Keywords: Obesity, Thermogenesis, Adipogenesis, OVOL2, UCP1, C/EBPα

Theboh phenotype

A phenotype observed among third-generation (G3) C57BL/6J mice heterozygous or homozygous for mutations induced byN-ethyl-N-nitrosourea (ENU), termedboh (Figure 1A), was characterized by increased body weight first detected at 10 weeks of age (Figure 1B) and approximately 20% increased at 12 weeks of age (Figure 1C). Heterozygous and homozygousboh mice were born at expected Mendelian frequencies and survived into adulthood. Magnetic resonance spectroscopy (MRS) showed thatboh/boh mice had an extremely high fat weight and low lean weight; indeed, higher than the comparatively small increase in total body weight at this age (Figures 1C–1E) would suggest. Approximately 556% increase in fat weight (Figure 1D) was accompanied by a 20% reduction in lean weight compared to wild type (WT) littermates (Figure 1E). Necropsy revealed that adipose tissue beds ofboh/boh mice were increased in size relative to those in WT littermates, including interscapular brown adipose tissue (iBAT), epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) (Figures 1F andS1A–S1C). An enlargement of both iBAT and the outside covering WAT was associated with a “hump” and a hairless region on the backs ofboh/boh mice (Figures 1A and1F). Hematoxylin and eosin (H&E) staining revealed enlarged adipocytes, indicative of hypertrophy (Figures 1G–1L,S1D, andS1E). In addition to the increased adipocyte size, plotting of adipocyte volume versus adipose tissue weight showed thatboh/boh WAT contained an increased number of adipocytes compared to WT WAT, indicative of hyperplasia (Figures S1F andS1G). At 12 weeks of age, fasting blood glucose levels were comparable betweenboh/boh mice and WT littermates, while fasting insulin levels were dramatically increased inboh/boh mice (Figures 1M and1N). Glucose intolerance (Figure 1O) and insulin resistance (Figure 1P) were both observed in 12-week-oldboh/boh mice. Interestingly, fasting blood glucose (Figure S1H), fasting insulin levels (Figure S1I), glucose tolerance (Figure S1J), and insulin tolerance (Figure S1K) were normal in 5-week-oldboh/boh mice when body weight was not increased. Presumably as a result of increased adiposity,boh/boh mice had increased leptin in the serum (Figure 1Q). Interestingly, while 12-week-oldboh/boh mice had increased fasting cholesterol levels (Figure 1R), no significant differences in the fasting triglyceride levels were observed (Figure 1S). In addition, 16-week-oldboh/boh mice had large, pallid livers and significantly increased body weight (Figures 1T–1V); H&E staining and Oil Red O (ORO) staining of liver sections showed an abundance of stored lipid (Figures 1W–1Z). Transmission electron microscope (TEM) images of liver sections showed “adipocyte-like” abnormal hepatocytes, each containing a single large lipid droplet (Figures S1L andS1M).

Theboh phenotype is caused by anOvol2 mutation

Theboh phenotype was mapped as a quantitative trait. A total of 92 G3 mice were weighed, and weights were scaled with respect to age and sex (Figure 2A). Automated mapping (Wang et al., 2015b) implicated a missense allele ofOvo like zinc finger 2 (Ovol2) as the causative mutation, displaying strongest linkage in a recessive model of inheritance (P=1.086×10⁻¹²) (Figures 2A and2B). Theboh mutation was a single nucleotide transition from G to A, causing substitution of a tyrosine for a highly conserved cysteine at position 120 (C120Y) in OVOL2 isoform A (OVOL2-A) and position 87 (C87Y) in the shorter OVOL2 isoform B (OVOL2-B), within the first zinc finger domain of both isoforms (Figures 2C andS2A). Overexpression of the WT andboh mutant forms of OVOL2 in 293T cells revealed a similar expression level by immunoblot, suggesting that theboh mutation does not affect the stability of OVOL2 protein (Figure 2D).

