Senp2 regulates adipose lipid storage by de-SUMOylation of Setdb1

Senp2 regulates adipose lipid storage by de-SUMOylation of Setdb1 Abstract One major function of adipocytes is to store excess energy in the form of triglycerides. Insufficient adipose lipid storage is associated 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 feeding. 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. lipid storage, Senp2, Setdb1, H3K9me3, Pparg and Cebpa Introduction Adipose tissue has an important role in nutrient homeostasis by either storing excess energy in the form of triglycerides (TGs) or hydrolyzing TGs to provide fuels for the rest of the body under fasting condition (Rosen and Spiegelman, 2014; Cohen and Spiegelman, 2016). Excess or insufficient lipid storage in adipose tissues would impair nutrient homeostasis, which is associated with many pathological conditions such as hyperlipidemia, insulin resistance, and type 2 diabetes (Garg, 2004; Agarwal and Garg, 2006; Rosen and Spiegelman, 2006; Liu et al., 2014; Patni and Garg, 2015). Under physiological conditions, the maintenance of normal adipose tissue mass is mainly the result of a balance of lipid storage and lipolysis (Bouchard et al., 1993). Cellular uptake of fatty acids and following storage in the form of TGs in adipocytes are key steps in lipid storage. These processes occur primarily through protein-mediated pathways consisting of a number of fatty acid transporters such as fatty acid translocase (Cd36) and fatty acid transport protein-1 (Fatp1) (Hajri and Abumrad, 2002). Lipolysis proceeds through successive steps involving the release of a single fatty acid chain and the generation of the intermediate species of diacylglycerol (DAG) and monoacylglycerol (MAG) (Young and Zechner, 2013). As a master regulator of adipogenesis, peroxisome proliferator-activated receptors γ (PPARγ) can modulate adipose tissue mass by regulating lipogenesis-related genes (Ahmadian et al., 2013). PPARγ mutation has been shown to link to familial partial lipodystrophy, a clinical disorder characterized by the loss of adipose tissues (Dunnigan et al., 1974; Savage et al., 2003). Selective disruption of Pparγ2 or adipocyte-specific Pparγ knockout leads to severe lipodystrophy and induces insulin resistance in mice (He et al., 2003; Zhang et al., 2004; Duan et al., 2007). Recent studies have shown that SUMO-specific protease 2 (Senp2) is involved in myogenesis (Qi et al., 2014a) and adipogenesis (Chung et al., 2010). Senp2 also regulates fatty acid metabolism in skeletal muscle (Koo et al., 2015). In this report, we observed a defect in adipose lipid storage in adipocyte-specific Senp2 knockout mice fed with high-fat diets (HFD). This mouse also showed an ectopic lipid distribution and insulin resistance. Therefore, adipocyte-specific Senp2 knockout mice exhibited a lipodystrophy-like phenotype. Mechanistically, adipocyte Senp2 deficiency caused the downregulation of Pparg and Cebpa as well as their downstream target genes related to lipid storage. We found that SET domain bifurcated 1 (Setdb1) was a SUMOylated protein and that Senp2 de-SUMOylated and regulated Setdb1 action in trimethylation at histone 3 lysine 9 (H3K9me3). Senp2 deficiency triggered a hyper-SUMOylation of Setdb1, which enhanced its occupancy on Pparg and Cebpa promoter loci to suppress their transcriptions. Our findings unravel that Senp2 is essential for regulating lipid metabolism in adipose tissues. Results Senp2 deficiency decreases adipose lipid storage Since Senp2 expression was dramatically increased in adipose tissues in several obese mouse models (Figure 1A), suggesting that Senp2 may play a role in the pathogenesis of obesity in adipose tissues. To investigate it, we generated a mouse model in which Senp2 was specifically knockout in adipose tissues (Senp2adqcKO) by crossing Senp2f/f with Adiponectin-cre mice (Lee et al., 2013; Jeffery et al., 2014). Senp2 expression was significantly downregulated in brown and white adipose tissues (BAT and WAT) (Supplementary Figure S1A and B) but was not in heart, liver, and skeletal muscle of Senp2adqcKO mice (Supplementary Figure S1B). Senp2adqcKO mice showed almost similar growth and fertility when compared to Senp2f/f mice fed with normal chow diets (NCD) (Supplementary Figure S1C). Nuclear magnetic resonance (NMR) analysis detected slightly less fat mass in Senp2adqcKO mice than in Senp2f/f mice at age of 24 weeks old (Supplementary Figure S1D). Fluorescence-activated cell sorting (FACS) analysis demonstrated that there was no difference in the population and proliferation of CD45−PDGFRα+ adipocyte progenitor cells (Lee et al., 2012) between Senp2adqcKO and Senp2f/f mice (Supplementary Figure S1E), indicating that adipocyte Senp2 deficiency in our model did not affect adipocyte progenitors. Figure 1 View largeDownload slide Senp2 deficiency decreases adipose lipid storage on HFD. (A) Senp2 expression was upregulated in adipose tissues of obese mice (n = 6–10/group). (B) The body weights of Senp2f/f and Senp2adqcKO male mice during 15 weeks of HFD feeding (n = 10/group). (C) The size of BAT, inguinal and epididymal fat pads of Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding. (D) The organ weights / body weight of Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (E) H&E staining of representative sections of BAT, iWAT, and eWAT from Senp2f/f and Senp2adqcKO 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). (G) Flow cytometry analysis of adipose-associated macrophages (F4/80+CD11b+ or F4/80+CD11b+CD11c+) in Senp2f/f and Senp2adqcKO 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. Figure 1 View largeDownload slide Senp2 deficiency decreases adipose lipid storage on HFD. (A) Senp2 expression was upregulated in adipose tissues of obese mice (n = 6–10/group). (B) The body weights of Senp2f/f and Senp2adqcKO male mice during 15 weeks of HFD feeding (n = 10/group). (C) The size of BAT, inguinal and epididymal fat pads of Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding. (D) The organ weights / body weight of Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (E) H&E staining of representative sections of BAT, iWAT, and eWAT from Senp2f/f and Senp2adqcKO 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). (G) Flow cytometry analysis of adipose-associated macrophages (F4/80+CD11b+ or F4/80+CD11b+CD11c+) in Senp2f/f and Senp2adqcKO 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. We then tested the role of adipose Senp2 in obesity by feeding Senp2f/f and Senp2adqcKOmice with HFD. During the first 9 weeks of HFD feeding, no significant difference in the gained body weight was observed between Senp2adqcKO and Senp2f/f mice. From 10 weeks after feeding with HFD, Senp2f/f mice continued to increase body weight, while Senp2adqcKO mice showed a decrease in the gained body weight (Figure 1B). Consistently, both BAT and WAT in Senp2adqcKO mice were over 50% smaller than that in Senp2f/f male mice (Figure 1C and D). Histologically, we observed much smaller size of adipocytes in Senp2adqcKO mice than in Senp2f/f mice fed with HFD (Figure 1E and F), indicating that the less fat mass in Senp2adqcKO mice might be a result of less adipose lipid storage during obesity. Additionally, we found that Senp2adqcKO mice had much more inflammation-related crown-like structures (Lee et al., 2014) in WAT than Senp2f/f did (Figure 1E). FACS analysis also confirmed this result by showing more macrophages infiltration in Senp2adqcKO WAT than in Senp2f/f WAT (Figure 1G and Supplementary Figure S1F). Although less fat storage was shown in Senp2adqcKO adipose tissues, the increased inflammation indicates that HFD induced adipocyte death would remain in adipose tissues. Senp2adqcKO mice exhibit an ectopic lipid accumulation and insulin resistance Since no difference in food intake was observed between Senp2adqcKO and Senp2f/f mice fed either with NCD or HFD (Supplementary Figure S2A). We next