molecular cascade accompanies HF transition, comprising inhibited BMP and activated Shh and Wnt/β-catenin/Lef-1 signaling (Sato et al, 1999; Botchkarev et al, 2001; Huelsken et al, 2001; Reddy et al, 2001). The BMP signal maintains HFs in the telogen phase (Blanpain et al, 2004). If BMP is antagonized by an inhibitory signal, such as de novo Noggin expression (Botchkarev et al, 2001), a new HF cycle is initiated that coincides with the telogen-to-anagen transition. Here, Shh signaling activates the Wnt/β-catenin/Lef-1pathway, which boosts cell proliferation and hair shaft formation (Botchkarev et al, 2002; Alonso & Fuchs, 2003; Andl et al, 2004; Zhang et al, 2006). During the anagen-to-catagen transition, TGF-β1 signaling promotes HF degeneration by arresting keratinocyte proliferation arrest and inducing apoptosis. The HF then returns to a quiescent state (Foitzik et al, 2000; Botchkarev & Kishimoto, 2003). Hair thinning and loss are prominent characteristics of aging. These features are largely attributable to HFSC reservoir depletion, functional decline, and extended dormancy (Lei & Chuong, 2016). In aged mice, HFSCs usually fail or delay to start a new hair cycle owing to an imbalance between quiescence and activation signaling in HFs. Numerous transcription factors, including Sox9, Tbx1, Lhx2, TCF3/4, Foxc1 and Nfatc1, help maintain a quiescent and undifferentiated HFSC status (Blanpain et al, 2004; Tumbar et al, 2004; Horsley et al, 2008). For example, Foxc1 deletion compromises quiescence by suppressing BMP and Nfatc1 signaling; this effect causes premature HFSC activation (Lay et al, 2016; Wang et al, 2016). NFATc1 is essential for HFSC quiescence (Horsley et al, 2008): During aging, hyper-accumulation of nuclear Nfatc1 extends the dormant period of HFSCs (Keyes et al, 2013). The precise mechanisms governing NFATc1 dynamics in normal adult hair cycling and aging, however, are poorly understood. Understanding the mechanisms that evoke quiescent HFSCs is equally important to help treat or prevent age-associated hair thinning and loss. The sirtuin protein family is involved in regulating longevity (Wu et al, 2018). Data from phenotypic analyses of knockout mice suggest that Sirt1, Sirt6, and Sirt7 are potential longevity-associated genes (Vakhrusheva et al, 2008; Kawahara et al, 2009; Kanfi et al, 2012; Satoh et al, 2013; Vazquez et al, 2016). Sirt7 is an NAD+-dependent deacylase and its expression declines in senescent cells (Blank & Grummt, 2017; Wu et al, 2018). Many studies have uncovered links between Sirt7 and aging: Sirt7−/− mice have a shortened lifespan by up to 50% and exhibit multiple aging phenotypes, including hepatic steatosis, gonadal fat pad content depletion, cardiac hypertrophy, and hematopoietic stem cell reduction (Shin et al, 2013; Ryu et al, 2014; Yoshizawa et al, 2014; Araki et al, 2015; Mohrin et al, 2015; Tang, 2015; Vazquez et al, 2016; Tang et al, 2019). Here, using genetically modified mice, we aimed to explore the role of Sirt7 in HFSCs and hair loss with aging. Results Loss of Sirt7 delays telogen-to-anagen transition in murine HFs Loss of Sirt7 accelerates aging in mice (Vazquez et al, 2016). Hair dysfunction is a common characteristic in the elderly. Remarkably, we observed a notable decrease in Sirt7 expression in hair follicles (HFs) in aged mice (18 months) compared to that of young mice (3 months). By contrast, Sirt1 and Sirt6 exhibited only a marginal change in expression level (Appendix Fig S1A). We thus focused our attention on Sirt7. First, to examine the role of Sirt7 in aging-related hair regeneration, we generated whole-body Sirt7 knockout (KO) mice. Immunohistochemical (IHC) staining and Western blotting confirmed that Sirt7 was expressed in Sirt7+/+ HFs (skin) but was absent in Sirt7−/− HFs (skin) (Appendix Fig S1B and C). Sirt7−/− and Sirt7+/+ mice showed only a marginal difference in the first postnatal hair cycle (postnatal days P6, P10, P14, to