Developmental Cell Article

A Multifunctional , EWS, Is Essential for Early Brown Fat Lineage Determination

Jun Hong Park,1 Hong Jun Kang,1 Soo Im Kang,1 Ji Eun Lee,2 Jamie Hur,1 Kai Ge,2 Elisabetta Mueller,1 Hongjie Li,1,5 Byeong-Chel Lee,3 and Sean Bong Lee1,4,* 1Genetics of Development and Disease Branch 2Molecular Endocrinology Branch National Institute of Diabetes and Digestive and Kidney Diseases, 9000 Rockville Pike, Bethesda, MD 20892, USA 3Department of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA 4Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1700 Tulane Avenue, New Orleans, LA 70112, USA 5Present address: Department of Environmental Medicine, New York University Langone Medical Center, Tuxedo, NY 10987, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2013.07.002

SUMMARY to heat is accomplished in mitochondria via actions of Uncou- pling Protein 1 (UCP1), a BAT-specific mitochondrial protein. The recent surge in obesity has provided an impetus BAT is highly vascularized and densely innervated by the sympa- to better understand the mechanisms of adipo- thetic nervous system. In response to a cold environment or diet, genesis, particularly in brown adipose tissue (BAT) BAT is activated by b-adrenergic stimulation to expend energy because of its potential utilization for antiobesity and maintain the thermal or energy balance, a process termed therapy. Postnatal brown adipocytes arise from early adaptive thermogenesis. Given recent findings of active BAT in muscle progenitors, but how brown fat lineage is adult humans (Nedergaard et al., 2007) and an inverse corre- lation between high BAT activity and obesity (Cypess et al., determined is not completely understood. Here, we 2009; van Marken Lichtenbelt et al., 2009), the utilization of show that a multifunctional protein, Ewing Sarcoma BAT as an antiobesity therapy and possibly for other metabolic (EWS), is essential for determining brown fat lineage disorders holds great promise. during development. BATs from Ews null embryos BAT exists in two distinct types that differ in their cell of origin. and newborns are developmentally arrested. Ews During embryogenesis, brown preadipocytes arise from early mutant brown preadipocytes fail to differentiate muscle progenitors (Atit et al., 2006; Timmons et al., 2007) that due to loss of Bmp7 expression, a critical early brown express the myogenic Myf5 (Seale et al., adipogenic factor. We demonstrate that EWS, along 2008) and form mature classical BAT in newborns. A second with its binding partner Y-box binding protein 1 type of brown fat cells is generated during adaptive thermo- (YBX1), activates Bmp7 transcription. Depletion of genesis in response to b-adrenergic stimulation. These brown- either Ews or Ybx1 leads to loss of Bmp7 expres- fat-like cells (termed brite or beige cells) are generated from Myf5-negative cells residing in perivascular regions of various sion and brown adipogenesis. Remarkably, Ews WAT depots (Gupta et al., 2012; Tran et al., 2012). Interestingly, null BATs and brown preadipocytes ectopically a recent study showed that WAT depots contain bipotent adipo- express myogenic . These results demonstrate cyte progenitors that express PDGFRa and give rise to either that EWS is essential for early brown fat lineage brown or white adipocytes in response to b-adrenergic stimula- determination. tion or a high-fat diet, respectively (Lee et al., 2012). The development of classical BAT has been intensely studied and many transcription factors, such as peroxisome prolif- INTRODUCTION erator-activated g (PPARg), PPARg coactivator 1 alpha (PGC-1a), CCAAT-enhancer-binding protein-a (CEBP-a), Chronic obesity driven by excessive food intake, lack of exer- CEBP-b, CEBP-d, and PRD1-BF1-RIZ1 homologous domain- cise, and/or defects in metabolism will ultimately result in containing 16 (PRDM16) (Farmer, 2008; Kajimura et al., 2010), metabolic disorders such as coronary failure, type 2 diabetes, and even microRNAs (e.g., MiR-193b-365; Sun et al., 2011), hypertension, stroke, and cancer (Li et al., 2005). White adipose have been identified as important regulators of classical BAT tissue (WAT) comprises the majority of adipose tissues in development and function. However, not much is known about humans and is the depot of unutilized fat in the form of triglyc- the upstream factors that determine the brown fat lineage. It has erides (Cristancho and Lazar, 2011). During starvation, stored been demonstrated that Bone-Morphogenic Protein 7 (BMP7) is triglycerides in WAT are utilized to provide energy. In contrast, critical for mesenchymal progenitor cells to commit to brown brown adipose tissue (BAT) oxidizes fat to provide heat to main- fat lineage (Tseng et al., 2008); however, the factors that regu- tain core body temperature and is rich in mitochondria (Frontini late BMP7 expression during the early commitment period are and Cinti, 2010; Tseng et al., 2010). Conversion of triglycerides unknown.

Developmental Cell 26, 393–404, August 26, 2013 ª2013 Elsevier Inc. 393 Developmental Cell EWS Is Essential for Brown Fat Cell Determination

Figure 1. Ews Is Essential for BAT Development (A) Photographs of interscapular BAT in newborns (P0.5). Arrows point to BAT. (B) Sagittal section and H&E staining of E18.5 embryos and newborns. Boxed area is shown at higher magnification in the insets. Asterisks indicate interscapular BAT. (C) Immunostaining of P0.5 interscapular BAT with anti-EWS, anti-UCP1, anti-PGC1a, or anti-PRDM16. Insets show higher magnification (4003). Scale bars are indicated. (D) Western blot analysis of interscapular BATs from Ews+/+ and Ews/ newborns (P0.5; n = 3). See also Figure S1.

