258 j Journal of Molecular Cell Biology (2018), 10(3), 258–266 doi:10.1093/jmcb/mjx055 Published online February 14, 2018 Article Senp2 regulates adipose lipid storage by de-SUMOylation of Setdb1 1,2,† 1,2,† 1,2 3 1,2 1,2 Quan Zheng , Ying Cao , Yalan Chen , Jiqiu Wang , Qiuju Fan , Xian Huang , 4 1,2 1,2 1,2 1,2, Yiping Wang , Tianshi Wang , Xiuzhi Wang , Jiao Ma , and Jinke Cheng Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inﬂammation, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China Department of Endocrinology and Metabolism, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China Cancer Metabolism Laboratory, Institutes of Biomedical Science, Shanghai Medical College, Fudan University, Shanghai 200032, China These authors contributed equally to this work. * Correspondence to: Jinke Cheng, E-mail: firstname.lastname@example.org Edited by Feng Liu One major function of adipocytes is to store excess energy in the form of triglycerides. Insufﬁcient adipose lipid storage is asso- ciated with many pathological conditions including hyperlipidemia, insulin resistance, and type 2 diabetes. In this study, we observed the overexpression of SUMO-speciﬁc protease 2 (Senp2) in adipose tissues during obesity. Adipocyte Senp2 deﬁciency resulted in less adipose lipid storage accompanied by an ectopic fat accumulation and insulin resistance under high-fat diet feed- ing. We further found that SET domain bifurcated 1 (Setdb1) was a SUMOylated protein and that SUMOylation promoted Setdb1 occupancy on the promoter locus of Pparg and Cebpa genes to suppress their expressions by H3K9me3. Senp2 could suppress Setdb1 function by de-SUMOylation. In adipocyte Senp2-deﬁciency mice, accumulation of the SUMOylated Setdb1 suppressed the expression of Pparg and Cebpa genes as well as lipid metabolism-related target genes, which would decrease the ability of lipid storage in adipocytes. These results revealed the crucial role of Senp2–Setdb1 axis in controlling adipose lipid storage. Keywords: lipid storage, Senp2, Setdb1,H3K9me3, Pparg and Cebpa Introduction occur primarily through protein-mediated pathways consisting of a Adipose tissue has an important role in nutrient homeostasis number of fatty acid transporters such as fatty acid translo- by either storing excess energy in the form of triglycerides (TGs) case (Cd36) and fatty acid transport protein-1 (Fatp1)(Hajri or hydrolyzing TGs to provide fuels for the rest of the body and Abumrad, 2002). Lipolysis proceeds through successive under fasting condition (Rosen and Spiegelman, 2014; Cohen steps involving the release of a single fatty acid chain and the and Spiegelman, 2016). Excess or insufﬁcient lipid storage in generation of the intermediate species of diacylglycerol (DAG) adipose tissues would impair nutrient homeostasis, which is and monoacylglycerol (MAG) (Young and Zechner, 2013). associated with many pathological conditions such as hyperlipid- As a master regulator of adipogenesis, peroxisome proliferator- emia, insulin resistance, and type 2 diabetes (Garg, 2004; Agarwal activated receptors γ (PPARγ) can modulate adipose tissue mass and Garg, 2006; Rosen and Spiegelman, 2006; Liu et al., 2014; by regulating lipogenesis-related genes (Ahmadian et al., 2013). Patni and Garg, 2015). Under physiological conditions, the main- PPARγ mutation has been shown to link to familial partial lipody- tenance of normal adipose tissue mass is mainly the result of a strophy, a clinical disorder characterized by the loss of adipose balance of lipid storage and lipolysis (Bouchard et al., 1993). tissues (Dunnigan et al., 1974; Savage et al., 2003). Selective Cellular uptake of fatty acids and following storage in the form of disruption of Pparγ2 or adipocyte-speciﬁc Pparγ knockout leads TGs in adipocytes are key steps in lipid storage. These processes to severe lipodystrophy and induces insulin resistance in mice (He et al., 2003; Zhang et al., 2004; Duan et al., 2007). Recent Received September 6, 2017. Revised October 27, 2017. Accepted December 19, studies have shown that SUMO-speciﬁc protease 2 (Senp2)is involved in myogenesis (Qi et al., 2014a) and adipogenesis (Chung © The Author (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. et al., 2010). Senp2 also regulates fatty acid metabolism in skeletal Downloaded from https://academic.oup.com/jmcb/article-abstract/10/3/258/4763638 by Ed 'DeepDyve' Gillespie