P20), with comparable levels of hair growth and color (Appendix Fig S1D and E). This finding indicates that the initial HF morphogenesis per se is not affected by Sirt7 loss. To assess whether Sirt7 regulates adult hair growth, we shaved the hair coat of female Sirt7+/+ and Sirt7−/− mice on postnatal day (P) 60, when the HFs of both genotypes were in the second telogen phase (Fig 1A). We evaluated hair coat recovery based on skin pigmentation and hair shaft growth as described (Stenn & Paus, 2001; Chai et al, 2019). By P120, Sirt7+/+ mice recovered an almost full hair coat, while the Sirt7−/− mice showed only ~ 25% recovery (Fig 1A and B). In parallel, hematoxylin and eosin (H&E) staining of the unshaved skin showed that at P90, a large population of Sirt7+/+ HFs had developed to a full size, indicating the entry of anagen phase, while Sirt7−/− HFs remained in telogen phase (Fig 1C, left). By the time, the Sirt7−/− HFs had progressed into mid-anagen phase, and at around P130, the Sirt7+/+ HFs had already moved to the next telogen phase (Fig 1C, right). Once they enter anagen phase, P-cadherin+ cells in the hair germ (HG) respond to growth signals and become proliferative, as indicated by Ki67 expression (Horsley et al, 2008). Consistently, we observed high Ki67 expression at P74 (early anagen) in Sirt7+/+ HGs, but low-to-no Ki67 in Sirt7−/− HFs even at P90 (Fig 1D and E). Figure 1. Sirt7 promotes HF telogen-to-anagen transition The hair coats of Sirt7−/− and Sirt7+/+ mice were clipped on P60 in the mid-second telogen phase. Images were captured at P60, P120, and P150. The postnatal day when the hair coat recovered by 25, 50, 75, and 100% in shaved Sirt7−/− and Sirt7+/+ mice. n = 6 mice/genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum, and maximum. H&E staining of the skin in unshaved Sirt7−/− and Sirt7+/+ mice on P90 and P130. Representative immunofluorescence images showing Ki67 and p-cadherin (Pcad, labeling HG cells) expression in Sirt7−/− and Sirt7+/+ HFs on P74 and P90. The HF boundary and dermal papilla (DP) are denoted by the dotted and dashed lines, respectively. Percent Ki67-positive cells in each hair germ in D; n = 3 mice and 10 HGs were counted in each mouse. Telogen duration in Sirt7−/− and Sirt7+/+ mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum, and maximum. The hair coats of Sirt7-TG and WT mice were clipped on P45, and images were captured on P56 and P90. The postnatal day when the hair coat recovered by 25, 50, 75, and 100% in shaved Sirt7-TG and WT mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum, and maximum. Representative immunofluorescence images showing Ki67 and Pcad expression in Sirt7-TG and WT HFs at P60 and P65. The HF boundary and DP are noted by the dotted and dashed lines, respectively. The percentage of Ki67-positive cells in each HG; n = 3 mice, and 10 HGs were counted in each mouse. H&E staining of skin tissues in unshaved Sirt7-TG and WT mice on P65. Telogen duration in Sirt7-TG and WT mice. n = 6 mice per genotype.Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum, and maximum. Abstracted HF cycling in Sirt7−/−, Sirt7+/+, Sirt7-TG, and WT mice mentioned above. The red star indicates anagen entry in Sirt7−/− and Sirt7-TG mice; the black arrows show the date of doxycycline feeding. Data information: Scale bar, 50 μm for immunofluorescence images; 200 μm for H&E staining images. The data represent the means ± s.e.m. **P < 0.01, ***P < 0.001, determined by Student's t-test. Download figure Download PowerPoint To confirm our findings, we generated a well-described depilation model to induce homogenous and synchronized HF cycling (Plasari et al, 2010). Seven days after depilation, the dorsal skin of Sirt7+/+ mice showed deep pigmentation, indicating anagen entry, while that of Sirt7−/− mice remained pink, indicative of telogen phase (Appendix Fig S1F). Indeed, Sirt7−/− mice showed an extended telogen phase by almost 30 days (Fig 1F). Together, these