RESULTS tantly, expression of UCP1, a marker of mature and functional BAT, as well as PGC1a and PRDM16, was nearly absent or Classical BAT Development Is Disrupted in the Absence greatly diminished in all newborn mutant BATs (Figure 1C). West- of Ews ern blot analysis further revealed markedly reduced levels We have previously shown that loss of Ews in a mixed 129SvEv/ of PPARg, CEBP-a, CEBP-b, and CEBP-d, and confirmed Black Swiss strain results in defects in B cell development and the near-complete absence of UCP1 expression (Figure 1D). meiosis, and causes premature cellular senescence in fibro- Expression of the EWS homolog, Fused in Sarcoma (FUS, also blasts and hematopoietic stem cells, as well as high postnatal called TLS), was not altered. Ews null newborns (P0.5) in the mortality (90%) (Cho et al., 2011; Li et al., 2007). Subsequently, 129SvEv/Black Swiss strain also displayed developmentally we backcrossed Ews+/ to the C57BL6/N strain for 12 genera- arrested BATs with lack of lipids and UCP1 expression (Fig- tions and generated Ews/ congenic mice. These mice were ure S1D). These results reveal an essential and unexpected born at near-Mendelian ratios, but all of the mutant mice died role for Ews in BAT development. Of note, breeding these mutant within 24 hr after birth (data not shown). Gross visual examination mice at thermoneutrality (32C) did not rescue the early postnatal revealed no major organ defects except for drastically reduced lethality of Ews null pups (n = 4). interscapular BATs in all Ews mutants compared with littermates (Figure 1A). As shown by western blot analysis of wild-type Ews Is Essential for Brown Adipocyte Differentiation mouse tissues, EWS is abundantly expressed in BAT as well To investigate the mechanism by which Ews regulates BAT as in WAT and most other tissues (Figure S1A available online). development, we derived immortalized brown preadipocytes Hematoxylin and eosin (H&E) staining confirmed the diminished from Ews wild-type and mutant newborns (Figure 2A). Following size of interscapular and subscapular BATs in Ews null embryos the addition of adipogenic cocktail, wild-type preadipocytes (embryonic day 18.5 [E18.5]) and newborns (postnatal day 0.5 differentiated robustly into brown adipocytes (Figure 2B). How- [P0.5]; Figures 1B and S1B). Further examination with oil red O ever, Ews null preadipocytes failed to differentiate into mature revealed a striking absence of multiloculated lipid droplets in brown adipocytes, and Ews+/ cells showed a slight reduction Ews mutant BATs compared with wild-type (Figure S1C). Impor- in brown fat differentiation (Figure 2B). Depletion of Ews with

394 Developmental Cell 26, 393–404, August 26, 2013 ª2013 Elsevier Inc. Developmental Cell EWS Is Essential for Brown Fat Cell Determination

Figure 2. Ews Is Required for Expression of Bmp7 and Brown Fat Differentiation in Brown Preadipocytes (A) Western blot analysis of immortalized brown preadipocytes with anti-EWS and anti-Actin. (B) Representative images of oil red O staining of brown adipocytes 8 days after addition of adipogenic cocktail. (Images in D–F images are higher magnification, 403). (C) qRT-PCR analysis of indicated transcripts was performed (in triplicate) in brown adipocytes at 0, 3, or 8 days after addition of adipogenic cocktail. Black bar: Ews+/+; white bar: Ews/. (D) qRT-PCR analysis of brown preadipocytes at the indicated hours (0–24 hr) after addition of adipogenic cocktail was performed in triplicates. For (C) and (D), three independent experiments were performed and data are represented as means ± SEM. Two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S2. two independent small hairpin RNAs (shRNAs) also blocked any physical interactions between EWS and these two fac- brown fat differentiation (Figures 2B and S2A). Upon adipogenic tors, nor did we observe any effects of EWS on the PPARg-or stimulation, wild-type brown preadipocytes expressed high CEBPa-mediated transcriptional reporter assays (data not levels of the adipogenic transcription factors Cebpa and Pparg, shown). EWS also did not interact with the upstream transcrip- as well as Ews (Figure 2C). In contrast, Ews mutant preadipo- tion factors CEBPb and CEBPd (data not shown). cytes failed to induce expression of the critical adipogenic fac- Subsequently, we reasoned that EWS might function up- tors Pparg and Prdm16, and expressed significantly reduced stream of these adipogenic factors, and examined the expres- levels of Cebpa. Expression of Pgc1a and other brown fat sion of very early adipogenic regulators. In wild-type brown markers (Ucp1, Elongation of very long chain fatty acids protein3 preadipocytes, addition of adipogenic cocktail induced a rapid [Elovl3], and cell-death-inducing DFFA-like effect A [Cidea]) was and continuous expression of Bmp7 and other upstream regula- also significantly dampened in the mutant cells, but expression tors (Figure 2D). Strikingly, Ews-deficient preadipocytes failed to of a nonadipogenic , Liver kinase B1 (Lkb1), was not express Bmp7 following the adipogenic stimulation. Expression suppressed. of the upstream transcription factor Cebpb was also significantly diminished in the absence of Ews, whereas Cebpd expression Loss of Ews Leads to a Block in Early Brown Fat was not affected. Expression of other early transcriptional Differentiation and Absence of Bmp7 Expression regulators (Krox20 [also known as Egr2] and Kruppel-like factor Because PPARg and CEBPa are critical transcriptional regula- 5 [Klf5]) was modestly reduced, whereas expression of an tors of adipogenesis, we sought to determine whether EWS upstream inhibitor of adipogenesis, Wnt10a (Kang et al., 2005; interacts with PPARg or CEBPa. However, we did not observe Tseng et al., 2005), was not altered.