user on 26 June 2018 Senp2 regulates adipose lipid storage by de-SUMOylation of Setdb1 j 259 muscle (Koo et al., 2015). In this report, we observed a defect in adi- (Figure 1G and Supplementary Figure S1F). Although less fat stor- adqcKO pose lipid storage in adipocyte-speciﬁc Senp2 knockout mice fed age was shown in Senp2 adipose tissues, the increased with high-fat diets (HFD). This mouse also showed an ectopic lipid inﬂammation indicates that HFD induced adipocyte death would distribution and insulin resistance. Therefore, adipocyte-speciﬁc remain in adipose tissues. Senp2 knockout mice exhibited a lipodystrophy-like phenotype. adqcKO Mechanistically, adipocyte Senp2 deﬁciency caused the downregula- Senp2 mice exhibit an ectopic lipid accumulation and tion of Pparg and Cebpa as well as their downstream target genes insulin resistance related to lipid storage. We found that SET domain bifurcated 1 Since no difference in food intake was observed between adqcKO f/f (Setdb1) was a SUMOylated protein and that Senp2 de-SUMOylated Senp2 and Senp2 mice fedeitherwithNCD or HFD and regulated Setdb1 action in trimethylation at histone 3 lysine 9 (Supplementary Figure S2A). We next assessed the serum triglycer- (H3K9me3). Senp2 deﬁciency triggered a hyper-SUMOylation of ide, cholesterol, and non-esteriﬁed fatty acid (NEFA) in aforemen- Setdb1, which enhanced its occupancy on Pparg and Cebpa pro- tioned mice fed with HFD. Figure 2A demonstrates that their adqcKO moter loci to suppress their transcriptions. Our ﬁndings unravel that concentrations were much higher in Senp2 serum than in f/f Senp2 is essential for regulating lipid metabolism in adipose tissues. Senp2 serum. We further stained liver sections and showed more adqcKO f/f severe hepatic steatosis in Senp2 mice than in Senp2 mice Results (Figure 2B). These data suggest that an ectopic lipid distribution was adqcKO Senp2 deﬁciency decreases adipose lipid storage accompanied with the less adipose lipid storage in Senp2 mice. Since Senp2 expression was dramatically increased in adipose To further demonstrate a view of fat distribution, we performed an tissues in several obese mouse models (Figure 1A), suggesting in vivo analysis for C-labeled oleate uptake. An equal injection of that Senp2 may play a role in the pathogenesis of obesity in initial radiation dose was determined by measuring C-oleate counts adipose tissues. To investigate it, we generated a mouse model in theplasma(SupplementaryFigureS2B). As demonstrated in in which Senp2 was speciﬁcally knockout in adipose tissues Figure 2C, we observed a lower level of C-oleate counts in adipose adqcKO f/f adqcKO (Senp2 ) by crossing Senp2 with Adiponectin-cre mice (Lee tissues of Senp2 mice including BAT, iWAT (inguinal WAT), and et al., 2013; Jeffery et al., 2014). Senp2 expression was signiﬁ- eWAT (epididymal WAT), but a higher level in the liver, heart, kidney, adqcKO cantly downregulated in brown and white adipose tissues (BAT and muscle tissues of Senp2 mice, 15 min post injection. The and WAT) (Supplementary Figure S1A and B) but was not in heart, data conﬁrmed the less adipose fat storage accompanied by an adqcKO adqcKO liver, and skeletal muscle of Senp2 mice (Supplementary ectopic fat accumulation in other non-adipose tissues of Senp2 adqcKO Figure S1B). Senp2 mice showed almost similar growth and mice. More importantly, the data also showed the defect in fat f/f adqcKO fertility when compared to Senp2 mice fed with normal chow uptake existed only in adipose tissues in Senp2 mice. diets (NCD) (Supplementary Figure S1C). Nuclear magnetic reson- We would expect insulin resistance accompanied with adqcKO adqcKO ance (NMR) analysis detected slightly less fat mass in Senp2 ectopic fat accumulation (Guilherme et al., 2008)in Senp2 f/f mice than in Senp2 mice at age of 24 weeks old (Supplementary mice. We thus assessed blood glucose and insulin intolerance. Figure S1D). Fluorescence-activated cell sorting (FACS) analysis Supplementary Figure S2C and D showed that the responses of adqcKO demonstrated that there was no difference in the population glucose and insulin tolerance in Senp2 were similar to that − + f/f and proliferation of CD45 PDGFRα adipocyte progenitor cells in Senp2 mice fed with NCD. However, under HFD, the ability adqcKO f/f adqcKO (Lee et al., 2012) between Senp2 and Senp2 mice for glucose clearance as well as insulin sensitivity in