data suggest that loss of Sirt7 delays the telogen-to-anagen transition in murine HFs. Induced Sirt7 expression shortens telogen phase and facilitates anagen entry We next asked whether forced Sirt7 expression in HFs in telogen phase would promote anagen entry. We used an inducible Sirt7 transgenic mouse line (Sirt7-TG) (Tang et al, 2017), in which Sirt7 expression could be induced by doxycycline (Dox) at the desired time. Sirt7-TG and wild-type (WT) littermate control mice were exposed to Dox (2 mg/ml, dissolved in water) at P42, when the HFs of both genotypes were both in telogen phase. We first confirmed Sirt7 overexpression by IHC staining at P56 (Appendix Fig S1G). Then, we examined hair coat recovery after shaving the telogen dorsal hair at P45. Strikingly, by P100, Sirt7-TG mice had recovered an almost full hair coat, while their WT littermates recovered only up to 50% hair (Fig 1G and H). Consistently, Sirt7-TG HGs showed prominent Ki67 expression by P65 (Fig 1I and J), significantly ahead of Ki67 expression in WT HGs, which was detectable from P74. Morphological analysis of the unshaved dorsal skin also suggested early anagen entry of Sirt7-TG HFs compared to WT HFs (Fig 1K). Thus, forced Sirt7 overexpression shortened the HF telogen phase by almost 20 days compared to WT mice (Fig 1L). Taken together, we detected an unaltered first hair cycle but an altered second hair cycle in our Sirt7 mutants: Sirt7−/− mice showed an extended telogen phase and Sirt7-TG mice showed a shortened telogen phase (Fig 1M). Sirt7 deletion delays HFSC activation HFSCs reside in the HF bulge: During hair regeneration in adulthood, these HFSCs undergo sequential activation and quiescence. HF cycling is regulated by the amount of dynamic signals (Hsu et al, 2011). We noted that a burst of Sirt7 expression first appeared in the HFSCs residing in the late telogen bulge. The expression levels progressively increased during the telogen-to-anagen shift in Sirt7+/+ HFs, regardless of normal hair cycling or induction by depilation (Fig 2A and B, and Appendix Fig S2A). By contrast, Sirt7 levels remained constant in the interfollicular epidermis (IFE) and intradermal white adipose tissue (dWAT), and almost comparable in the sebaceous gland (SG) (Appendix Fig S2B–D). Thus, Sirt7 expression in HFSCs was specifically coupled with hair cycling progression. Consistently, anagen bulge HFSCs showed a 2-fold increase in Sirt7 expression compared to those telogen bulge HFSCs (Appendix Fig S2E). Figure 2. Sirt7 deletion delays HFSC activation A, B. IHC labeling of Sirt7 and Ki67 expression in the skin at different HF phases in the second hair cycle and in the depilation-induced hair cycle in WT mice. P56, P69, P74, and P84 indicate the postnatal day in the second hair cycle. Pd, post-depilation. Dashed line indicates bulge (Bu) of a hair follicle, and solid line indicates the Ki-67-positive staining region. C. The hair coats of Sirt7f/f and Sirt7f/f;K15-Cre mice were clipped at P60, and images were captured on P60, P125, and P150. D. The postnatal day of hair coat recovery by 25, 50, 75, and 100% in shaved Sirt7f/f and Sirt7f/f;K15-Cre mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum, and maximum. E. H&E staining of the skin in Sirt7f/f and Sirt7f/f;K15-Cre mice at P100. F. Representative immunofluorescence images showing Ki67/Pcad expression (upper) and Cd34/Ki67 expression (lower) in Sirt7 fl/fl and Sirt7f/f;K15-Cre HFs on P56, P74, and P85. The white dashed line indicates bulge of hair follicle. G. The percentage of Ki67-positive cells per HG; n = 3 mice and 10 HGs were counted in each mouse. H. Ki67-positive cells per bulge; n = 3 mice and 10 bulges were counted in each mouse. I. Schematic illustrating the HF cycle in Sirt7f/f and Sirt7f/f;K15-Cre mice after mifepristone-induced Cre activation. The arrow indicates the time of mifepristone treatment (details in Materials and Methods); the red star indicates the end