Developmental Cell 26, 393–404, August 26, 2013 ª2013 Elsevier Inc. 395 Developmental Cell EWS Is Essential for Brown Fat Cell Determination

Figure 3. EWS and YBX1 Form a Complex upon Adipogenic Stimulation and Activate Bmp7 Transcription (A) IP-western blot analysis of U2OS cells co- transfected with CMV-EWS and CMV-YBX1 plas- mids (top panel). Lower panels show IP-western analysis of endogenous EWS and YBX1 interac- tion without (middle) or with (bottom) adipogenic stimulation (4 hr). (B) U2OS cells stably transfected with murine Bmp7 promoter-Luc (1,395 bp) were transfected with CMV-EWS, CMV-YBX1, or both along with Renilla luciferase, and luciferase activities were calculated after normalization with Renilla lucif- erase. Three independent experiments were per- formed in triplicate. Data are represented as means ± SEM. One-way ANOVA, ***p < 0.001. (C) ChIP analysis of murine Bmp7 promoter region with anti-EWS and anti-YBX1 with or without adi- pogenic stimulation (4 hr); 1% and 2% of total chromatin were used as input. Primers are indi- cated with arrows. Numbers are relative to the upstream of the translation start site (+1). (D) Wild-type brown preadipocytes transfected with siRNAs against Ews, Ybx1, or scrambled control were cultured for 8 days in media con- taining adipogenic cocktail and stained with oil red O. (E) Western blot analysis of brown preadipocytes transfected with Ews, Ybx1 or scrambled control siRNAs. (F) qRT-PCR analysis of differentiated brown adipocytes transfected with Ybx1 or scrambled control siRNA. (G) Bmp7 transcript was analyzed by qRT-PCR at indicated times (0–24 hr) following adipogenic stimulation in brown preadipocytes transfected with Ybx1 or scrambled control siRNA. Three independent experiments were performed and data are represented as means ± SEM. *p < 0.05, ***p < 0.001. See also Figure S3.

YBX1 interactions in brown preadipo- cytes with and without adipogenic stimu- lation. In the basal state, endogenous EWS interacted weakly with YBX1 (Fig- ure 3A, middle panels), but following a Ews Forms a Complex with Ybx1, and the Complex short adipogenic stimulation (4 hr), the EWS-YBX1 interaction Activates Bmp7 Transcription was greatly enhanced (Figure 3A, lower panels). BMP7 is the earliest known brown cell-fate determination factor, EWS encodes a potent transactivation domain in the amino and Bmp7 null newborns display smaller BAT size and markedly terminus (May et al., 1993) and is frequently involved in chromo- reduced UCP1 expression (Tseng et al., 2008), similar to Ews somal translocations that generate aberrant transcription factors mutants. Recently, YBX1 (also called YB1), a multifunctional (Sankar and Lessnick, 2011). Thus, we tested the effects of EWS protein with roles in transcription and translation (Kohno et al., on the transcriptional regulation of Bmp7 using a 1,395 bp 2003), was shown to bind and activate the mouse Bmp7 pro- (1392 to +3) mouse Bmp7 promoter-reporter construct moter (Wang and Hirschberg, 2011). This was intriguing given that contains an YBX1-binding site in the proximal region that EWS and YBX1 physically and functionally interact (Chansky (192 to +3) (Wang and Hirschberg, 2011). Expression of EWS et al., 2001; Dutertre et al., 2010). We first confirmed the EWS- or YBX1 alone resulted in a modest activation of the Bmp7 pro- YBX1 interaction by ectopic expression of EWS and YBX1 in moter (1.6-fold) compared with the empty vector control (Fig- U2OS cells followed by immunoprecipitation (IP) and western ure 3B). However, coexpression of EWS and YBX1 resulted in blot analysis (Figure 3A, top panels). To further demonstrate an additive activation (2.6-fold) of the Bmp7 promoter, which the physiological interaction, we examined endogenous EWS- was statistically significant compared with that observed with