Ovol2 is highly expressed in the testis (Masuet al., 1998). Although the expression ofOvol2 is low in adipose tissues compared to testis, it is detectable and furthermore, a relatively higher expression level ofOvol2 was detected in the stromal vascular fraction (SVF) than in fat cells themselves (Figure 2E). In addition, SVF isolated from eWAT and iWAT expressed moreOvol2 compared with SVF from iBAT (Figure 2E). The expression ofOvol2 mRNA was increased in the iWAT ofboh/boh mice, suggesting possible feedback regulation ofOvol2 transcription (Figure S2B). By CRISPR/Cas9 gene targeting, a null allele ofOvol2 was made. No phenotype was apparent in heterozygotes for the null allele (Ovol2^+/−). TheOvol2^−/− genotype caused fully penetrant embryonic lethality as previously reported (Mackayet al., 2006;Unezakiet al., 2007). However, compound heterozygotes with theboh allele and a null allele ofOvol2 (Ovol2^boh/−) were viable and displayed all aspects of theboh phenotype (Figures 2F–2P andS2C–S2W).Ovol2^boh/− mice had increased body weight beginning at 9 weeks of age (Figure 2F), when body weights of homozygousboh mice were still normal (Figure 1B). At 12 weeks of age,Ovol2^boh/− mice had overall stronger phenotypes thanOvol2^boh/boh mice. These data are consistent with the interpretation that theboh allele confers a viable hypomorphic effect.

Obesity in OVOL2 deficient mice develops with normal food intake and decreased energy expenditure

Food intake ofOvol2^boh/boh andOvol2^boh/− mice was monitored beginning at 6 weeks of age (Figures 3A–3D). No significant differences in food intake were found betweenOvol2^boh/boh mice and WT littermates before 10 weeks of age (Figure 3A), when body weight was also similar (Figure 1B). Beginning at 10 weeks of age, increased body weight ofOvol2^boh/boh mice was accompanied by slightly increased food intake per mouse per day (Figure 3A); however, the cumulative food intake until 13 weeks of age was not significantly different from that of WT mice (Figure 3B). No significant differences in food intake were found betweenOvol2^boh/− mice and WT littermates before 9 weeks of age (Figure 3C), when body weight was also similar (Figure 2F). Beginning at 9 weeks of age, significantly increased body weight ofOvol2^boh/− mice was accompanied by significantly increased food intake per mouse per day (Figure 3C), and the cumulative food intake ofOvol2^boh/− mice was also significantly increased (Figure 3D). To check if the increased food intake was the cause of the obesity in OVOL2 deficient mice,Ovol2^boh/boh mice were pair-fed with the same amount of food as WT littermate mice beginning at 7 weeks of age when there were no differences in body weight, fat weight, and lean weight (Figures 3E–3G). Four weeks after pair-feeding,Ovol2^boh/boh mice had gained significantly more fat weight than WT littermate mice (Figure 3F), suggesting a contribution of factors other than increased food intake to the obesity in OVOL2 deficient mice. Indeed, metabolic cage experiments revealed thatOvol2^boh/boh mice had decreased energy expenditure during the dark phase of the light cycle (Figures 3H and3I). In addition,Ovol2^boh/boh mice had slightly increased respiratory exchange ratio (RER) (Figure 3J) during the light phase, suggesting a decreased use of lipid in the energy production. No consistent change of activities betweenOvol2^boh/boh mice and WT littermate mice could be determined.Ovol2^boh/boh mice had slightly increased central fine movement (Figure 3K) during the dark phase with no change in central ambulatory movement (Figure 3L).Ovol2^boh/boh mice also presented decreased peripheral fine and ambulatory movement during the dark phase and slightly increased peripheral fine and ambulatory movement during the light phase (Figures 3M and3N). Together, these data suggest that decreased energy expenditure might contribute to the development of theboh obese phenotype.