of the telogen phase in Sirt7f/f;K15-Cre HFs. Data information: Scale bar, 50 μm for IHC and immunofluorescence images, 200 μm for H&E staining images. The data represent the means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, obtained by Student's t-test. Download figure Download PowerPoint Of note, CD34 staining and FACS analysis showed that the number of HFSCs was comparable between Sirt7+/+ and Sirt7−/− mice (Appendix Fig S2F and G). This finding was consistent with our observation of normal HF morphogenesis in Sirt7−/− mice and encouraged us to explore whether Sirt7 promotes HFSC activation. To that end, we generated epidermal bulge stem cell-specific Sirt7 KO mice (Sirt7f/f;K15-Cre) by crossing Keratin 15 (K15)-CrePGR (Cre activation induced by mifepristone) mice to Sirt7 floxed mice (Sirt7f/f) (Morris et al, 2004; Oshimori & Fuchs, 2012). At P42, we treated Sirt7f/f;K15-Cre mice and Sirt7f/f littermates with mifepristone and confirmed Sirt7 deletion in bulge stem cells by IHC staining at P74 (Appendix Fig S2H). Compared to other organs and tissues, only the skin exhibited prominent Cre expression and a significant Sirt7 reduction by 50%, thus ruling out possible off-target effects (Appendix Fig S2I). Notably, Sirt7f/f;K15-Cre mice possessed an equivalent number of HFSCs compared to their littermate controls (Appendix Fig S2J). To assess the impact of Sirt7 loss in epidermal stem cells on HF cycling, we clipped the dorsal hair of both genotypes at P60 (at terlogen phase, Fig 2C) and monitored the subsequent hair growth. Sirt7f/f control mice fully recovered the hair coat by P125, while Sirt7f/f;K15-Cre mice presented an almost 50% recovery (Fig 2C and D). This extension in telogen phase in Sirt7f/f;K15-Cre mice was evidenced by H&E and low Ki67 staining in HG and bulge cells in the Sirt7f/f;K15-Cre skin (Fig 2E–H). Additionally, the HFSC number was comparable between Sirt7f/f;K15-Cre and Sirt7f/f mice (marked by CD34) (Fig 2F, lower panel and Appendix Fig S2K), again implicating that Sirt7 loss has little impact on HF development but shifts HF cycling (Fig 2I). To further confirm that Sirt7 contributes to HFSC activation, we conducted a BrdU pulse-chase experiment in Sirt7+/+ and Sirt7−/− mice following depilation-induced hair cycle synchronization at telogen phase. Depilation removes an inner bulge layer of BMP6 and FGF18-expressing cells that consequently activates quiescent HFSCs (Hsu et al, 2011). On post-depilation days 2 and 4, we found that much fewer Sirt7−/− bulge cells incorporated BrdU, indicating less activation of HFSCs at early anagen phase (Appendix Fig S3A and B). Despite this apparent delay, the number of BrdU-labeled bulge cells eventually increased in Sirt7−/− HFs, indicating that Sirt7 deletion most likely delays rather blocks HFSC activation. IFE/SG growth and dWAT expansion are highly synchronized with the hair cycle (Li et al, 2001; Kruglikov & Scherer, 2016; Reichenbach et al, 2018). Consistently, we observed a similar delay in IFE/SG proliferation and dWAT expansion in Sirt7−/− mice (Appendix Fig S3C–G). To assess whether the effects of Sirt7 on HFSCs are cell autonomous, we labeled bulge SCs from Sirt7+/+, Sirt7−/−, and Sirt7-TG mice as CD34+/integrin-α6+ and purified them by FACS from the telogen phase HFs. The cells were allowed to grow for 14 days, as described (Blanpain et al, 2004). We found that the Sirt7 deficiency resulted in a lower number of colonies; by contrast, Sirt7 overexpression significantly boosted colony formation efficiency (CFE) (Fig 3A and B). The Sirt7 KO colonies were also smaller in size than the WT colonies; conversely, forced Sirt7 expression promoted colony expansion (Fig 3C). Of note, we observed a minimal change in the Annexin-V/PI signals between Sirt7+/+ and Sirt7−/− colonies (Appendix Fig S3H). Further, the expression of the stem cell marker Lgr5 (Jaks et al, 2008) was equivalent in Sirt7+/+ and Sirt7−/− colonies, arguing against