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either one alone. The cooperative transcriptional nature of the Ews Null Embryos Display Markedly Reduced Bmp7 EWS-YBX1 complex was further examined by chromatin IP Expression in Early Brown Fat Progenitors (ChIP) analysis. In unstimulated brown preadipocytes, a small Next, we examined early embryonic BAT development. In amount of YBX1 was present on the proximal P1 region (443 contrast to WAT, which is generated postnatally, the develop- to 13) of Bmp7 promoter, along with barely detectable levels ment of classical BAT starts between E14.5 and E15.5 post- of EWS (Figure 3C), but following a short adipogenic stimulation coitus (Hirning et al., 1989) and completes differentiation prior (4 hr), EWS and YBX1 were rapidly recruited to the P1 region. to birth (Cristancho and Lazar, 2011; Gesta et al., 2007). In Less abundant but detectable levels of EWS and YBX1 were wild-type E15.5 embryos, interscapular BAT comprised of also found in the upstream regions. Interestingly, in Ews null densely packed patches of preadipocytes was readily detect- brown preadipocytes, a small amount of YBX1 that was present able (Figure 5Aa) and expanded rapidly through E17.5 (Fig- on the P1 region of Bmp7 promoter in the basal condition was ure 5Ab) with clearly visible multiloculated lipid droplets at not further enriched upon adipogenic stimulation (Figure S3A). E18.5 (Figure 5Ad). In contrast, E15.5 Ews null embryos dis- YBX1 has been shown to shuttle between the nucleus and cyto- played drastically reduced interscapular BAT comprised of plasm (Sutherland et al., 2005). However, inactivation of Ews did markedly smaller and fewer patches of brown preadipocytes not alter the nucleus/cytoplasm ratio or the expression of YBX1 that were loosely organized (Figure 5Ai). There was a limited (Figure S3B). These results strongly suggest that the recruitment expansion of brown adipocytes at E17.5 and E18.5 (Figures of both factors to the Bmp7 promoter is dependent on the forma- 5Aj and 5Ak), but mutant BATs still contained considerably fewer tion of EWS-YBX1 complex. adipocytes and lacked multiloculated lipid droplets (Figure 5Al). Immunostaining with Ki-67 antibodies revealed significantly YBX1 Is Essential for Bmp7 Expression during Brown fewer proliferating preadipocytes in the mutant embryonic Adipocyte Differentiation In Vitro BATs compared with wild-type (Figures 5B and 5C). In contrast, The above findings suggest an unexpected role for YBX1 in immunostaining with activated CASP3 antibodies revealed very brown fat development. To test this, we examined the effects few, if any, apoptotic cells in embryonic BATs of either genotype of small interfering RNA (siRNA)-mediated depletion of Ybx1 or (data not shown). Transmission electron microscopy (TEM) anal- Ews on brown fat differentiation. Silencing of Ybx1 or Ews led ysis of newborn BATs (P0.5) further confirmed the absence of to severe inhibition of brown adipocyte differentiation, with lipid droplets in mutant adipocytes (compare Figure 5Ah and significantly reduced expression of critical BAT genes such 5Ap). Expression of the critical BAT genes Pparg, Cox7a, as Ucp1, Pgc1a, Prdm16, and others (Figures 3D–3F). Impor- Ucp1, and Otopetrin1 was significantly reduced in E18.5 Ews tantly, depletion of Ybx1 resulted in a complete loss of Bmp7 null BATs (Figures S6A and S6B). expression following adipogenic stimulation (Figure 3G). These Next, we examined Bmp7 expression in E15.5–E18.5 embryos results demonstrate that both EWS and YBX1 are critical for by RNA in situ hybridization. In wild-type E15.5 BATs, Bmp7 was Bmp7 expression during the early steps of brown adipocyte not ubiquitously expressed in all cells, but was expressed in differentiation. intermittently dispersed aggregates of preadipocytes (Figures 5Ae and S6C). By E17.5, Bmp7 expression was greatly reduced Complementation of Ews or Bmp7 Restores Brown Fat (Figure 5Af) and became undetectable at E18.5 (Figure 5Ag). In Differentiation contrast, Bmp7 expression was dramatically diminished in early To determine whether restoring Ews expression in the mutant brown preadipocytes of E15.5 Ews null embryos (Figure 5Am) brown preadipocytes can rescue adipogenesis, we transduced and became undetectable at E17.5 (Figure 5An). There was no Ews null preadipocytes with a lentivirus expressing Ews or detectable signal with a sense Bmp7 probe (Figure S6D). As re- GFP as a control. Complementation of Ews in the mutant pre- vealed by immunostaining analysis, YBX1 was highly expressed adipocytes resulted in a partial restoration of adipogenesis, in the early brown fat precursors of E15.5 Ews+/+ and Ews/ whereas the GFP-expressing mutant cells failed to undergo dif- embryos (Figure S6E). ferentiation (Figure S4A). Expression of the downstream adipo- genic factors Pparg, Prdm16, and Ucp1 was significantly Elevated Myogenic Gene Expression in Ews Null Brown increased upon Ews complementation (Figure 4A). Ectopic Preadipocytes expression of CEBPb or PPARg also rescued adipogenesis in Because brown preadipocytes share a common lineage with the Ews null cells (Figures 4C and S4B), suggesting that these myogenic progenitors (Atit et al., 2006; Seale et al., 2008), we factors function downstream of Ews. Remarkably, re-expression examined the expression of myogenic genes in Ews mutant of Ews in the mutant cells restored Bmp7 expression following BATs. As expected, quantitative RT-PCR (qRT-PCR) showed a adipogenic stimulation (Figure 4B) and the expression of other significant reduction in the expression of BAT markers (Ucp1 early adipogenic factors (Figure S5A). To further demonstrate and Otopetrin1)(Seale et al., 2007) in the mutant BAT compared that BMP7 is the critical missing factor, Ews null brown preadipo- with the wild-type control (Figure 6A). Surprisingly, BATs from cytes were grown in the absence or presence of BMP7. Addition Ews null newborns expressed significantly elevated levels of of recombinant BMP7 along with adipogenic cocktail in Ews null myogenic genes, embryonic skeletal muscle Myosin heavy chain cells resulted in a robust brown fat differentiation (Figure 4C) and 3 (Myh3), Tnnt2, Myf5, Myf6, and (Myg)(Figure 6A). Im- restoration of critical adipogenic genes (Figures 4D and S5B), munostaining analysis further confirmed the dramatic increase in whereas the mutant cells grown in the adipogenic cocktail the expression of TNNT2 and MYH3 in Ews null newborns BATs without BMP7 failed to differentiate or express the critical adipo- compared with wild-type BATs (Figure 6B). MyoD and Myg tran- genic genes. scripts were also moderately elevated in E18.5 BATs of Ews null

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Figure 4. Complementation of Ews or BMP7 Rescues Brown Adipogenesis in Ews–/– Preadipocytes (A and B) Ews+/+ or Ews/ preadipocytes transduced with a lentivirus expressing either Ews or GFP (control) were stimulated with adipogenic cocktail for 8 days (A) or for the indicated hours (0–24 hr) (B), and the indicated transcripts were analyzed by qRT-PCR. Three independent experiments were performed and data are represented as means ± SEM. Two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S4. (C and D) Ews+/+ or Ews/ brown preadipocytes were cultured in media containing adipogenic cocktail with or without recombinant BMP7 (100 ng/ml) for 2 days, and continuously cultured in adipogenic cocktail-only containing media for 6 more days. Subsequently, cells were either stained with oil red O (C) or analyzed for BAT markers by qRT-PCR analysis (D). Three independent experiments were performed. Data in (D) are represented as means ± SEM. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S4 and S5. embryos (Figure S6B). Furthermore, following adipogenic stimu- Expression of Ews, Ybx1, and Bmp7 Is Induced in Mature lation, we observed increased expression of muscle-specific BAT in Response to Cold or a High-Fat Diet Tnnt2 and Myg genes in Ews null brown preadipocytes (Fig- To examine whether EWS might also play a role in the expan- ure S7A). Intriguingly, when cultured in myogenic differentiation sion of mature BAT in response to thermogenic stress, we media (2% horse serum), Ews null brown preadipocytes, but analyzed the expression of EWS, YBX1, and BMP7 along with not wild-type cells, expressed elevated levels of myogenic genes other brown fat markers in interscapular BAT of mice under (Figures 6C and 6D). However, unlike C2C12 myoblasts, mutant two different thermogenic stimuli: cold or a high-fat diet. In cells did not form fused muscle fibers. Similarly, depletion of either thermogenic stimulation, expression of Ews was Ews, Ybx1,orBmp7 by lentivirus-mediated shRNA in wild-type modestly induced compared with controls (Figures 7A and brown preadipocytes also resulted in elevated myogenic gene 7B). Expression of Bmp7 in BAT was markedly increased in expression following myogenic stimulation (Figure S7B). These response to cold along with other brown fat markers (Figure 7A). results suggest that Ews, along with Ybx1, is critical for brown In response to a 12-week high-fat diet, a 2-fold increase in Ybx1 fat lineage determination in early myogenic progenitors. Never- expression was observed, whereas Bmp7 expression was not theless, the development of muscle, bone, and kidney appeared significantly altered (Figure 7B). These results indicate that grossly normal in Ews null newborns (Figure S7C), and expres- EWS, YBX1, and their transcriptional target, BMP7, might play sion of Bmp7 in the kidneys of Ews null animals was unaltered a role in the expansion of mature BAT during adaptive compared with controls (Figure S7D). thermogenesis.