assessed the serum triglyceride, cholesterol, and non-esterified fatty acid (NEFA) in aforementioned mice fed with HFD. Figure 2A demonstrates that their concentrations were much higher in Senp2adqcKO serum than in Senp2f/f serum. We further stained liver sections and showed more severe hepatic steatosis in Senp2adqcKO mice than in Senp2f/f mice (Figure 2B). These data suggest that an ectopic lipid distribution was accompanied with the less adipose lipid storage in Senp2adqcKO mice. Figure 2 View largeDownload slide Senp2adqcKO mice exhibit an ectopic lipid accumulation and insulin resistance. (A) The concentrations of serum TGs, cholesterol, and NEFA in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (B) H&E staining of representative liver sections from Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding. Scale bar, 100 μm. (C) The amount of 14C-labeled oleate uptake in the indicated organs during 15 min after intravenous injection of 14C-labeled oleate in Senp2f/f and Senp2adqcKO male mice (n = 5/group). (D) Glucose tolerance test in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (E) Insulin tolerance test in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (F) The concentrations of blood glucose or serum insulin in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (G) AKT and phosphorylated AKT were blotted in lysates of eWAT, gastrocnemius muscle, or liver from Senp2f/f and Senp2adqcKO 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. Figure 2 View largeDownload slide Senp2adqcKO mice exhibit an ectopic lipid accumulation and insulin resistance. (A) The concentrations of serum TGs, cholesterol, and NEFA in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (B) H&E staining of representative liver sections from Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding. Scale bar, 100 μm. (C) The amount of 14C-labeled oleate uptake in the indicated organs during 15 min after intravenous injection of 14C-labeled oleate in Senp2f/f and Senp2adqcKO male mice (n = 5/group). (D) Glucose tolerance test in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (E) Insulin tolerance test in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (F) The concentrations of blood glucose or serum insulin in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (G) AKT and phosphorylated AKT were blotted in lysates of eWAT, gastrocnemius muscle, or liver from Senp2f/f and Senp2adqcKO 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. To further demonstrate a view of fat distribution, we performed an in vivo analysis for 14C-labeled oleate uptake. An equal injection of initial radiation dose was determined by measuring 14C-oleate counts in the plasma (Supplementary Figure S2B). As demonstrated in Figure 2C, we observed a lower level of 14C-oleate counts in adipose tissues of Senp2adqcKO mice including BAT, iWAT (inguinal WAT), and eWAT (epididymal WAT), but a higher level in the liver, heart, kidney, and muscle tissues of Senp2adqcKO mice, 15 min post injection. The data confirmed the less adipose fat storage accompanied by an ectopic fat accumulation in other non-adipose tissues of Senp2adqcKO mice. More importantly, the data also showed the defect in fat uptake existed only in adipose tissues in Senp2adqcKO mice. We would expect insulin resistance accompanied with ectopic fat accumulation (Guilherme et al., 2008) in Senp2adqcKO mice. We thus assessed blood glucose and insulin intolerance. Supplementary Figure S2C and D showed that the responses of glucose and insulin tolerance in Senp2adqcKO were similar to that in Senp2f/f mice fed with NCD. However, under HFD, the ability for glucose clearance as well as insulin sensitivity in Senp2adqcKO mice were markedly reduced compared with Senp2f/f mice (Figure 2D and E). In consistence, fasting glucose and insulin levels were elevated more in Senp2adqcKO mice than in Senp2f/f mice (Figure 2F). We also examined insulin signaling in both Senp2f/f and Senp2adqcKO mice fed with HFD. Following insulin injection, Senp2f/f mice displayed a robust increase in AKT phosphorylation (at T308 and S473 sites) in eWAT, gastrocnemius muscle, and liver. However, this response was greatly attenuated in Senp2adqcKO mice (Figure 2G), indicating that insulin signaling was impaired in Senp2adqcKO mice, which resulted in insulin resistance observed in these mice. Senp2 modulates Pparγ and Cebpα function in adipose tissues To characterize the molecular basis of less adipose lipid storage observed in Senp2adqcKO mice, we analyzed the expression of the genes related to lipid metabolism in adipose tissues, which include the genes that involved in fatty acid uptake and trafficking (e.g. Lpl, Cd36, Fabp4), and genes in lipid synthesis (e.g. Fasn, Srebp, Dgat2, and Slc25a1). We detected a minor reduction in the expression of some genes in adipose tissues of Senp2adqcKO mice on NCD (Supplementary Figure S3A). However, the most of the genes were significantly downregulated in adipose tissues of Senp2adqcKO mice when fed with HFD (Figure 3A). In addition, Adiponectin (Adipoq) were also downregulated in Senp2adqcKO mice (Figure 3A and B). Figure 3 View largeDownload slide Senp2 regulates Pparg and Cebpa expression through SETDB1-mediated H3K9me3 in adipose tissues. (A) RT-PCR analysis of genes related to lipid metabolism in adipose tissues from Senp2f/f or Senp2adqcKO mice at the end of 15 weeks of HFD feeding (n = 4/group). (B) The concentration of serum adiponectin in Senp2f/f or Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 3/group). (C) RT-PCR analysis of Pparg and Cebpa genes in adipose tissues from Senp2f/f or Senp2adqcKO mice at the end of 15 weeks of HFD feeding (n = 4/group). (D) Pparγ and Cebpα were blotted in adipose lysates from Senp2f/f or Senp2adqcKO male mice at the end of 15 weeks of HFD feeding. (E) ChIP analysis of H3K4me3, H3K9me3, or H3K27me3 occupancy in the locus of Pparg and Cebpa promoters in the adipose of Senp2f/f or Senp2adqcKO male mice on HFD (n = 4/group). (F) ChIP analysis of Setdb1 occupancy in the locus of Pparg and Cebpa promoters in the adipose of Senp2f/f or Senp2adqcKO 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. Figure 3 View largeDownload slide Senp2 regulates Pparg and Cebpa expression through SETDB1-mediated H3K9me3 in adipose tissues. (A) RT-PCR analysis of genes related to lipid metabolism in adipose tissues from Senp2f/f or Senp2adqcKO mice at the end of 15 weeks of HFD feeding (n = 4/group). (B) The concentration of serum adiponectin in Senp2f/f or Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 3/group). (C) RT-PCR analysis of Pparg and Cebpa genes in adipose tissues from Senp2f/f or Senp2adqcKO mice at the end of 15 weeks of HFD feeding (n = 4/group). (D) Pparγ and Cebpα were blotted in adipose lysates from Senp2f/f or Senp2adqcKO male mice at the end of 15 weeks of HFD feeding. (E) ChIP analysis of H3K4me3, H3K9me3, or H3K27me3 occupancy in the locus of Pparg and Cebpa promoters in the adipose of Senp2f/f or Senp2adqcKO male mice on HFD (n = 4/group). (F) ChIP analysis of Setdb1 occupancy in the locus of Pparg and Cebpa promoters in the adipose of Senp2f/f or Senp2adqcKO 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. Interestingly, almost all these genes are mainly controlled by master transcription factors Pparγ and Cebpα in adipocytes (Lefterova et al., 2008). We therefore determined whether adipocyte Senp2 deficiency could alter Pparγ and Cebpα in adipose tissues. As shown in Figure 3C and D, Senp2adqcKO mice had much lower levels of both Pparg and Cebpa than Senp2f/f mice did, suggesting an essential role of Senp2 in regulation of the expression of both Pparγ and Cebpα. Additionally, we detected accumulation of SUMOylated Pparγ in adipose tissues of Senp2adqcKO mice (Supplementary Figure S3B). Since SUMOylation suppresses Pparγ transcription activity (Dutchak et al., 2012), the accumulation of SUMOylated Pparγ might contribute to the downregulation of