Senp2 f/f (Supplementary Figure S1E), indicating that adipocyte Senp2 mice were markedly reduced compared with Senp2 mice deﬁciency in our model did not affect adipocyte progenitors. (Figure 2D and E). In consistence, fasting glucose and insulin adqcKO f/f We then tested the role of adipose Senp2 in obesity by feeding levels were elevated more in Senp2 mice than in Senp2 f/f adqcKO Senp2 and Senp2 mice with HFD. During the ﬁrst 9 weeks mice (Figure 2F). We also examined insulin signaling in both f/f adqcKO of HFD feeding, no signiﬁcant difference in the gained body Senp2 and Senp2 mice fed with HFD. Following insulin adqcKO f/f f/f weight was observed between Senp2 and Senp2 mice. injection, Senp2 mice displayed a robust increase in AKT f/f From 10 weeks after feeding with HFD, Senp2 mice continued to phosphorylation (at T308 and S473 sites) in eWAT, gastrocne- adqcKO increase body weight, while Senp2 mice showed a decrease mius muscle, and liver. However, this response was greatly adqcKO in the gained body weight (Figure 1B). Consistently, both BAT attenuated in Senp2 mice (Figure 2G), indicating that adqcKO adqcKO and WAT in Senp2 mice were over 50% smaller than that in insulin signaling was impaired in Senp2 mice, which f/f Senp2 male mice (Figure 1C and D). Histologically, we observed resulted in insulin resistance observed in these mice. adqcKO much smaller size of adipocytes in Senp2 mice than in f/f Senp2 mice fed with HFD (Figure 1E and F), indicating that the Senp2 modulates Pparγ and Cebpα function in adipose tissues adqcKO less fat mass in Senp2 mice might be a result of less adi- To characterize the molecular basis of less adipose lipid storage adqcKO pose lipid storage during obesity. Additionally, we found that observed in Senp2 mice, we analyzed the expression of the adqcKO Senp2 mice had much more inﬂammation-related crown-like genes related to lipid metabolism in adipose tissues, which include f/f structures (Lee et al., 2014)inWAT than Senp2 did (Figure 1E). the genes that involved in fatty acid uptake and trafﬁcking (e.g. FACS analysis also conﬁrmed this result by showing more macro- Lpl, Cd36,Fabp4), and genes in lipid synthesis (e.g. Fasn, Srebp, adqcKO f/f phages inﬁltration in Senp2 WAT than in Senp2 WAT Dgat2,and Slc25a1). We detected a minor reduction in the Downloaded from https://academic.oup.com/jmcb/article-abstract/10/3/258/4763638 by Ed 'DeepDyve' Gillespie user on 26 June 2018 260 j Zheng et al. Figure 1 Senp2 deﬁciency decreases adipose lipid storage on HFD. (A) Senp2 expression was upregulated in adipose tissues of obese mice f/f adqcKO (n = 6–10/group). (B) The body weights of Senp2 and Senp2 male mice during 15 weeks of HFD feeding (n = 10/group). (C) The size f/f adqcKO of BAT, inguinal and epididymal fat pads of Senp2 and Senp2 male mice at the end of 15 weeks of HFD feeding. (D) The organ f/f adqcKO weights / body weight of Senp2 and Senp2 male mice at the end of 15 weeks of HFD feeding (n = 5/group). (E) H&E staining of rep- f/f adqcKO resentative sections of BAT, iWAT, and eWAT from Senp2 and Senp2 male mice at the end of 15 weeks of HFD feeding. Scale bar, 100 μm. (F) The size of adipocytes in the above iWAT and eWAT sections were analyzed by using Image J software (n > 200 cells/group). + + + + + f/f adqcKO (G) Flow cytometry analysis of adipose-associated macrophages (F4/80 CD11b or F4/80 CD11b CD11c )in Senp2 and Senp2 mice fed on HFD (n = 5/group). Data presented in A, B, D, F, and G are mean ± SEM, Student’s t-test, *P < 0.05,**P < 0.01. adqcKO expression of some genes in adipose tissues of Senp2 addition, Adiponectin (Adipoq) were also downregulated in adqcKO mice on NCD (Supplementary Figure S3A).However,the most Senp2 mice (Figure 3Aand B). of the genes were signiﬁcantly downregulated in adipose tis- Interestingly, almost all these genes are mainly controlled by adqcKO sues of Senp2 mice when fed with HFD (Figure 3A). In master transcription factors Pparγ and Cebpα in adipocytes Downloaded from https://academic.oup.com/jmcb/article-abstract/10/3/258/4763638 by Ed 'DeepDyve' Gillespie user on 26 June 2018 Senp2 regulates adipose lipid storage by de-SUMOylation of Setdb1 j 261 adqcKO Figure 2 Senp2 mice exhibit an ectopic lipid accumulation and insulin resistance. (A) The concentrations of serum TGs, cholesterol, and f/f adqcKO NEFA in Senp2 and Senp2 male mice at the end of 15 weeks of HFD feeding (n = 