398 Developmental Cell 26, 393–404, August 26, 2013 ª2013 Elsevier Inc. Developmental Cell EWS Is Essential for Brown Fat Cell Determination

Figure 5. Loss of Bmp7 Expression in E15.5 BATs of Ews Null Embryos (A) Ews+/+ and Ews/ littermate embryos were harvested at E15.5 (a, e, i, and m), E17.5 (b, f, j, and n), and E18.5 (c, g, k, and o), and paraffin- embedded for H&E (a–d and i–l) or RNA in situ analysis (e–g and m–o) with antisense mouse Bmp7 probe. Insets and panels (d) and (l) show higher-magnification images of the boxed areas. Scale bars are indicated. (h and p) Transmission electron micrographs of P0.5 newborn BAT from Ews+/+ (h) and Ews/ (p). (B) Interscapular BAT from E18.5 Ews+/+ and Ews/ littermate embryos were immunostained with Ki-67 and DAPI. Representative images from two independent embryos are shown. (C) Quantification of Ki-67-positive brown fat cells. Data are represented as means ± SEM. Student’s t test, two-tailed, ***p < 0.001. See also Figure S6.

glitazone or b3-adrenergic stimulation also induced expression of Ews, Ybx1, and Bmp7 in inguinal fat. In contrast, rosi- glitazone- or b3-agonist-induced expres- sion of Ews, Ybx1, Bmp7 Ucp1, and Cidea was significantly reduced in inguinal fat of Ews+/ mice compared with Ews+/+ littermates (Figures 7C–7E). Expression of the brite/beige cell-specific gene Tmem26 (Wu et al., 2012) was also significantly reduced (Figure S7E). These results suggest that EWS may play an important role in generating thermogeni- cally induced brown fat cells in res- ponse to rosiglitazone or b-adrenergic stimulation.

DISCUSSION

The obesity epidemic, driven by exces- sive energy intake and inactivity leading to accumulation of fat in mostly visceral A Role for EWS in Generating Thermogenically Induced WAT, has become a major global health threat. Recent discov- Brown Fat eries of active BATs in adults (Cypess et al., 2009; Nedergaard In response to b-adrenergic agonist or PPARg agonist stimula- et al., 2007; van Marken Lichtenbelt et al., 2009), coupled with tion, a distinctive type of brown fat cells is generated in WAT the energy-burning capacity of BAT in humans (Ouellet et al., depots (Frontini and Cinti, 2010; Wu et al., 2013). Thermogeni- 2012), have spurred excitement about the possibility of utilizing cally induced brown fat cells (brite or beige cells) are generated BAT expansion and/or activation as a therapeutic approach. more abundantly in subcutaneous WAT depots (e.g., inguinal fat) Thus, a precise understanding of the upstream regulators than in visceral WAT. A recent study showed that BMP7 is also of brown adipogenesis will facilitate the development of new important for generating brown adipocytes from progenitor cells approaches to counteract obesity and related metabolic disor- isolated from subcutaneous WAT and skeletal muscle (Schulz ders. BMP7 is the earliest known brown adipogenic determina- et al., 2011). To examine whether EWS might play a role in the tion factor (Tseng et al., 2008), but to date, the upstream genes formation of brite/beige cells in vivo, we administered rosiglita- that regulate BMP7 expression have not been identified. Our zone for 4 days or b3-agonist CL316,243 for 10 days to Ews+/+ study fills this void by demonstrating that EWS, along with and Ews+/ mice and analyzed the expression of brown fat YBX1, directly activates BMP7 expression during the early markers in inguinal WAT. In wild-type mice, expression of brown adipocyte development. The transcriptional regulation Ucp1 and Cidea was significantly increased following rosiglita- of BMP7 by EWS appears to be specific to brown fat tissue, as zone or CL316,243 treatment (Figures 7C–7E). Intriguingly, rosi- BMP7 expression in kidneys of Ews null mice was unaltered.

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Figure 6. Ews Deficiency Leads to Ectopic Expression of Myogenic Genes in BATs (A) SYBR Green qRT-PCR analysis of brown fat (Ucp1 and Otopetrin1) and myogenic (Mhy3, Tnnt2, MyoD, Myf5, Myf6, and Myg) genes in Ews+/+ and Ews/ newborn BATs (P0.5; n = 5 per genotype). Relative transcript levels were calculated after normalization with b-Actin. Data are represented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Immunohistochemistry of Ews+/+ and Ews/newborn BATs (P0.5) with antibodies against TNNT2 and MYH3. Scale bar is indicated. (C) qRT-PCR analysis of Ews+/+ or Ews/ brown preadipocytes cultured for 6 days in 2% horse serum media (myogenic stimulation). Three independent experiments were performed and data are represented as means ± SEM. *p < 0.05, ***p < 0.001. (D) Immunostaining of Ews+/+, Ews/, or C2C12 cells grown for 6 days in 2% horse serum with anti-MYHC antibody and DAPI (blue). Two representative fields are shown. See also Figure S7.