Pparγ activation in Senp2adqcKO mice. Senp2 deficiency enhances H3K9 trimethylation in locus of Pparg and Cebpa H3K4/H3K9me3 bivalent domains have been shown as a repressive mechanism for Pparg and Cebpa expression during adipogenesis (Matsumura et al., 2015). We asked whether this mechanism would work in Senp2 regulation of Pparg and Cebpa expressions either. Indeed, chromatin immunoprecipitation (ChIP) assay detected a significant increase of H3K9me3, but not H3K4me3 or H3K27me3 in the promoter locus of Pparg and Cebpa in Senp2adqcKO adipocytes (Figure 3E). This result indicated that Pparg and Cebpa expressions could be suppressed by the increased H3K9me3. Since Setdb1 as a histone methyltransferase for H3K9 trimethylation regulates Cebpa and Pparg transcriptions in adipocytes (Matsumura et al., 2015), we thus asked whether Setdb1 would control H3K9 trimethylation in Senp2adqcKO adipocytes. ChIP assay detected a significant increase in Setdb1 occupancy in the locus of Pparg and Cebpa promoters in Senp2adqcKO adipocytes (Figure 3F), suggesting that Senp2 deficiency would modulate Setdb1 action on H3K9 trimethylation to suppress Pparg and Cebpa expressions in adipocytes. Senp2 controls Pparg and Cebpa expression by de-SUMOylation of Setdb1 We next asked how Senp2 regulates Setdb1. Since Setdb1 has been reported as a SUMOylated protein (although date not shown) (Hendriks et al., 2015), we proposed that Setdb1 would be de-SUMOylated by Senp2. To test it, we first co-transfected Setdb1 and SUMO plasmids in 293T cells and confirmed that Setdb1 was conjugated by either SUMO1 or SUMO2 (Figure 4A). Interestingly, Senp2 could selectively de-conjugate SUMO2-conjugated but not SUMO1-conjugated Setdb1 (Figure 4B). We further mapped Lys1050 as a SUMO2 acceptor site on Setdb1. Mutation of K1050R completely depleted SUMO2 conjugation on Setdb1 (Figure 4C). Moreover, the accumulation of SUMO2-Setdb1 was shown in the adipocytes of Senp2adqcKO mice and Senp2-knockdown 3T3-L1-Tet-on-shSenp2 cells (treated by doxycycline) (Figure 4D and E). These data suggested that Setdb1 could be regulated by Senp2-dependent de-SUMOylation. Figure 4 View largeDownload slide 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 followed 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-transfected 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 from Senp2f/f or Senp2adqcKO 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 differentiation 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 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-Pparγ, anti-Cebpα, 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 presented are mean ± SD, **P < 0.01. Figure 4 View largeDownload slide 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 followed 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-transfected 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 from Senp2f/f or Senp2adqcKO 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 differentiation 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 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-Pparγ, anti-Cebpα, 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 presented are mean ± SD, **P < 0.01. To further determine the role of SUMOylation in Setdb1 activity, we generated Flag-Setdb1-WT or Flag-Setdb1-K1050R stably expressed 3T3-L1-Tet-on-shSenp2 cells (Supplementary Figure S4A). SUMO2-conjugated Flag-Setdb1 were detected only in Flag-Setdb1-WT but not in Flag-Setdb1-K1050R expressed cells (Supplementary Figure S4B). ChIP assay demonstrated that much more Flag-Setdb1-WT than K1050R mutant could be recruited to the promoter locus of Pparg and Cebpa in 3T3-L1-Tet-on-shSenp2 cells (Figure 4F). We then silenced Setdb1 in Senp2-knockdown 3T3-L1 cells and detected an increase in Pparγ and Cebpα expressions in these cells (Figure 4G and H). Interestingly, re-expression of Setdb1-WT but not Setdb1-K1050R could reduce Pparg and Cebpa expressions in Senp2 and Setdb1 double silenced cells (Figure 4G and H). Consistent with this, knockdown of endogenous Setdb1 increased the expression of Cd36, Fasn, and Dgat2 genes (Figure 4H) and lipid droplets accumulation in Senp2-silenced 3T3-L1 cells (Supplementary Figure S4C). Re-expression of Setdb1-WT in these cells reduced the expression of CD36, Fasn, and Dgat2 genes, while Setdb1-K1050R could not change their expression (Figure 4H). Taken together, these data revealed that SUMOylation promoted Setdb1 recruitment to the locus of Pparg and Cebpa, which subsequently inhibited Pparg and Cebpa expressions in adipocytes. Discussion In this study, we utilized adipocyte Senp2-deficiency mice model to determine the role of Senp2 in adipose lipid storage. Adipose Senp2 deficiency resulted in less adipose lipid storage as well as an ectopic fat accumulation and insulin resistance in mice fed with HFD. Interestingly, Senp2 expression was increased in obese adipose tissues. These data suggest an essential role of Senp2 for storing excess fat in adipose tissues during obesity. This function would benefit the body by protecting from lipotoxicity during obesity. We found that Senp2 is essential for Pparγ and Cebpα expressions by suppressing Setdb1-mediated H3K9me3. Setdb1 is a SUMOylatd protein (Hendriks et al., 2015; Figure 4). We mapped SUMO acceptor site at K1050 of Setdb1. Setdb1 has a structure composed of evolutionarily conserved SET, pre-SET, and post-SET domain, while the SET domain of Setdb1 is interrupted by the insertion of several hundred amino acids (Kang, 2015). K1050 locates in the insertion sequence of Setdb1. We showed that SUMOylation was essential for Setdb1 occupancy to the promoter of Pparg and Cebpa genes. However, the global H3K9me3 levels did not change in Setdb1 K1050R expression cells (data not shown). These data suggest that SUMOylation mainly affect Setdb1 binding to chromatin but not its catalytic activity. Setdb1 has been shown to form a complex with MBD1 and MCAF1 (MBD1-containing chromatin-associated factor 1) to catalyze H3K9me3 on Pparg and Cebpa loci (Matsumura et al., 2015). It would be possible that SUMOylation may regulate the interaction between Setdb1 and MBD1/MCAF1 to promote Setdb1 bound to the promoter of Pparg and Cebpa genes. Senp2 can reverse the Setdb1 suppression by de-SUMOylation. Interestingly, Setdb1 can be SUMOylated by SUMO1 and SUMO2. However, Senp2 only de-SUMOylates SUMO2-conjugated but not SUMO1-conjugated Setdb1. Although SENP2 has been reported to be prone to SUMO2-conjugated protein, it is unknown how Senp2 to distinguish SUMO1- or SUMO2-conjugated proteins for de-SUMOylation. If Senp2 activity is reduced, the expression of Pparg and Cebpa genes would be suppressed by Setdb1 SUMOylation through increased H3K9me3. Therefore, we define a Senp2-Setdb1 axis in controlling adipose lipid metabolism by reversible SUMOylation. The disruption of Pparγ2 or adipocyte-specific Pparγ knockout has been shown to cause severe lipodystrophy and insulin resistance in mice (He et al., 2003; Zhang et al., 2004; Duan et al., 2007). In human, PPARγ mutation also links to familial partial lipodystrophy. Although adipose Senp2 dysfunction has been shown to result in lipodystrophy-like phenotype by suppression of Pparγ expression in mice, it is unknown whether SENP2 dysfunction occurs in human lipodystrophy case. Thus, it is worth to find out whether SENP2 mutation or other SENP2 dysfunction events occur in adipocytes in lipodystrophy patients. Materials and methods Mice Senp2f/f mice were generated as previously described (Qi et al., 2014b). Adiponectin-cre mice were from Dr Jiqiu Wang (Ruijin Hospital, Shanghai Jiao Tong University School of Medicine). Mice were maintained at room temperature (22°C), with a 12-h light-dark cycle and free access to food and water. For diet experiments, 7-week-old male mice were fed HFD with 60% kcal fat (Research Diets, D12492) for 16 weeks. Minispec TD-NMR Analysers (Bruker Instruments) were performed to