5/group). (B) H&E staining of representative liver sec- f/f adqcKO 14 tions from Senp2 and Senp2 male mice at the end of 15 weeks of HFD feeding. Scale bar, 100 μm. (C) The amount of C-labeled oleate 14 f/f adqcKO uptake in the indicated organs during 15 min after intravenous injection of C-labeled oleate in Senp2 and Senp2 male mice (n = 5/group). f/f adqcKO (D) Glucose tolerance test in Senp2 and Senp2 male mice at the end of 15 weeks of HFD feeding (n = 5/group). (E) Insulin tolerance test f/f adqcKO in Senp2 and Senp2 male mice at the end of 15 weeks of HFD feeding (n = 5/group). (F) The concentrations of blood glucose or serum insu- f/f adqcKO lin in Senp2 and Senp2 male mice at the end of 15 weeks of HFD feeding (n = 5/group). (G) AKT and phosphorylated AKT were blotted in f/f adqcKO lysates of eWAT, gastrocnemius muscle, or liver from Senp2 and Senp2 male mice with or without insulin injection at the end of 15 weeks of HFD feeding. Data presented in A and C–F are mean ± SEM, Student’s t-test, *P < 0.05,**P < 0.01. (Lefterova et al., 2008). We therefore determined whether adi- adipogenesis (Matsumura et al., 2015). We asked whether this pocyte Senp2 deﬁciency could alter Pparγ and Cebpα in adipose mechanism would work in Senp2 regulation of Pparg and Cebpa adqcKO tissues. As shown in Figure 3C and D, Senp2 mice had expressions either. Indeed, chromatin immunoprecipitation f/f much lower levels of both Pparg and Cebpa than Senp2 mice did, (ChIP) assay detected a signiﬁcant increase of H3K9me3, but suggesting an essential role of Senp2 in regulation of the expres- not H3K4me3 or H3K27me3 in the promoter locus of Pparg and adqcKO sion of both Pparγ and Cebpα. Additionally, we detected accumula- Cebpa in Senp2 adipocytes (Figure 3E). This result indi- adqcKO tion of SUMOylated Pparγ in adipose tissues of Senp2 mice cated that Pparg and Cebpa expressions could be suppressed (Supplementary Figure S3B). Since SUMOylation suppresses Pparγ by the increased H3K9me3. transcription activity (Dutchak et al., 2012), the accumulation of Since Setdb1 as a histone methyltransferase for H3K9 tri- SUMOylated Pparγ might contribute to the downregulation of methylation regulates Cebpa and Pparg transcriptions in adipo- adqcKO Pparγ activation in Senp2 mice. cytes (Matsumura et al., 2015), we thus asked whether Setdb1 adqcKO would control H3K9 trimethylation in Senp2 adipocytes. ChIP assay detected a signiﬁcant increase in Setdb1 occupancy in adqcKO Senp2 deﬁciency enhances H3K9 trimethylation in locus of the locus of Pparg and Cebpa promoters in Senp2 adipo- Pparg and Cebpa cytes (Figure 3F), suggesting that Senp2 deﬁciency would modu- H3K4/H3K9me3 bivalent domains have been shown as a late Setdb1 action on H3K9 trimethylation to suppress Pparg and repressive mechanism for Pparg and Cebpa expression during Cebpa expressions in adipocytes. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/3/258/4763638 by Ed 'DeepDyve' Gillespie user on 26 June 2018 262 j Zheng et al. Figure 3 Senp2 regulates Pparg and Cebpa expression through SETDB1-mediated H3K9me3 in adipose tissues. (A) RT-PCR analysis of genes f/f adqcKO related to lipid metabolism in adipose tissues from Senp2 or Senp2 mice at the end of 15 weeks of HFD feeding (n = 4/group). f/f adqcKO (B) The concentration of serum adiponectin in Senp2 or Senp2 male mice at the end of 15 weeks of HFD feeding (n = 3/group). f/f adqcKO (C) RT-PCR analysis of Pparg and Cebpa genes in adipose tissues from Senp2 or Senp2 mice at the end of 15 weeks of HFD feeding f/f adqcKO (n = 4/group). (D) Pparγ and Cebpα were blotted in adipose lysates from Senp2 or Senp2 male mice at the end of 15 weeks of HFD feeding. (E) ChIP analysis of H3K4me3,H3K9me3,orH3K27me3 occupancy in the locus of Pparg and Cebpa promoters in the adipose of f/f adqcKO Senp2 or Senp2 male mice on HFD (n = 4/group). (F) ChIP analysis of Setdb1 occupancy in the locus of Pparg and Cebpa promoters f/f adqcKO in the adipose of Senp2 or Senp2 male mice on HFD (n = 4/group). Data presented in A–C, E, and F are mean ± SEM, Student’s t-test, *P < 0.05,**P < 0.01. Senp2 controls Pparg and Cebpa expression by de-SUMOylation Figure S4B). ChIP assay demonstrated that much more Flag- of Setdb1 Setdb1-WT than K1050R mutant could be recruited to the pro- We next asked how Senp2 regulates Setdb1. Since Setdb1 moter locus of Pparg and Cebpa in 3T3-L1-Tet-on-shSenp2 has been reported