Interestingly, adipogenic stimulation rapidly induces or stabilizes any features of muscle differentiation other than the elevated the EWS-YBX1 interaction, but the mechanism by which the myogenic gene expression. A similar observation was reported EWS-YBX1 complex is formed or stabilized is not clear. Identi- in a study of Prdm16 null mice (Seale et al., 2008), in which fying the upstream signaling pathways that lead to formation ectopic myogenic expression was detected in mutant BAT tis- of the EWS-YBX1 complex to induce brown adipogenesis will sues with no signs of muscle differentiation. These findings provide further mechanistic insights into early BAT development. indicate that in the absence of Ews or Prdm16, early Myf5+ Additionally, our findings reveal a role for YBX1 in brown fat brown fat progenitors will ectopically express myogenic genes, development. Lu et al. (2005) generated and characterized perhaps by default as a result of loss of brown fat specification, Ybx1-deficient mice, which died shortly after birth; however, but are incapable of generating fully differentiated muscle. they did not examine BAT development in these mice. Hence, This might be due to a lack of external myogenic signals in vivo confirmation of the role of YBX1 in BAT development required to generate myofibers, or it could indicate that Ews awaits further studies. null or Prdm16 null preadipocytes have partially committed to- Loss of Ews in early brown preadipocytes not only leads to a ward a nonmyogenic lineage. It is also possible that EWS might block in BAT development but also results in elevated myogenic suppress myogenic differentiation in the Myf5+ progenitors. gene expression. Interestingly, shRNA-mediated depletion of However, ectopic expression of EWS in the C2C12 myoblast either Ybx1 or Bmp7 in brown preadipocytes also led to cell line did not inhibit myogenic differentiation (data not shown). increased expression of myogenic genes. These findings pro- A role for the EWS-YBX1-BMP7 axis in thermogenesis is vide further evidence that EWS, along with YBX1 and its tran- suggested by their increased expression under different ther- scriptional target BMP7, is critical for brown fat specification in mogenic stimuli, and by the reduced expression of brown- early Myf5+ progenitors. Although Ews null brown preadipocytes fat-specific genes in inguinal WAT of Ews heterozygous mice expressed myogenic genes in response to myogenic stimula- stimulated with b3-agonist or rosiglitazone. However, to unequiv- tion, these cells were incapable of forming fused muscle fibers ocally demonstrate a role for EWS in adaptive thermogenesis, a in culture. Moreover, BATs from Ews null mice did not show conditional Ews null mouse is needed. It also remains to be

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Figure 7. Expression of Ews, Ybx1, and Bmp7 Is Induced in Response to Thermogenic Stimulation, and Ews Haploinsufficiency Leads to Reduced Brown Fat Gene Expression following Rosiglitazone or b3-Agonist Stimulation (A and B) qRT-PCR analysis of interscapular BAT from C57BL6 mice after 8 hr exposure to ambient or cold (4C) temperature (n = 3; A) or after consuming a normal or high-fat diet for 12 weeks (n = 3; B). (C) qRT-PCR analysis of inguinal fat fads from Ews+/+ or Ews+/ mice following four daily i.p. injections with PBS or rosiglitazone (10 mg/kg; n = 3). (D) Immunostaining of UCP1 in inguinal fat from (C). Representative images are shown; 1003 magnification. (E) qRT-PCR analysis of inguinal fat fads from Ews+/+ or Ews+/ mice following ten daily i.p. injections with PBS or CL316,243 (1 mg/kg; n = 3). Data are represented as means ± SEM. Two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S7. determined whether EWS requires YBX1 and subsequent activa- balanced chromosomal translocation t(12;16)(q13;p11) is found tion of BMP7 for the formation of brite/beige cells in response to that results in a fusion of FUS to DDIT3 (FUS-DDIT3, also called rosiglitazone or b-adrenergic stimulation. FUS/TLS-CHOP)(Crozat et al., 1993; Rabbitts et al., 1993), It is evident that EWS has additional functions outside of BAT and in minor cases, a t(12;22)(q13;q12) translocation is found, development, as our previous studies demonstrated its roles in B leading to a fusion of EWS-DDIT3 (Panagopoulos et al., 1996). cell development, meiosis, and premature senescence (Cho These findings, along with others (Zinszner et al., 1994), suggest et al., 2011; Li et al., 2007). EWS encodes a multifunctional that the N-terminal domains of EWS and FUS are functionally RNA binding protein that, despite lacking a sequence-specific interchangeable. DDIT3 (CHOP) encodes a member of the double-stranded DNA binding domain, functions in transcription CEBP family of transcription factors, and by virtue of forming a (Araya et al., 2003) as well as in splicing (Dutertre et al., 2010; heterodimer with other CEBP family , it functions in a Paronetto et al., 2011). This is similar to other RNA binding pro- dominant-negative manner (Ron and Habener, 1992). Notably, teins such as PGC1a (Girnun, 2012) and FUS/TLS (Tan et al., it has been shown that FUS-DDIT3 forms a complex with CEBPb 2012). Given that FUS and EWS are paralogs with sequence and inhibits adipogenesis by blocking CEBPb transcriptional and functional similarities, it will be interesting to examine activity (Adelmant et al., 1998; Kuroda et al., 1997). The findings whether FUS also has a role in adipogenesis. However, our find- in this study suggest a mechanism by which EWS-DDIT3 ings clearly show that FUS does not compensate for the loss of (or FUS-DDIT3) might further interfere with adipogenesis by EWS during classical BAT development. Interestingly, in the inhibiting the functions of remaining EWS (e.g., regulation of majority of myxoid liposarcomas (a subtype of liposarcoma), a BMP7).

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EXPERIMENTAL PROCEDURES tiation media (exchanged every 2 days) for 6 days (Fasshauer et al., 2001). For BMP7-induced adipogenesis, Ews+/+ or Ews/ preadipocytes were treated Cell Lines and siRNA Transfection with induction media containing recombinant mouse BMP7 (R&D Systems) U2OS, HKE293, and C2C12 cells were cultured in Dulbecco’s modified for 2 days (100 ng/ml) and cultured in differentiation media for 6 days as Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), described above. Cells were fixed with 4% formalin and stained for 2 hr with