evaluate body composition. The experiments were not randomized, and the investigators were not blinded to allocation during experiments or outcome assessments. No statistical analysis was applied to predetermine sample size. All procedures were evaluated and approved by the Animal Care Committee of Shanghai Jiaotong University School of Medicine. GTT, ITT, and blood glucose For glucose tolerance test, mice were fasted for 14 h and injected with D-glucose (2 g/kg) intraperitoneally. For insulin tolerance test, mice were fasted for 6 h and injected with recombinant human insulin (0.75 U/kg) intraperitoneally. Blood was drawn from the tail vein and measured by glucose meters (OneTouch Ultra). Serum insulin and adiponectin Mice were fasted for 6 h before blood samples were collected through retro-orbital bleeding and then centrifuged for 20 min. Serum insulin and adiponectin levels were measured using commercial ELISA kits (CrystalChem, Cayman Chemical). Serum triglyceride, cholesterol, and NEFA Total serum triglyceride, cholesterol, and NEFA were enzymatically measured with Serum Triglyceride Determination Kit (Sigma), Mouse Cholesterol ELISA Kit (MyBioSource), and Free Fatty Acid Quantification Kit (BioVision), respectively, according to the manufacturer’s instructions. Organ 14C-oleate uptake studies Organ 14C-oleate uptake was determined as described previously (Schlein et al., 2016). In brief, oleate containing 14C-oleate tracer (0.5MBq/kg body weight) diluted in PBS (33 mg/ml) within 5-fold molar excess to fatty acid-free BSA was injected intravenously 10 min prior to necropsy. Plasma samples were collected through retro-orbital bleeding before sacrificed, and organs were harvested after systemic perfusion with PBS-heparin (10 U/ml). Tissues were minced and digested by Solvable (PerkinElmer) for counting using Tricarb2910TR (PerkinElmer). Histological analysis Tissues were harvested and fixed with 4% paraformaldehyde, dehydrated, paraffin-embedded, and sectioned (5 μm) prior to hematoxylin and eosin (H&E) staining. For cell size measurements, cross-sectional areas of adipocytes were calculated by tracing the adipocyte periphery in ImageJ. Stromal vascular cells isolation and flow cytometry WAT was minced, digested, and filtered, and SVCs were isolated as described (Church et al., 2014). For adipose precursor cell analysis, SVCs were stained with anti-CD45 (eBiosciences), anti-PDGFRα (eBiosciences), and anti-Ki67 (eBiosciences) simultaneously. For myeloid cell analysis, SVCs were stained with anti-F4/80 (eBiosciences), anti-CD11b (eBiosciences) and anti-CD11c (eBiosciences). Cells were analyzed using FACS Verse (BD Bioscience) and FlowJo software. Plasmid and stable cell line construction To generate stable 3T3-L1-Tet-on-shSenp2 cells, shRNA-targeting Senp2 gene was constructed in pLKO-Tet-On vector, the shRNA-targeting sequences are shown in Supplementary Table S1. The shRNA lentivirus were packaged and infected in 3T3-L1 cells, then stable clones were selected with 3 μg/ml puromycin for 1 week. Setdb1 and Setdb1 mutant constructs were cloned into pFlag-CMV2 expression vectors and pCDH-GFP lentiviral backbone using standard PCR-based cloning strategies and verified by DNA sequencing. To generate 3T3-L1-Tet-on-shSenp2 cells stably expressing wild-type or K1050R-mutated Flag-Setdb1, Setdb1 and Setdb1 mutant lentivirus were packaged and infected in 3T3-L1-Tet-on-shSenp2 cells. The cells were selected by FACS. Cell culture and Oil Red O staining HEK-293T and 3T3-L1 cells were cultured in DMEM (Hyclone) supplemented with 10% FBS (Gibco) and 100 μg/ml penicillin/streptomycin. All cells were maintained in a humidified 37°C/5% CO2 incubator. For adipocyte differentiation assay, 3T3-L1-Tet-on-shSenp2 cells were cultured and reached at full confluency for 2 days. Then cells were induced by the addition of insulin (5 μg/ml, Sigma), dexamethasone (1 μM, Sigma), and isobutyl-1-methylxanthine (0.5 mM, Sigma). After 2 days, the medium was replaced by growth medium supplemented with insulin for 10 days. This medium was changed every 2 days until 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 knockdown of endogenous Setdb1, three pairs of siRNA were designed based on 3′ UTR of Setdb1 mRNA. The siRNA sequences are presented in Supplementary Table S1. siRNAs (20 μM) were transfected into differentiated cells by TurboFect Transfection Reagent (Thermo Scientific). For Oil Red O staining, fully differentiated 3T3-L1 cells were fixed with 4% paraformaldehyde for 20 min followed by Oil Red O incubation for 30 min. RNA isolation and quantitative RT-PCR Total RNA was extracted from cells or tissues using TRIzol reagent (Roche). 1 μg RNA was reverse transcribed into complementary DNA with a cDNA synthesis kit (Takara) as per instruction. Real-time PCR was performed on the LC480system (Roche) using SYBER Green Supermix (Takara). Values were normalized to ribosomal protein S18 (Rps18) levels using the ΔΔCt method. Primers used in this study were listed in Supplementary Table S1. Chromatin immunoprecipitation assay Adipose tissues were minced into small pieces (1–3 mm3) in PBS supplemented with protease inhibitors and crosslinked with 1% formaldehyde. Tissues were centrifuged at 1350× g for 5 min at room temperature. The lipid-rich tissue pieces (upper layer) were washed with PBS and subjected into ChIP analysis. 3T3-L1 cells were crosslinked with 1% paraformaldehyde. ChIP–qPCR assays were performed as described previously (Wang et al., 2015). Briefly, cross-linked cells were sonicated. Solubilized chromatin was immunoprecipitated with antibodies against H3K4me3, H3K9me3, H3K27me3, SETDB1, Flag or negative control IgG. Antibody–chromatin complexes were pulled down using protein A-sepharose (Millipore). Beads were then washed, and eluted. After crosslink reversal and proteinase K treatment, immunoprecipitated DNA was extracted with phenol–chloroform followed by ethanol precipitation. The DNA fragments were further analyzed by qPCR. Primer sequences are provided in Supplementary Table S1. Immunoprecipitation and immunoblotting Cells or tissues were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM PMSF, 10 mM N-ethylmaleimide, and protease inhibitors). Cell lysates were then sonicated and centrifuged at 20000× g for 15 min at 4°C, and the supernatants were added to the appropriate antibody coupled with protein A/G beads. After incubation for 6 h at 4°C, beads were washed with RIPA buffer and eluted in 2% SDS solution then analyzed by Immunoblotting. Proteins were detected by antibodies, including anti-SENP2 (sc-67075), anti-PPARγ (2430), anti-C/EBPα (ab40764), anti-SUMO2/3 (ab81371), anti-GAPDH (ab8245), anti-TUBULIN (T9026), anti-pAKT T308 (9275), anti-pAKT S473 (ab18206), anti-AKT (9272), anti-HA (H9658), anti-Flag (F3162), anti-Myc (9402), and anti-SETDB1(ab12317). Statistical analysis All data derived from cells are represented as mean ± standard deviation (SD), data from tissues are represented as mean ± standard error of the mean (SEM). Student’s t-test was performed to analyze significance (*P < 0.05; **P < 0.01). Graphs were generated by Prism software packages. Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. Funding This work was supported by grants from the National Natural Science Foundation of China (91019021 and 81430069 to J.C.), the National Basic Research Program of China (973 Program) (2013CB910902 to J.C.), Shanghai Committee of Science and Technology (15ZR1424500 to T.W. and 15140904300), Shanghai Municipal Education Commission (ZZjdyx15003 to T.W. and 2017-01-07-00-01-E00050 to J.C.), and Shanghai Jiao Tong University School of Medicine (14XJ10001 to T.W.). Conflict of interest: none declared. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Cell Biology Oxford University Press

Senp2 regulates adipose lipid storage by de-SUMOylation of Setdb1

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Oxford University Press