as a SUMOylated protein (although date not cells (Figure 4F). We then silenced Setdb1 in Senp2-knockdown shown) (Hendriks et al., 2015), we proposed that Setdb1 would 3T3-L1 cells and detected an increase in Pparγ and Cebpα be de-SUMOylated by Senp2. To test it, we ﬁrst co-transfected expressions in these cells (Figure 4G and H). Interestingly, re- Setdb1 and SUMO plasmids in 293T cells and conﬁrmed that expression of Setdb1-WT but not Setdb1-K1050R could reduce Setdb1 was conjugated by either SUMO1 or SUMO2 (Figure 4A). Pparg and Cebpa expressions in Senp2 and Setdb1 double Interestingly, Senp2 could selectively de-conjugate SUMO2-con- silenced cells (Figure 4G and H). Consistent with this, knock- jugated but not SUMO1-conjugated Setdb1 (Figure 4B). We fur- down of endogenous Setdb1 increased the expression of Cd36, ther mapped Lys1050 as a SUMO2 acceptor site on Setdb1. Fasn, and Dgat2 genes (Figure 4H) and lipid droplets accumula- Mutation of K1050R completely depleted SUMO2 conjugation tion in Senp2-silenced 3T3-L1 cells (Supplementary Figure S4C). on Setdb1 (Figure 4C). Moreover, the accumulation of SUMO2- Re-expression of Setdb1-WT in these cells reduced the expression adqcKO Setdb1 was shown in the adipocytes of Senp2 mice and of CD36, Fasn,and Dgat2 genes, while Setdb1-K1050R could not Senp2-knockdown 3T3-L1-Tet-on-shSenp2 cells (treated by doxy- change their expression (Figure 4H). Taken together, these data cycline) (Figure 4D and E). These data suggested that Setdb1 could revealed that SUMOylation promoted Setdb1 recruitment to the be regulated by Senp2-dependent de-SUMOylation. locus of Pparg and Cebpa, which subsequently inhibited Pparg To further determine the role of SUMOylation in Setdb1 activ- and Cebpa expressions in adipocytes. ity, we generated Flag-Setdb1-WT or Flag-Setdb1-K1050R stably expressed 3T3-L1-Tet-on-shSenp2 cells (Supplementary Figure S4A). Discussion SUMO2-conjugated Flag-Setdb1 were detected only in Flag-Setdb1- In this study, we utilized adipocyte Senp2-deﬁciency mice model WT but not in Flag-Setdb1-K1050R expressed cells (Supplementary to determine the role of Senp2 in adipose lipid storage. Adipose Downloaded from https://academic.oup.com/jmcb/article-abstract/10/3/258/4763638 by Ed 'DeepDyve' Gillespie user on 26 June 2018 Senp2 regulates adipose lipid storage by de-SUMOylation of Setdb1 j 263 Figure 4 Senp2 regulates SETDB1 activity by de-SUMOylation. (A) 293T cells were cotransfected with Flag-Setdb1, HA-SUMO1, or HA- SUMO2 plasmids as indicated. The cell lysates were immunoprecipitated (IP) with anti-Flag antibody and followed by blot (IB) with anti-HA or anti-Flag antibody. Whole-cell lysates were blotted (IB) with anti-HA antibody. (B) 293T cells were cotransfected with Flag-Setdb1, HA-SUMO1, HA-SUMO2, Myc-Senp2, or Myc-Senp2m (catalytic mutant) as indicated. The cell lysates were IP with anti-Flag antibody and fol- lowed by IB with anti-HA or anti-Flag antibody. Whole-cell lysates were IB with anti-HA or anti-Myc antibody. (C) 293T cells were co-trans- fected with Flag-Setdb1-WT, Flag-Setdb1-K355R, Flag-Setdb1-K1050R, or HA-SUMO2 as indicated. The cell lysates were IP with anti-Flag antibody and followed by IB with anti-HA or anti-Flag antibody. Whole-cell lysates were IB with anti-HA antibody. (D) Adipose tissues extract f/f adqcKO from Senp2 or Senp2 mice were IP with anti-SUMO2 antibody and followed by IB with anti-SETDB1 antibody. Whole lysates were IB with anti-SETDB1 or anti-SUMO2 antibody. (E) 3T3-L1-Tet-on-shSenp2 cell were treated with or without doxycycline at day 12 of differenti- ation procedure. The cell lysates were IP with anti-SUMO2 antibody and followed by IB with anti-Setdb1 antibody. Whole lysates were IB with anti-Setdb1 or anti-SUMO2 antibodies. (F) Flag-Setdb1-WT or Flag-Setdb1-K1050R occupancy was analyzed by ChIP with anti-Flag Downloaded from https://academic.oup.com/jmcb/article-abstract/10/3/258/4763638 by Ed 'DeepDyve' Gillespie user on 26 June 2018 264 j Zheng et al. Senp2 deﬁciency resulted in less adipose lipid storage as well as Materials and methods an ectopic fat accumulation and insulin resistance in mice fed with Mice f/f HFD. Interestingly, Senp2 expression was increased in obese adi- Senp2 mice were generated as previously described pose tissues. These data suggest an essential role of Senp2 for (Qi et al., 2014b). Adiponectin-cre mice were from Dr Jiqiu Wang storing