100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen) at 5% CO2. For 0.5% oil red O solution (in 70% isopropyl alcohol). siRNA transfection, cells were transfected with 50 nM of pooled siRNAs against Ews (three independent), Ybx1 (two independent), or scrambled Histology, Immunohistochemistry, and Transmission Electron control using Lipofectamine RNAiMAX (Invitrogen). The following siRNAs Microscopy were purchased and mixed before transfection: Wild-type and mutant embryos (E15.5, E17.5, and E18.5) and newborns (P0.5) were euthanized, fixed in 10% neutral formalin (Sigma), embedded in paraffin, mEws-1: 50-GCA GUU ACU CUC AGC AGA AdTdT-30 (Sigma) and sectioned for H&E staining. Oil red O staining was performed on freshly mEws-2: 50-GAG ACU AGU CAA CCU CAA UdTdT-30 (Sigma) frozen newborn brown fat tissues. Immunohistochemistry was performed us- mEws-3: 50-CUG ACA ACA GUG CAA UUU AdTdT-30 (Sigma) ing the ABC elite kit and ImmPACT DAB peroxidase substrate (Vector Labs). mYbx1-1: 50-AGA AGG UCA UCG CAA CGA AdTdT-30 (Ambion) Samples were counterstained by hematoxylin GS (Vector Labs), dehydrated, mYbx1-2: 50-CCA CGC AAU UAC CAG CAA AdTdT-30 (Ambion) and mounted for light microscopy analysis (Leica Microsystems). Ki-67 immu- Scrambled Silencer Negative Control#1 (Ambion) nostaining was performed with mouse anti-Ki67 and Alexa594-conjugated secondary antibodies and mounted with DAPI-containing mounting media Antibodies (Vector Labs). Randomly chosen fields were photographed (at least four fields Rabbit polyclonal EWS antibody has been described previously (Li et al., 2007) per embryo) and Ki-67-positive cells were counted and quantified against the and was purchased from Bethyl Laboratories. The following antibodies were total number of nuclei. For transmission electron microscopy (TEM), freshly used: mouse anti-GAPDH, goat anti-PGC1a, rabbit anti-PRDM16, and mouse isolated BAT (P0.5) was immediately fixed for 4 hr in 2.5% glutaraldehyde anti-PPARg (Santa Cruz Biotechnology); rabbit anti-YBX1 (Bethyl Labora- and processed for TEM analysis. tories); mouse anti-TLS (BD Biosciences); rabbit anti-CEBP/a, rabbit anti- CEBP/b, and rabbit anti-CEBP/d (Cell Signaling); mouse anti-a-Actin (Sigma); IP-Western Blot Analysis rabbit anti-UCP1 (Abcam); mouse anti-Ki67 clone TEC-3 (DAKO); and rabbit Ews+/+ and Ews/ preadipocytes were grown in the absence or presence of anti-TNNT2 (Proteintech). Anti-MYHC and anti-MYH3 antibodies were pro- adipogenic cocktail for 4 hr and nuclear extracts were prepared. EWS or YBX1 vided by Alexandra McPherron (NIDDK). was immunoprecipitated with either anti-EWS or anti-YBX1 antibody, followed by SDS-PAGE, and western blotting was performed with anti-EWS or anti- Lentivirus Vectors YBX1. The same procedure was used for U2OS cells transfected with CMV- Lentivirus shRNAs against mouse Ews, Ybx1, and Bmp7 were purchased from Ews and CMV-Ybx1. Sigma. Full-length mouse Ews complementary DNA (cDNA) was cloned into the EcoRI/NotI sites of pCDH-GFP-puro lentivirus vector (SBI). Lentivirus vec- ChIP Assay tor harboring PPARg was provided by Kai Ge (NIDDK), and lentivirus vector ChIP was performed as previously described (Kim et al., 2007) using rabbit containing CEBPb was purchased from Addgene (#15712). anti-EWS (Li et al., 2007) or anti-YBX1 antibody, the Magna chip G kit (Milli- pore), and the Taq PCR Master kit (QIAGEN). The primers used for ChIP are Animal Handling listed in the Supplemental Experimental Procedures. Ews+/ mice in mixed background (129SvEv/Black Swiss) were crossed with C57BL6 for 12 generations. Ews+/ mice (C57BL6N12) were intercrossed to Gene-Expression Analysis by qRT-PCR generate Ews null mice. A multiplex PCR was used for genotyping (see Supple- All tissues were harvested, immediately stored in RNAlater (Ambion), and kept mental Experimental Procedures for primers). All animal procedures were at 80C. Total RNA was isolated using the RNeasy mini kit (QIAGEN) and approved and performed according to the guidelines provided by the NIH quantified using NanoDrop (NanaDrop Technology). cDNAs were synthesized Animal Research Advisory Committee and the Tulane University Institutional using the SuperScript cDNA synthesis kit (Invitrogen), and analyzed by real- Animal Care and Use Committee. time PCR with either TaqMan probes or the SYBR Green method (Applied Biosystems). The relative quantity of each transcript was calculated by the Expression Analysis of BAT and Inguinal Fat under Different comparative Ct method normalized against either Gapdh or b-Actin. Thermogenic Challenges Interscapular BATs from C57BL6 mice housed individually at ambient temper- Mouse Bmp7 Promoter-Reporter Assay ature or at 4C for 8 hr, and fed a normal or high-fat diet for 12 weeks, were A 1.4-kb mouse Bmp7 promoter-reporter plasmid (pGL3-Bmp7-luc-1395) isolated and the expression of the indicated genes was analyzed by qRT- (Wang and Hirschberg, 2011) was kindly provided by Dr. Raimund Hirschberg PCR (n = 3 for each group). Ews+/+ or Ews+/ mice were injected daily (i.p.) (Los Angeles Biomedical Research Institute, Los Angeles, CA). Stable U2OS with rosiglitazone (10 mg/kg) or PBS for 4 days (n = 3 per genotype), and cells harboring pGL3-Bmp7-luc-1395 plasmid was generated using puromy- inguinal fat pads were isolated and analyzed by qRT-PCR for expression of cin selection. Stable U2OS cells were transfected with empty pCMVSport6 the indicated genes. Similar experiments were performed with ten daily injec- (control), pCMVSport6-EWS, or pCMVSport6-YBX1, or both, along with tions (i.p.) of the b3-agonist CL316,243 (1 mg/kg). Renilla luciferase plasmid (Promega). After 48 hr, cells were harvested and measured for luciferase activity using the Dual-Luciferase Reporter assay sys- Generation of Brown Preadipocyte and Differentiation tem (Promega). Brown preadipocytes were isolated from P0.5 Ews+/+, Ews+/,orEws/ mouse interscapular brown fat pads and immortalized using a retrovirus RNA In Situ Hybridization expressing SV40 large T antigen as previously described (Fasshauer et al., A 270 bp mouse Bmp7 cDNA was amplified (forward: 50-CGA GAC CTT CCA 2000). Immortalized brown preadipocytes were cultured in DMEM supple- GAT CAC AGT-30; reverse: 50-ATG AAG GGT TGC TTG TTC TG-30), cloned into mented with 10% FBS. To induce differentiation into mature brown adipo- pCRII plasmid (Invitrogen), and verified by sequencing. The antisense or sense cytes, preadipocytes were cultured for 4 days in differentiation media probes were synthesized using the DIG RNA labeling kit (Roche). Embryos supplemented with insulin and triiodothyronine (T3; Sigma), followed by a were harvested at different times (E15.5, E17.5, and E18.5), fixed immedi- 2 day incubation with induction media (the beginning of this induction period ately in 10% formalin, and prospectively genotyped (tail biopsy). Paraffin- was considered as differentiation day 0) supplemented with insulin, triiodothy- embedded embryos were sectioned (10 mm), hybridized with either antisense ronine, indomethacin, dexamethasone, troglitazone, and IBMX (this is referred or sense Bmp7 RNA at 55C overnight, and visualized using alkaline phospha- to as the adipogenic cocktail). After induction, cells were cultured in differen- tase-coupled anti-digoxigenin antibody and BCIP-NBT substrate (Roche).