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© The Author (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
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Abstract

Abstract One major function of adipocytes is to store excess energy in the form of triglycerides. Insufficient adipose lipid storage is associated 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 feeding. 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. lipid storage, Senp2, Setdb1, H3K9me3, Pparg and Cebpa Introduction Adipose tissue has an important role in nutrient homeostasis by either storing excess energy in the form of triglycerides (TGs) or hydrolyzing TGs to provide fuels for the rest of the body under fasting condition (Rosen and Spiegelman, 2014; Cohen and Spiegelman, 2016). Excess or insufficient lipid storage in adipose tissues would impair nutrient homeostasis, which is associated with many pathological conditions such as hyperlipidemia, insulin resistance, and type 2 diabetes (Garg, 2004; Agarwal and Garg, 2006; Rosen and Spiegelman, 2006; Liu et al., 2014; Patni and Garg, 2015). Under physiological conditions, the maintenance of normal adipose tissue mass is mainly the result of a balance of lipid storage and lipolysis (Bouchard et al., 1993). Cellular uptake of fatty acids and following storage in the form of TGs in adipocytes are key steps in lipid storage. These processes occur primarily through protein-mediated pathways consisting of a number of fatty acid transporters such as fatty acid translocase (Cd36) and fatty acid transport protein-1 (Fatp1) (Hajri and Abumrad, 2002). Lipolysis proceeds through successive steps involving the release of a single fatty acid chain and the generation of the intermediate species of diacylglycerol (DAG) and monoacylglycerol (MAG) (Young and Zechner, 2013). As a master regulator of adipogenesis, peroxisome proliferator-activated receptors γ (PPARγ) can modulate adipose tissue mass by regulating lipogenesis-related genes (Ahmadian et al., 2013). PPARγ mutation has been shown to link to familial partial lipodystrophy, a clinical disorder characterized by the loss of adipose tissues (Dunnigan et al., 1974; Savage et al., 2003). Selective disruption of Pparγ2 or adipocyte-specific Pparγ knockout leads to severe lipodystrophy and induces insulin resistance in mice (He et al., 2003; Zhang et al., 2004; Duan et al., 2007). Recent studies have shown that SUMO-specific protease 2 (Senp2) is involved in myogenesis (Qi et al., 2014a) and adipogenesis (Chung et al., 2010). Senp2 also regulates fatty acid metabolism in skeletal muscle (Koo et al., 2015). In this report, we observed a defect in adipose lipid storage in adipocyte-specific Senp2 knockout mice fed with high-fat diets (HFD). This mouse also showed an ectopic lipid distribution and insulin resistance. Therefore, adipocyte-specific Senp2 knockout mice exhibited a lipodystrophy-like phenotype. Mechanistically, adipocyte Senp2 deficiency caused the downregulation of Pparg and Cebpa as well as their downstream target genes related to lipid storage. We found that SET domain bifurcated 1 (Setdb1) was a SUMOylated protein and that Senp2 de-SUMOylated and regulated Setdb1 action in trimethylation at histone 3 lysine 9 (H3K9me3). Senp2 deficiency triggered a hyper-SUMOylation of Setdb1, which enhanced its occupancy on Pparg and Cebpa promoter loci to suppress their transcriptions. Our findings unravel that Senp2 is essential for regulating lipid metabolism in adipose tissues. Results Senp2 deficiency decreases adipose lipid storage Since Senp2 expression was dramatically increased in adipose tissues in several obese mouse models (Figure 1A), suggesting that Senp2 may play a role in the pathogenesis of obesity in adipose tissues. To investigate it, we generated a mouse model in which Senp2 was specifically knockout in adipose tissues (Senp2adqcKO) by crossing Senp2f/f with Adiponectin-cre mice (Lee et al., 2013; Jeffery et al., 2014). Senp2 expression was significantly downregulated in brown and white adipose tissues (BAT and WAT) (Supplementary Figure S1A and B) but was not in heart, liver, and skeletal muscle of Senp2adqcKO mice (Supplementary Figure S1B). Senp2adqcKO mice showed almost similar growth and fertility when compared to Senp2f/f mice fed with normal chow diets (NCD) (Supplementary Figure S1C). Nuclear magnetic resonance (NMR) analysis detected slightly less fat mass in Senp2adqcKO mice than in Senp2f/f mice at age of 24 weeks old (Supplementary Figure S1D). Fluorescence-activated cell sorting (FACS) analysis demonstrated that there was no difference in the population and proliferation of CD45−PDGFRα+ adipocyte progenitor cells (Lee et al., 2012) between Senp2adqcKO and Senp2f/f mice (Supplementary Figure S1E), indicating that adipocyte Senp2 deficiency in our model did not affect adipocyte progenitors. Figure 1 View largeDownload slide Senp2 deficiency decreases adipose lipid storage on HFD. (A) Senp2 expression was upregulated in adipose tissues of obese mice (n = 6–10/group). (B) The body weights of Senp2f/f and Senp2adqcKO male mice during 15 weeks of HFD feeding (n = 10/group). (C) The size of BAT, inguinal and epididymal fat pads of Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding. (D) The organ weights / body weight of Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (E) H&E staining of representative sections of BAT, iWAT, and eWAT from Senp2f/f and Senp2adqcKO 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). (G) Flow cytometry analysis of adipose-associated macrophages (F4/80+CD11b+ or F4/80+CD11b+CD11c+) in Senp2f/f and Senp2adqcKO 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. Figure 1 View largeDownload slide Senp2 deficiency decreases adipose lipid storage on HFD. (A) Senp2 expression was upregulated in adipose tissues of obese mice (n = 6–10/group). (B) The body weights of Senp2f/f and Senp2adqcKO male mice during 15 weeks of HFD feeding (n = 10/group). (C) The size of BAT, inguinal and epididymal fat pads of Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding. (D) The organ weights / body weight of Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (E) H&E staining of representative sections of BAT, iWAT, and eWAT from Senp2f/f and Senp2adqcKO 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). (G) Flow cytometry analysis of adipose-associated macrophages (F4/80+CD11b+ or F4/80+CD11b+CD11c+) in Senp2f/f and Senp2adqcKO 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. We then tested the role of adipose Senp2 in obesity by feeding Senp2f/f and Senp2adqcKOmice with HFD. During the first 9 weeks of HFD feeding, no significant difference in the gained body weight was observed between Senp2adqcKO and Senp2f/f mice. From 10 weeks after feeding with HFD, Senp2f/f mice continued to increase body weight, while Senp2adqcKO mice showed a decrease in the gained body weight (Figure 1B). Consistently, both BAT and WAT in Senp2adqcKO mice were over 50% smaller than that in Senp2f/f male mice (Figure 1C and D). Histologically, we observed much smaller size of adipocytes in Senp2adqcKO mice than in Senp2f/f mice fed with HFD (Figure 1E and F), indicating that the less fat mass in Senp2adqcKO mice might be a result of less adipose lipid storage during obesity. Additionally, we found that Senp2adqcKO mice had much more inflammation-related crown-like structures (Lee et al., 2014) in WAT than Senp2f/f did (Figure 1E). FACS analysis also confirmed this result by showing more macrophages infiltration in Senp2adqcKO WAT than in Senp2f/f WAT (Figure 1G and Supplementary Figure S1F). Although less fat storage was shown in Senp2adqcKO adipose tissues, the increased inflammation indicates that HFD induced adipocyte death would remain in adipose tissues. Senp2adqcKO mice exhibit an ectopic lipid accumulation and insulin resistance Since no difference in food intake was observed between Senp2adqcKO and Senp2f/f mice fed either with NCD or HFD (Supplementary Figure S2A). We next assessed the serum triglyceride, cholesterol, and non-esterified fatty acid (NEFA) in aforementioned mice fed with HFD. Figure 2A demonstrates that their concentrations were much higher in Senp2adqcKO serum than in Senp2f/f serum. We further stained liver sections and showed more severe hepatic steatosis in Senp2adqcKO mice than in Senp2f/f mice (Figure 2B). These data suggest that an ectopic lipid distribution was accompanied with the less adipose lipid storage in Senp2adqcKO mice. Figure 2 View largeDownload slide Senp2adqcKO mice exhibit an ectopic lipid accumulation and insulin resistance. (A) The concentrations of serum TGs, cholesterol, and NEFA in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (B) H&E staining of representative liver sections from Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding. Scale bar, 100 μm. (C) The amount of 14C-labeled oleate uptake in the indicated organs during 15 min after intravenous injection of 14C-labeled oleate in Senp2f/f and Senp2adqcKO male mice (n = 5/group). (D) Glucose tolerance test in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (E) Insulin tolerance test in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (F) The concentrations of blood glucose or serum insulin in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (G) AKT and phosphorylated AKT were blotted in lysates of eWAT, gastrocnemius muscle, or liver from Senp2f/f and Senp2adqcKO 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. Figure 2 View largeDownload slide Senp2adqcKO mice exhibit an ectopic lipid accumulation and insulin resistance. (A) The concentrations of serum TGs, cholesterol, and NEFA in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (B) H&E staining of representative liver sections from Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding. Scale bar, 100 μm. (C) The amount of 14C-labeled oleate uptake in the indicated organs during 15 min after intravenous injection of 14C-labeled oleate in Senp2f/f and Senp2adqcKO male mice (n = 5/group). (D) Glucose tolerance test in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (E) Insulin tolerance test in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (F) The concentrations of blood glucose or serum insulin in Senp2f/f and Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 5/group). (G) AKT and phosphorylated AKT were blotted in lysates of eWAT, gastrocnemius muscle, or liver from Senp2f/f and Senp2adqcKO 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. To further demonstrate a view of fat distribution, we performed an in vivo analysis for 14C-labeled oleate uptake. An equal injection of initial radiation dose was determined by measuring 14C-oleate counts in the plasma (Supplementary Figure S2B). As demonstrated in Figure 2C, we observed a lower level of 14C-oleate counts in adipose tissues of Senp2adqcKO mice including BAT, iWAT (inguinal WAT), and eWAT (epididymal WAT), but a higher level in the liver, heart, kidney, and muscle tissues of Senp2adqcKO mice, 15 min post injection. The data confirmed the less adipose fat storage accompanied by an ectopic fat accumulation in other non-adipose tissues of Senp2adqcKO mice. More importantly, the data also showed the defect in fat uptake existed only in adipose tissues in Senp2adqcKO mice. We would expect insulin resistance accompanied with ectopic fat accumulation (Guilherme et al., 2008) in Senp2adqcKO mice. We thus assessed blood glucose and insulin intolerance. Supplementary Figure S2C and D showed that the responses of glucose and insulin tolerance in Senp2adqcKO were similar to that in Senp2f/f mice fed with NCD. However, under HFD, the ability for glucose clearance as well as insulin sensitivity in Senp2adqcKO mice were markedly reduced compared with Senp2f/f mice (Figure 2D and E). In consistence, fasting glucose and insulin levels were elevated more in Senp2adqcKO mice than in Senp2f/f mice (Figure 2F). We also examined insulin signaling in both Senp2f/f and Senp2adqcKO mice fed with HFD. Following insulin injection, Senp2f/f mice displayed a robust increase in AKT phosphorylation (at T308 and S473 sites) in eWAT, gastrocnemius muscle, and liver. However, this response was greatly attenuated in Senp2adqcKO mice (Figure 2G), indicating that insulin signaling was impaired in Senp2adqcKO mice, which resulted in insulin resistance observed in these mice. Senp2 modulates Pparγ and Cebpα function in adipose tissues To characterize the molecular basis of less adipose lipid storage observed in Senp2adqcKO mice, we analyzed the expression of the genes related to lipid metabolism in adipose tissues, which include the genes that involved in fatty acid uptake and trafficking (e.g. Lpl, Cd36, Fabp4), and genes in lipid synthesis (e.g. Fasn, Srebp, Dgat2, and Slc25a1). We detected a minor reduction in the expression of some genes in adipose tissues of Senp2adqcKO mice on NCD (Supplementary Figure S3A). However, the most of the genes were significantly downregulated in adipose tissues of Senp2adqcKO mice when fed with HFD (Figure 3A). In addition, Adiponectin (Adipoq) were also downregulated in Senp2adqcKO mice (Figure 3A and B). Figure 3 View largeDownload slide Senp2 regulates Pparg and Cebpa expression through SETDB1-mediated H3K9me3 in adipose tissues. (A) RT-PCR analysis of genes related to lipid metabolism in adipose tissues from Senp2f/f or Senp2adqcKO mice at the end of 15 weeks of HFD feeding (n = 4/group). (B) The concentration of serum adiponectin in Senp2f/f or Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 3/group). (C) RT-PCR analysis of Pparg and Cebpa genes in adipose tissues from Senp2f/f or Senp2adqcKO mice at the end of 15 weeks of HFD feeding (n = 4/group). (D) Pparγ and Cebpα were blotted in adipose lysates from Senp2f/f or Senp2adqcKO male mice at the end of 15 weeks of HFD feeding. (E) ChIP analysis of H3K4me3, H3K9me3, or H3K27me3 occupancy in the locus of Pparg and Cebpa promoters in the adipose of Senp2f/f or Senp2adqcKO male mice on HFD (n = 4/group). (F) ChIP analysis of Setdb1 occupancy in the locus of Pparg and Cebpa promoters in the adipose of Senp2f/f or Senp2adqcKO 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. Figure 3 View largeDownload slide Senp2 regulates Pparg and Cebpa expression through SETDB1-mediated H3K9me3 in adipose tissues. (A) RT-PCR analysis of genes related to lipid metabolism in adipose tissues from Senp2f/f or Senp2adqcKO mice at the end of 15 weeks of HFD feeding (n = 4/group). (B) The concentration of serum adiponectin in Senp2f/f or Senp2adqcKO male mice at the end of 15 weeks of HFD feeding (n = 3/group). (C) RT-PCR analysis of Pparg and Cebpa genes in adipose tissues from Senp2f/f or Senp2adqcKO mice at the end of 15 weeks of HFD feeding (n = 4/group). (D) Pparγ and Cebpα were blotted in adipose lysates from Senp2f/f or Senp2adqcKO male mice at the end of 15 weeks of HFD feeding. (E) ChIP analysis of H3K4me3, H3K9me3, or H3K27me3 occupancy in the locus of Pparg and Cebpa promoters in the adipose of Senp2f/f or Senp2adqcKO male mice on HFD (n = 4/group). (F) ChIP analysis of Setdb1 occupancy in the locus of Pparg and Cebpa promoters in the adipose of Senp2f/f or Senp2adqcKO 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. Interestingly, almost all these genes are mainly controlled by master transcription factors Pparγ and Cebpα in adipocytes (Lefterova et al., 2008). We therefore determined whether adipocyte Senp2 deficiency could alter Pparγ and Cebpα in adipose tissues. As shown in Figure 3C and D, Senp2adqcKO mice had much lower levels of both Pparg and Cebpa than Senp2f/f mice did, suggesting an essential role of Senp2 in regulation of the expression of both Pparγ and Cebpα. Additionally, we detected accumulation of SUMOylated Pparγ in adipose tissues of Senp2adqcKO mice (Supplementary Figure S3B). Since SUMOylation suppresses Pparγ transcription activity (Dutchak et al., 2012), the accumulation