excess fat in adipose tissues during obesity. This function (Ruijin Hospital, Shanghai Jiao Tong University School of Medicine). would beneﬁt the body by protecting from lipotoxicity during Mice were maintained at room temperature (22°C), with a 12-h obesity. light-dark cycle and free access tofoodand water. Fordietexperi- We found that Senp2 is essential for Pparγ and Cebpα expres- ments, 7-week-old male mice were fed HFD with 60%kcalfat sions by suppressing Setdb1-mediated H3K9me3. Setdb1 is a (Research Diets, D12492)for 16 weeks. Minispec TD-NMR SUMOylatd protein (Hendriks et al., 2015;Figure 4). We mapped Analysers (Bruker Instruments) were performed to evaluate SUMO acceptor site at K1050 of Setdb1.Setdb1 has a structure body composition. The experiments were not randomized, composed of evolutionarily conserved SET, pre-SET, and post-SET and the investigators were not blinded to allocation during domain, while the SET domain of Setdb1 is interrupted by the experiments or outcome assessments. No statistical analysis insertion of several hundred amino acids (Kang, 2015). K1050 was applied to predetermine sample size. All procedures locates in the insertion sequence of Setdb1. We showed that were evaluated and approved by the Animal Care Committee SUMOylation was essential for Setdb1 occupancy to the promoter of Shanghai Jiaotong University School of Medicine. of Pparg and Cebpa genes. However, the global H3K9me3 levels did not change in Setdb1 K1050R expression cells (data not GTT, ITT, and blood glucose shown). These data suggest that SUMOylation mainly affect For glucose tolerance test, mice were fasted for 14 h and Setdb1 binding to chromatin but not its catalytic activity. Setdb1 injected with D-glucose (2 g/kg) intraperitoneally. For insulin has been shown to form a complex with MBD1 and MCAF1 tolerance test, mice were fasted for 6 h and injected with recom- (MBD1-containing chromatin-associated factor 1) to catalyze binant human insulin (0.75 U/kg) intraperitoneally. Blood was H3K9me3 on Pparg and Cebpa loci (Matsumura et al., 2015). It drawn from the tail vein and measured by glucose meters would be possible that SUMOylation may regulate the interaction (OneTouch Ultra). between Setdb1 and MBD1/MCAF1 to promote Setdb1 bound to the promoter of Pparg and Cebpa genes. Serum insulin and adiponectin Senp2 can reverse the Setdb1 suppressionbyde-SUMOylation. Mice were fasted for 6 h before blood samples were collected Interestingly, Setdb1 canbeSUMOylatedbySUMO1 and SUMO2. through retro-orbital bleeding and then centrifuged for 20 min. However, Senp2 only de-SUMOylates SUMO2-conjugated but not Serum insulin and adiponectin levels were measured using com- SUMO1-conjugated Setdb1.Although SENP2 has been reported to mercial ELISA kits (CrystalChem, Cayman Chemical). be prone to SUMO2-conjugated protein, it is unknown how Senp2 to distinguish SUMO1-orSUMO2-conjugated proteins for de- Serum triglyceride, cholesterol, and NEFA SUMOylation. If Senp2 activity is reduced, the expression of Pparg Total serum triglyceride, cholesterol, and NEFA were enzyma- and Cebpa genes would be suppressed by Setdb1 SUMOylation tically measured with Serum Triglyceride Determination Kit through increased H3K9me3.Therefore, we deﬁne aSenp2-Setdb1 (Sigma), Mouse Cholesterol ELISA Kit (MyBioSource), and Free axis in controlling adipose lipid metabolism by reversible Fatty Acid Quantiﬁcation Kit (BioVision), respectively, according SUMOylation. to the manufacturer’s instructions. The disruption of Pparγ2 or adipocyte-speciﬁc Pparγ knockout has been shown to cause severe lipodystrophy and insulin resist- ance in mice (He et al., 2003; Zhang et al., 2004; Duan et al., Organ C-oleate uptake studies 2007). In human, PPARγ mutation also links to familial partial Organ C-oleate uptake was determined as described previ- lipodystrophy. Although adipose Senp2 dysfunction has been ously (Schlein et al., 2016). In brief, oleate containing C-oleate shown to result in lipodystrophy-like phenotype by suppression tracer (0.5MBq/kg body weight) diluted in PBS (33 mg/ml) within of Pparγ expression in mice, it is unknown whether SENP2 dys- 5-fold molar excess to fatty acid-free BSA was injected intraven- function occurs in human lipodystrophy case. Thus, it is worth to ously 10 min prior to necropsy. Plasma samples were collected ﬁnd out whether SENP2 mutation or other SENP2 dysfunction through retro-orbital bleeding before sacriﬁced, and organs were events occur in adipocytes in lipodystrophy patients. harvested after systemic perfusion with PBS-heparin (10 U/ml). antibody in the locus of Pparg and Cebpa promoters in 3T3-L1-Tet-on-shSenp2 cells stably transfected with GFP, Setdb1-WT, or Setdb1- K1050R at Day 12 of differentiation procedure (n = 4/group). Data presented are mean ± SD, **P < 0.01.