Obesity ofLep^ob/ob mice is enhanced byOvol2^boh/boh

Lep^ob/ob mutant mice (C57BL/6J background) (Ingalls et al., 1950;Zhang et al., 1994) were crossed toOvol2^boh/boh mice and double heterozygotes intercrossed to determine whether homozygosity for both mutations would result in a more severe obesity phenotype than homozygosity for either single mutation. At 16 weeks of age,Lep^ob/ob; Ovol2^+/+ mice displayed a 252% increase in body weight with respect to WT mice.Lep^+/+;Ovol2^boh/boh mice showed a 163% increase in body weight.Lep^ob/ob;Ovol2^boh/boh mice showed a 285% increase in body weight, significantly more than mice homozygous for either mutation alone (Figure S3A). Similarly, the fat/lean body weight ratio was higher inLep^ob/ob;Ovol2^boh/boh mice than in either of the single homozygotes (Figure S3B). A significant difference was also observed for total body fat (Figure S3C). However, whileLep^ob/ob; Ovol2^+/+ mice showed increased lean weight,Lep^+/+;Ovol^boh/boh mice had decreased lean weight, both in comparison to WT mice. The lean weights ofLep^ob/ob;Ovol2^boh/boh mice were comparable to those of WT littermates (Figure S3D).Lep^ob/ob;Ovol2^boh/boh mice also showed elevated levels of fasting blood glucose (Figure S3E), fasting insulin (Figure S3F), fasting triglycerides (Figure S3G), fasting cholesterol (Figure S3H), and liver lipid (Figures S3I–S3P) compared with the single homozygotes. The exacerbated weight, fat, and metabolic phenotypes observed inLep;Ovol2 double mutant mice compared with either single mutant suggest that leptin and OVOL2 function in distinct signaling pathways to control obesity. Whereas obesity in the classicob model is caused by increased caloric consumption and reduced energy expenditure,Ovol2^boh/− andOvol2^boh/boh mice show at worse a mild increase in food intake over their WT littermates, which did not fully account for the increase in fat and body weight caused by OVOL2 deficiency.

Defective thermogenesis in OVOL2 deficient mice

Since we observed decreased energy expenditure in OVOL2 deficient mice but no reduction in physical activities, we moved on to measure cold tolerance in these animals. When housed at room temperature (23 °C), 16-week-oldOvol2^boh/− mice had normal basal core body temperature (Figure 4A). During an acute cold stress experiment (6 °C) in the absence of food,Ovol2^boh/− mice failed to maintain their core body temperature as compared with WT littermate control mice (Figure 4A). We noticed a progressive hair loss on the back of both male and femaleOvol2^boh/boh mice (Figures S4A andS4H) which could reduce the skin insulation. Indeed, H&E staining showed disrupted hair follicles inOvol2^boh/boh mice which was consistent with the progressive hair loss phenotype (Figures S4B–S4G andS4I–S4N). On the other hand,Ovol2^boh/boh mice had significantly increased dermal adipose tissues which could provide better insulation against cold (Figures S4B–S4G andS4I–S4N). We shaved bothOvol2^boh/− mice and WT littermate mice to eliminate the effect of hair in the acute cold stress experiment (Figure 4B). The core temperature ofOvol2^boh/− mice dropped dramatically to 23.5 °C at 45 min while the core temperature of WT littermate mice was maintained at 32.7 °C (Figure 4B). These data suggest that although theboh mutation clearly affects the development of hair follicles, the reduction in hair growth is not solely responsible for the cold intolerance of OVOL2 deficient mice. Moreover, the increased dermal adipose tissue inOvol2^boh/boh mice is insufficient for these animals to maintain a normal body temperature under acute cold exposure.

When the housing temperature was decreased from 30 °C to 6 °C within a 12 h period,Ovol2^boh/boh mice increased their energy expenditure similarly to WT mice for the first 2 h but then expended less energy than WT littermate mice for the remainder of the experiment (Figures 4C and4D). This finding suggested that as the temperature fell below the lower limit of the thermoneutral zone (~28°C), mechanisms by which WT mice perform thermogenesis fail inOvol2^boh/boh mice. In addition to cold, we monitored energy expenditure as an indication of thermogenesis induced by noradrenaline (NA), a nonselective agonist of adrenergic receptors and an established inducer of BAT-dependent thermogenesis (Figures 4E and4F). As shown inFigure 4F, NA injection increased energy expenditure in WT mice but barely did so inOvol2^boh/boh mice. Together these data suggest that brown fat function may be compromised in OVOL2 deficient mice.