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Myogenic Differentiation Cristancho, A.G., and Lazar, M.A. (2011). Forming functional fat: a growing Ews+/+ and Ews / preadipocytes were grown in DMEM with 2% horse serum understanding of adipocyte differentiation. Nat. Rev. Mol. Cell Biol. 12, for 6 days, and total RNA was isolated and analyzed by qRT-PCR for the indi- 722–734. cated myogenic genes. For immunofluorescence with anti-MYHC, cells grown Crozat, A., Aman, P., Mandahl, N., and Ron, D. (1993). Fusion of CHOP to in DMEM containing 2% horse serum were fixed, blocked with 5% goat serum, a novel RNA-binding protein in human myxoid liposarcoma. Nature 363, incubated overnight with anti-MYHC, and visualized with Alexa Fluor 594 goat 640–644. anti-mouse secondary antibody (Molecular Probes). DAPI was used to visu- Cypess, A.M., Lehman, S., Williams, G., Tal, I., Rodman, D., Goldfine, A.B., alize nuclei. Kuo, F.C., Palmer, E.L., Tseng, Y.H., Doria, A., et al. (2009). 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Essential role of insulin receptor substrate-2 in insulin stimulation of Glut4 translocation and glucose uptake in brown adipocytes. SUPPLEMENTAL INFORMATION J. Biol. Chem. 275, 25494–25501. Fasshauer, M., Klein, J., Kriauciunas, K.M., Ueki, K., Benito, M., and Kahn, Supplemental Information includes Supplemental Experimental Procedures C.R. (2001). Essential role of insulin receptor substrate 1 in differentiation of and seven figures and can be found with this article online at http://dx.doi. brown adipocytes. Mol. Cell. Biol. 21, 319–329. org/10.1016/j.devcel.2013.07.002. Frontini, A., and Cinti, S. (2010). Distribution and development of brown adipo- cytes in the murine and human adipose organ. Cell Metab. 11, 253–256. ACKNOWLEDGMENTS Gesta, S., Tseng, Y.H., and Kahn, C.R. (2007). Developmental origin of fat: tracking obesity to its source. Cell 131, 242–256. We thank Raimund Hirschberg (Los Angeles Biomedical Research Institute) for Girnun, G.D. (2012). The diverse role of the PPARg coactivator 1 family of providing the murine Bmp7 promoter-Luc plasmids; Huiyan Lu (NIDDK) for transcriptional coactivators in cancer. Semin. Cell Dev. Biol. 23, 381–388. in vitro fertilization and generation of the Ews mutant 129SvEv/Black Swiss Gupta, R.K., Mepani, R.J., Kleiner, S., Lo, J.C., Khandekar, M.J., Cohen, P., strain; Oksana Gavrilova (NIDDK) for the temperature-controlled chamber; Frontini, A., Bhowmick, D.C., Ye, L., Cinti, S., and Spiegelman, B.M. (2012). Sandra E. Dunn (University of British Columbia), Alexandra McPherron and Zfp423 expression identifies committed preadipocytes and localizes to Yun Kyung Lee (NIDDK), and Hyeog Kang (NHLBI) for helpful discussions; adipose endothelial and perivascular cells. Cell Metab. 15, 230–239. and Jan Endlich (JFE Enterprises) for TEM. This research was supported in Hirning, U., Schmid, P., Schulz, W.A., Kozak, L.P., and Hameister, H. (1989). part by the Intramural Research Program of the NIDDK, NIH, and by startup In developing brown adipose tissue c- protooncogene expression is funds from Tulane University (to S.B.L.). J.H.P., H.J.K., S.I.K., J.H., and H.L. restricted to early differentiation stages. Cell Differ. Dev. 27, 243–248. performed experiments; J.E.L., K.G., E.M., and B.-C.L. contributed critical reagents and suggestions; and J.H.P. and S.B.L. designed experiments, Kajimura, S., Seale, P., and Spiegelman, B.M. (2010). Transcriptional control of analyzed data, and wrote the manuscript. brown fat development. Cell Metab. 11, 257–262. Kang, S., Bajnok, L., Longo, K.A., Petersen, R.K., Hansen, J.B., Kristiansen, K., Received: January 2, 2013 and MacDougald, O.A. (2005). Effects of Wnt signaling on brown adipocyte Revised: June 25, 2013 differentiation and metabolism mediated by PGC-1alpha. Mol. Cell. Biol. 25, Accepted: July 3, 2013 1272–1282. 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