of SUMOylated Pparγ might contribute to the downregulation of Pparγ activation in Senp2adqcKO mice. Senp2 deficiency enhances H3K9 trimethylation in locus of Pparg and Cebpa H3K4/H3K9me3 bivalent domains have been shown as a repressive mechanism for Pparg and Cebpa expression during adipogenesis (Matsumura et al., 2015). We asked whether this mechanism would work in Senp2 regulation of Pparg and Cebpa expressions either. Indeed, chromatin immunoprecipitation (ChIP) assay detected a significant increase of H3K9me3, but not H3K4me3 or H3K27me3 in the promoter locus of Pparg and Cebpa in Senp2adqcKO adipocytes (Figure 3E). This result indicated that Pparg and Cebpa expressions could be suppressed by the increased H3K9me3. Since Setdb1 as a histone methyltransferase for H3K9 trimethylation regulates Cebpa and Pparg transcriptions in adipocytes (Matsumura et al., 2015), we thus asked whether Setdb1 would control H3K9 trimethylation in Senp2adqcKO adipocytes. ChIP assay detected a significant increase in Setdb1 occupancy in the locus of Pparg and Cebpa promoters in Senp2adqcKO adipocytes (Figure 3F), suggesting that Senp2 deficiency would modulate Setdb1 action on H3K9 trimethylation to suppress Pparg and Cebpa expressions in adipocytes. Senp2 controls Pparg and Cebpa expression by de-SUMOylation of Setdb1 We next asked how Senp2 regulates Setdb1. Since Setdb1 has been reported as a SUMOylated protein (although date not shown) (Hendriks et al., 2015), we proposed that Setdb1 would be de-SUMOylated by Senp2. To test it, we first co-transfected Setdb1 and SUMO plasmids in 293T cells and confirmed that Setdb1 was conjugated by either SUMO1 or SUMO2 (Figure 4A). Interestingly, Senp2 could selectively de-conjugate SUMO2-conjugated but not SUMO1-conjugated Setdb1 (Figure 4B). We further mapped Lys1050 as a SUMO2 acceptor site on Setdb1. Mutation of K1050R completely depleted SUMO2 conjugation on Setdb1 (Figure 4C). Moreover, the accumulation of SUMO2-Setdb1 was shown in the adipocytes of Senp2adqcKO mice and Senp2-knockdown 3T3-L1-Tet-on-shSenp2 cells (treated by doxycycline) (Figure 4D and E). These data suggested that Setdb1 could be regulated by Senp2-dependent de-SUMOylation. Figure 4 View largeDownload slide 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 followed 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-transfected 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 from Senp2f/f or Senp2adqcKO 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 differentiation 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 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-Pparγ, anti-Cebpα, 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 presented are mean ± SD, **P < 0.01. Figure 4 View largeDownload slide 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 followed 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-transfected 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 from Senp2f/f or Senp2adqcKO 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 differentiation 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 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-Pparγ, anti-Cebpα, 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 presented are mean ± SD, **P < 0.01. To further determine the role of SUMOylation in Setdb1 activity, we generated Flag-Setdb1-WT or Flag-Setdb1-K1050R stably expressed 3T3-L1-Tet-on-shSenp2 cells (Supplementary Figure S4A). SUMO2-conjugated Flag-Setdb1 were detected only in Flag-Setdb1-WT but not in Flag-Setdb1-K1050R expressed cells (Supplementary Figure S4B). ChIP assay demonstrated that much more Flag-Setdb1-WT than K1050R mutant could be recruited to the promoter locus of Pparg and Cebpa in 3T3-L1-Tet-on-shSenp2 cells (Figure 4F). We then silenced Setdb1 in Senp2-knockdown 3T3-L1 cells and detected an increase in Pparγ and Cebpα expressions in these cells (Figure 4G and H). Interestingly, re-expression of Setdb1-WT but not Setdb1-K1050R could reduce Pparg and Cebpa expressions in Senp2 and Setdb1 double silenced cells (Figure 4G and H). Consistent with this, knockdown of endogenous Setdb1 increased the expression of Cd36, Fasn, and Dgat2 genes (Figure 4H) and lipid droplets accumulation in Senp2-silenced 3T3-L1 cells (Supplementary Figure S4C). Re-expression of Setdb1-WT in these cells reduced the expression of CD36, Fasn, and Dgat2 genes, while Setdb1-K1050R could not change their expression (Figure 4H). Taken together, these data revealed that SUMOylation promoted Setdb1 recruitment to the locus of Pparg and Cebpa, which subsequently inhibited Pparg and Cebpa expressions in adipocytes. Discussion In this study, we utilized adipocyte Senp2-deficiency mice model to determine the role of Senp2 in adipose lipid storage. Adipose Senp2 deficiency resulted in less adipose lipid storage as well as an ectopic fat accumulation and insulin resistance in mice fed with HFD. Interestingly, Senp2 expression was increased in obese adipose tissues. These data suggest an essential role of Senp2 for storing excess fat in adipose tissues during obesity. This function would benefit the body by protecting from lipotoxicity during obesity. We found that Senp2 is essential for Pparγ and Cebpα expressions by suppressing Setdb1-mediated H3K9me3. Setdb1 is a SUMOylatd protein (Hendriks et al., 2015; Figure 4). We mapped SUMO acceptor site at K1050 of Setdb1. Setdb1 has a structure composed of evolutionarily conserved SET, pre-SET, and post-SET domain, while the SET domain of Setdb1 is interrupted by the insertion of several hundred amino acids (Kang, 2015). K1050 locates in the insertion sequence of Setdb1. We showed that SUMOylation was essential for Setdb1 occupancy to the promoter of Pparg and Cebpa genes. However, the global H3K9me3 levels did not change in Setdb1 K1050R expression cells (data not shown). These data suggest that SUMOylation mainly affect Setdb1 binding to chromatin but not its catalytic activity. Setdb1 has been shown to form a complex with MBD1 and MCAF1 (MBD1-containing chromatin-associated factor 1) to catalyze H3K9me3 on Pparg and Cebpa loci (Matsumura et al., 2015). It would be possible that SUMOylation may regulate the interaction between Setdb1 and MBD1/MCAF1 to promote Setdb1 bound to the promoter of Pparg and Cebpa genes. Senp2 can reverse the Setdb1 suppression by de-SUMOylation. Interestingly, Setdb1 can be SUMOylated by SUMO1 and SUMO2. However, Senp2 only de-SUMOylates SUMO2-conjugated but not SUMO1-conjugated Setdb1. Although SENP2 has been reported to be prone to SUMO2-conjugated protein, it is unknown how Senp2 to distinguish SUMO1- or SUMO2-conjugated proteins for de-SUMOylation. If Senp2 activity is reduced, the expression of Pparg and Cebpa genes would be suppressed by Setdb1 SUMOylation through increased H3K9me3. Therefore, we define a Senp2-Setdb1 axis in controlling adipose lipid metabolism by reversible SUMOylation. The disruption of Pparγ2 or adipocyte-specific Pparγ knockout has been shown to cause severe lipodystrophy and insulin resistance in mice (He et al., 2003; Zhang et al., 2004; Duan et al., 2007). In human, PPARγ mutation also links to familial partial lipodystrophy. Although adipose Senp2 dysfunction has been shown to result in lipodystrophy-like phenotype by suppression of Pparγ expression in mice, it is unknown whether SENP2 dysfunction occurs in human lipodystrophy case. Thus, it is worth to find out whether SENP2 mutation or other SENP2 dysfunction events occur in adipocytes in lipodystrophy patients. Materials and methods Mice Senp2f/f mice were generated as previously described (Qi et al., 2014b). Adiponectin-cre mice were from Dr Jiqiu Wang (Ruijin Hospital, Shanghai Jiao Tong University School of Medicine). Mice were maintained at room temperature (22°C), with a 12-h light-dark cycle and free access to food and water. For diet experiments, 7-week-old male mice were fed HFD with 60% kcal fat (Research Diets, D12492) for 16 