(G) 3T3-L1-Tet-on-shSenp2 cells were stably co-transfected with GFP, Setdb1-WT, Setdb1-K1050R, siNC (non-speciﬁc siRNA), or siSetdb1 (against Setdb1 siRNA) as indicated. Whole proteins from these cells with or without Dox treatment were extracted and blotted with anti-Ppar, anti-Cebp, anti-Setdb1, anti- Senp2, or anti-TUBULIN antibody. (H) The mRNA levels of selected genes were analyzed by RT-PCR in the cells in G (n = 4/group). Data pre- sented are mean ± SD, **P < 0.01. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/3/258/4763638 by Ed 'DeepDyve' Gillespie user on 26 June 2018 Senp2 regulates adipose lipid storage by de-SUMOylation of Setdb1 j 265 Tissues were minced and digested by Solvable (PerkinElmer) for fully differentiated 3T3-L1 cellswereﬁxed with 4% paraformalde- counting using Tricarb2910TR (PerkinElmer). hyde for 20 min followed by Oil Red O incubation for 30 min. RNA isolation and quantitative RT-PCR Histological analysis Total RNA was extracted from cells or tissues using TRIzol Tissues were harvested and ﬁxed with 4% paraformaldehyde, reagent (Roche). 1 μg RNA was reverse transcribed into com- dehydrated, parafﬁn-embedded, and sectioned (5 μm) prior to plementary DNA with a cDNA synthesis kit (Takara) as per hematoxylin and eosin (H&E) staining. For cell size measure- instruction. Real-time PCR was performed on the LC480system ments, cross-sectional areas of adipocytes were calculated by (Roche) using SYBER Green Supermix (Takara). Values were tracing the adipocyte periphery in ImageJ. normalized to ribosomal protein S18 (Rps18) levels using the ΔΔCt method. Primers used in this study were listed in Stromal vascular cells isolation and ﬂow cytometry Supplementary Table S1. WAT was minced, digested, and ﬁltered, and SVCs were iso- lated as described (Church et al., 2014). For adipose precursor Chromatin immunoprecipitation assay cell analysis, SVCs were stained with anti-CD45 (eBiosciences), Adipose tissues were minced into small pieces (1–3 mm )in anti-PDGFRα (eBiosciences), and anti-Ki67 (eBiosciences) simul- PBS supplemented with protease inhibitors and crosslinked taneously. For myeloid cell analysis, SVCs were stained with with 1% formaldehyde. Tissues were centrifuged at 1350× g for anti-F4/80 (eBiosciences), anti-CD11b (eBiosciences) and anti- 5 min at room temperature. The lipid-rich tissue pieces (upper CD11c (eBiosciences). Cells were analyzed using FACS Verse layer) were washed with PBS and subjected into ChIP analysis. (BD Bioscience) and FlowJo software. 3T3-L1 cells were crosslinked with 1% paraformaldehyde. ChIP– qPCR assays were performed as described previously (Wang et al., Plasmid and stable cell line construction 2015). Brieﬂy, cross-linked cells were sonicated. Solubilized chro- To generate stable 3T3-L1-Tet-on-shSenp2 cells, shRNA-targeting matin was immunoprecipitated with antibodies against H3K4me3, Senp2 gene was constructed in pLKO-Tet-On vector, the shRNA- H3K9me3,H3K27me3,SETDB1, Flag or negative control IgG. targeting sequences are shown in Supplementary Table S1.The Antibody–chromatin complexes were pulled down using protein A- shRNA lentivirus were packaged and infected in 3T3-L1 cells, then sepharose (Millipore). Beads were then washed, and eluted. After stable clones were selected with 3 μg/ml puromycin for 1 week. crosslink reversal and proteinase K treatment, immunoprecipitated Setdb1 and Setdb1 mutant constructs were cloned into pFlag- DNA was extracted with phenol–chloroform followed by ethanol CMV2 expression vectors and pCDH-GFP lentiviral backbone precipitation. The DNA fragments were further analyzed by qPCR. using standard PCR-based cloning strategies and veriﬁed by DNA Primer sequences are provided in Supplementary Table S1. sequencing. To generate 3T3-L1-Tet-on-shSenp2 cells stably expressing wild-type or K1050R-mutated Flag-Setdb1, Setdb1 Immunoprecipitation and immunoblotting and Setdb1 mutant lentivirus were packaged and infected in 3T3- Cells or tissues were collected and lysed