weeks. Minispec TD-NMR Analysers (Bruker Instruments) were performed to evaluate body composition. The experiments were not randomized, and the investigators were not blinded to allocation during experiments or outcome assessments. No statistical analysis was applied to predetermine sample size. All procedures were evaluated and approved by the Animal Care Committee of Shanghai Jiaotong University School of Medicine. GTT, ITT, and blood glucose For glucose tolerance test, mice were fasted for 14 h and injected with D-glucose (2 g/kg) intraperitoneally. For insulin tolerance test, mice were fasted for 6 h and injected with recombinant human insulin (0.75 U/kg) intraperitoneally. Blood was drawn from the tail vein and measured by glucose meters (OneTouch Ultra). Serum insulin and adiponectin Mice were fasted for 6 h before blood samples were collected through retro-orbital bleeding and then centrifuged for 20 min. Serum insulin and adiponectin levels were measured using commercial ELISA kits (CrystalChem, Cayman Chemical). Serum triglyceride, cholesterol, and NEFA Total serum triglyceride, cholesterol, and NEFA were enzymatically measured with Serum Triglyceride Determination Kit (Sigma), Mouse Cholesterol ELISA Kit (MyBioSource), and Free Fatty Acid Quantification Kit (BioVision), respectively, according to the manufacturer’s instructions. Organ 14C-oleate uptake studies Organ 14C-oleate uptake was determined as described previously (Schlein et al., 2016). In brief, oleate containing 14C-oleate tracer (0.5MBq/kg body weight) diluted in PBS (33 mg/ml) within 5-fold molar excess to fatty acid-free BSA was injected intravenously 10 min prior to necropsy. Plasma samples were collected through retro-orbital bleeding before sacrificed, and organs were harvested after systemic perfusion with PBS-heparin (10 U/ml). Tissues were minced and digested by Solvable (PerkinElmer) for counting using Tricarb2910TR (PerkinElmer). Histological analysis Tissues were harvested and fixed with 4% paraformaldehyde, dehydrated, paraffin-embedded, and sectioned (5 μm) prior to hematoxylin and eosin (H&E) staining. For cell size measurements, cross-sectional areas of adipocytes were calculated by tracing the adipocyte periphery in ImageJ. Stromal vascular cells isolation and flow cytometry WAT was minced, digested, and filtered, and SVCs were isolated as described (Church et al., 2014). For adipose precursor cell analysis, SVCs were stained with anti-CD45 (eBiosciences), anti-PDGFRα (eBiosciences), and anti-Ki67 (eBiosciences) simultaneously. For myeloid cell analysis, SVCs were stained with anti-F4/80 (eBiosciences), anti-CD11b (eBiosciences) and anti-CD11c (eBiosciences). Cells were analyzed using FACS Verse (BD Bioscience) and FlowJo software. Plasmid and stable cell line construction To generate stable 3T3-L1-Tet-on-shSenp2 cells, shRNA-targeting Senp2 gene was constructed in pLKO-Tet-On vector, the shRNA-targeting sequences are shown in Supplementary Table S1. The shRNA lentivirus were packaged and infected in 3T3-L1 cells, then stable clones were selected with 3 μg/ml puromycin for 1 week. Setdb1 and Setdb1 mutant constructs were cloned into pFlag-CMV2 expression vectors and pCDH-GFP lentiviral backbone using standard PCR-based cloning strategies and verified by DNA sequencing. To generate 3T3-L1-Tet-on-shSenp2 cells stably expressing wild-type or K1050R-mutated Flag-Setdb1, Setdb1 and Setdb1 mutant lentivirus were packaged and infected in 3T3-L1-Tet-on-shSenp2 cells. The cells were selected by FACS. Cell culture and Oil Red O staining HEK-293T and 3T3-L1 cells were cultured in DMEM (Hyclone) supplemented with 10% FBS (Gibco) and 100 μg/ml penicillin/streptomycin. All cells were maintained in a humidified 37°C/5% CO2 incubator. For adipocyte differentiation assay, 3T3-L1-Tet-on-shSenp2 cells were cultured and reached at full confluency for 2 days. Then cells were induced by the addition of insulin (5 μg/ml, Sigma), dexamethasone (1 μM, Sigma), and isobutyl-1-methylxanthine (0.5 mM, Sigma). After 2 days, the medium was replaced by growth medium supplemented with insulin for 10 days. This medium was changed every 2 days until 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 knockdown of endogenous Setdb1, three pairs of siRNA were designed based on 3′ UTR of Setdb1 mRNA. The siRNA sequences are presented in Supplementary Table S1. siRNAs (20 μM) were transfected into differentiated cells by TurboFect Transfection Reagent (Thermo Scientific). For Oil Red O staining, fully differentiated 3T3-L1 cells were fixed with 4% paraformaldehyde for 20 min followed by Oil Red O incubation for 30 min. RNA isolation and quantitative RT-PCR Total RNA was extracted from cells or tissues using TRIzol reagent (Roche). 1 μg RNA was reverse transcribed into complementary DNA with a cDNA synthesis kit (Takara) as per instruction. Real-time PCR was performed on the LC480system (Roche) using SYBER Green Supermix (Takara). Values were normalized to ribosomal protein S18 (Rps18) levels using the ΔΔCt method. Primers used in this study were listed in Supplementary Table S1. Chromatin immunoprecipitation assay Adipose tissues were minced into small pieces (1–3 mm3) in PBS supplemented with protease inhibitors and crosslinked with 1% formaldehyde. Tissues were centrifuged at 1350× g for 5 min at room temperature. The lipid-rich tissue pieces (upper layer) were washed with PBS and subjected into ChIP analysis. 3T3-L1 cells were crosslinked with 1% paraformaldehyde. ChIP–qPCR assays were performed as described previously (Wang et al., 2015). Briefly, cross-linked cells were sonicated. Solubilized chromatin was immunoprecipitated with antibodies against H3K4me3, H3K9me3, H3K27me3, SETDB1, Flag or negative control IgG. Antibody–chromatin complexes were pulled down using protein A-sepharose (Millipore). Beads were then washed, and eluted. After crosslink reversal and proteinase K treatment, immunoprecipitated DNA was extracted with phenol–chloroform followed by ethanol precipitation. The DNA fragments were further analyzed by qPCR. Primer sequences are provided in Supplementary Table S1. Immunoprecipitation and immunoblotting Cells or tissues were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM PMSF, 10 mM N-ethylmaleimide, and protease inhibitors). Cell lysates were then sonicated and centrifuged at 20000× g for 15 min at 4°C, and the supernatants were added to the appropriate antibody coupled with protein A/G beads. After incubation for 6 h at 4°C, beads were washed with RIPA buffer and eluted in 2% SDS solution then analyzed by Immunoblotting. Proteins were detected by antibodies, including anti-SENP2 (sc-67075), anti-PPARγ (2430), anti-C/EBPα (ab40764), anti-SUMO2/3 (ab81371), anti-GAPDH (ab8245), anti-TUBULIN (T9026), anti-pAKT T308 (9275), anti-pAKT S473 (ab18206), anti-AKT (9272), anti-HA (H9658), anti-Flag (F3162), anti-Myc (9402), and anti-SETDB1(ab12317). Statistical analysis All data derived from cells are represented as mean ± standard deviation (SD), data from tissues are represented as mean ± standard error of the mean (SEM). Student’s t-test was performed to analyze significance (*P < 0.05; **P < 0.01). Graphs were generated by Prism software packages. Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. Funding This work was supported by grants from the National Natural Science Foundation of China (91019021 and 81430069 to J.C.), the National Basic Research Program of China (973 Program) (2013CB910902 to J.C.), Shanghai Committee of Science and Technology (15ZR1424500 to T.W. and 15140904300), Shanghai Municipal Education Commission (ZZjdyx15003 to T.W. and 2017-01-07-00-01-E00050 to J.C.), and Shanghai Jiao Tong University School of Medicine (14XJ10001 to T.W.). Conflict of interest: none declared. 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Journal of Molecular Cell BiologyOxford University Press

Published: Feb 14, 2018

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