in radioimmuno- L1-Tet-on-shSenp2 cells. The cells were selected by FACS. precipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 400 mM NaCl, 1%TritonX-100, 0.1%SDS, 1 mM PMSF, 10 Cell culture and Oil Red O staining mM N-ethylmaleimide, and protease inhibitors). Cell lysates HEK-293Tand 3T3-L1 cells were cultured in DMEM were then sonicated and centrifuged at 20000× g for 15 min (Hyclone) supplemented with 10%FBS (Gibco)and 100 μg/ml at 4°C, and the supernatants were added to the appropriate penicillin/streptomycin. All cells were maintained in a humidi- antibody coupled with protein A/G beads. After incubation for 6 h ﬁed 37°C/5%CO incubator. For adipocyte differentiation assay, at 4°C, beads were washed with RIPA buffer and eluted in 2%SDS 3T3-L1-Tet-on-shSenp2 cells were cultured and reached at full solution then analyzed by Immunoblotting. Proteins were detected conﬂuency for 2 days. Then cells were induced by the addition by antibodies, including anti-SENP2 (sc-67075), anti-PPARγ (2430), of insulin (5 μg/ml, Sigma), dexamethasone (1 μM, Sigma), and anti-C/EBPα (ab40764), anti-SUMO2/3 (ab81371), anti-GAPDH isobutyl-1-methylxanthine (0.5 mM, Sigma). After 2 days, the (ab8245), anti-TUBULIN (T9026), anti-pAKT T308 (9275), anti-pAKT medium was replaced by growth medium supplemented with S473 (ab18206), anti-AKT (9272), anti-HA (H9658), anti-Flag insulin for 10 days. This medium was changed every 2 days until (F3162), anti-Myc (9402), and anti-SETDB1(ab12317). the end of differentiation. To knockdown endogenous Senp2, 3T3-L1-Tet-on-shSenp2 cells were treated with doxycycline (0.1 μg/ml, Sigma) for 6 days after adipocyte differentiation. For Statistical analysis knockdown of endogenous Setdb1, three pairs of siRNA were All data derived from cells are represented as mean ± stand- designed based on 3′ UTR of Setdb1 mRNA. The siRNA ard deviation (SD), data from tissues are represented as mean ± sequences are presented in Supplementary Table S1.siRNAs standard error of the mean (SEM). Student’s t-test was performed (20 μM) were transfected into differentiated cells by TurboFect to analyze signiﬁcance (*P < 0.05;**P < 0.01). Graphs were gen- Transfection Reagent (Thermo Scientiﬁc). For Oil Red O staining, erated by Prism software packages. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/3/258/4763638 by Ed 'DeepDyve' Gillespie user on 26 June 2018 266 j Zheng et al. Hajri, T., and Abumrad, N.A. (2002). Fatty acid transport across membranes: rele- Supplementary material vance to nutrition and metabolic pathology. Annu. Rev. Nutr. 22, 383–415. Supplementary material is available at Journal of Molecular He, W., Barak, Y., Hevener, A., et al. (2003). Adipose-speciﬁc peroxisome Cell Biology online. proliferator-activated receptor γ knockout causes insulin resistance in fat and liver but not in muscle. Proc. Natl Acad. Sci. USA 100, 15712–15717. Hendriks, I.A., Treffers, L.W., Verlaan-de Vries, M., et al. (2015). SUMO-2 Funding orchestrates chromatin modiﬁers in response to DNA damage. Cell Rep. This work was supported by grants from the National Natural pii: S2211-1247(15)00179-5. Science Foundation of China (91019021 and 81430069 to J.C.), Jeffery, E., Berry, R., Church, C.D., et al. (2014). Characterization of Cre recom- the National Basic Research Program of China (973 Program) binase models for the study of adipose tissue. Adipocyte 3, 206–211. (2013CB910902 to J.C.), Shanghai Committee of Science and Kang, Y.K. (2015). SETDB1 in early embryos and embryonic stem cells. Curr. Issues Mol. Biol. 17, 1–10. Technology (15ZR1424500 to T.W. and 15140904300), Shanghai Koo, Y.D., Choi, J.W., Kim, M., et al. (2015). SUMO-speciﬁc protease 2 Municipal Education Commission (ZZjdyx15003 to T.W. and (SENP2) is an important regulator of fatty acid metabolism in skeletal mus- 2017-01-07-00-01-E00050 to J.C.), and Shanghai Jiao Tong cle. Diabetes 64, 2420–2431. University School of Medicine (14XJ10001 to T.W.). Lee, Y.S., Kim, J.W., Osborne, O., et al. (2014). Increased adipocyte O con- sumption triggers HIF-1α, causing inﬂammation and insulin resistance in Conﬂict of interest: none declared. obesity. Cell 157, 1339–1352. Lee, Y.H., Petkova, A.P., Mottillo, E.P., et al. (2012). 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Journal of Molecular Cell Biology – Oxford University Press
Published: Feb 14, 2018
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