OVOL2 is essential for brown/beige fat mediated thermogenesis

Compared to the iBAT of WT littermates, the color of iBAT from 16-week-oldOvol2^boh/boh andOvol2^boh/− mice appeared abnormally white (Figures 1F andS2C). H&E staining revealed that adipocytes withinOvol2^boh/boh andOvol2^boh/− iBAT were heterogeneous in size, with many containing a large unilocular lipid droplet (Figures 1G,1H,S2D, andS2E), suggesting the function of brown fat might be compromised. Immunohistochemical staining of UCP1 in iBAT ofOvol2^boh/− mice showed that UCP1 appeared to be absent from the larger adipocytes containing unilocular lipid droplets, while the smaller adipocytes with multilocular lipid droplets were positive for UCP1 expression (Figures 5A and5B). Beige adipocytes were visible as a cluster of UCP1 positive cells in the iWAT of WT mice (Figure 5C), but no UCP1 positive beige adipocytes were found in the iWAT ofOvol2^boh/− mice (Figure 5D). Consistent with the histological analysis, quantification ofUcp1,Prdm16,Cidea, andPpargc1a by real-time PCR showed a reduction of these brown/beige cell markers in iBAT of 16-week-oldOvol2^boh/− mice (Figure 5E), as well as in iWAT, where absolute levels of these mRNAs were lower than in iBAT (Figure 5F). To directly examine energy metabolismin vitro, we used Seahorse XF system to measure the oxygen consumption rate (OCR) of isolated brown adipose tissues. As shown inFigure 5G, OVOL2 deficient brown adipose tissues had a decreased OCR for all test conditions (ATP-linked, maximal, and non-mitochondrial) compared with WT brown adipose tissues. Moreover, addition of NA in the medium further increased OCR in WT brown adipose tissues, but not in OVOL2 deficient brown adipose tissues (Figure 5G). The reduced OCR in all conditions suggested a reduced number of mitochondria. Indeed, the iBAT of 16-week-oldOvol2^boh/− mice had decreased protein levels of different mitochondrial markers, including outermembrane protein (TOM20), innermembrane proteins (UCP1 and COX IV), and intermembrane proteins (AIF and OPA1) (Figure 5H). Lipolysis of WAT plays an important role in fueling thermogenesis of BAT. We checked the WAT lipolysis by stimulating tissue explants with CL316,243, an agonist of β3-adrenergic receptors. Consistent with the defective thermogenesis observed in OVOL2 deficient mice, the overall rate of WAT lipolysis in 16-week-oldOvol2^boh/− mice was decreased as indicated by increased EC50 (+/+, 4.575 ng/ul;Ovol2^boh/−, 8.753 ng/ul) and decreased maximum rate (+/+, 0.4735 mg/g;Ovol2^boh/−, 0.1975 mg/g) (Figure 5I).

Because obesity ofOvol2^boh/boh andOvol2^boh/− mice develops after 9 weeks of age and progresses with age, we examined thermogenic gene expression and resistance to cold stress in 5-week-oldOvol2^boh/boh mice, which had similar body weight, fat weight, and lean weight as WT littermates (Figures S5D–S5F). At 5 weeks of age,Ovol2^boh/boh iBAT and iWAT showed slightly reduced expression levels of thermogenic genes compared to iBAT and iWAT from WT littermates, but this was not statistically significant (Figures S5A andS5B); tolerance to acute cold stress was similar in 5-week-oldOvol2^boh/boh and WT mice (Figure S5C). After chronic (10 days) cold stress or treatment with CL316,243,Ovol2^boh/boh and WT mice had similar body weights and lean weight percentages, and showed similar reductions in fat weight percentages (Figures S5G–S5I). UCP1 mRNA and protein were upregulated similarly inOvol2^boh/boh and WT iBAT in response to both treatments (Figures 5J and5K), but this response was reduced in iWAT ofOvol2^boh/boh mice compared to WT mice (Figures 5J and5L). These data suggest that while perception of cold is preserved in 5-week-oldOvol2^boh/boh mice, their ability to induce browning of WAT is impaired in response to chronic stimuli. Together, these data suggest that reduced formation and functionality of brown and beige adipocytes resulted in a defect of thermogenesis in OVOL2 deficient mice.

OVOL2 directly interacts with C/EBPα

To investigate the mechanisms by which OVOL2 regulates adipocytes, we used mass spectrometry to analyze proteins that immunoprecipitated with 3xFlag tagged OVOL2-A (3xFlag-OVOL2-A) from differentiated 3T3-L1 adipocytes (Figure S6A). Several OVOL2 specific interacting proteins were identified, among which C/EBPα was the top candidate (Figure S6B). OVOL2 interacted with C/EBPα when expressed in 293T cells (Figure 6A).In vitro GST pull-down experiments supported a direct interaction between OVOL2 and C/EBPα (Figure 6B). Interestingly, OVOL2 specifically interacted with C/EBPα among other C/EBP family proteins (Figure S6C). We also examined the subcellular localization of Flag tagged OVOL2 (Flag-OVOL2) and HA tagged C/EBPα in undifferentiated 3T3-L1 pre-adipocytes (Day 0) (Figure 6C). Both OVOL2 and C/EBPα alone showed specific nuclear localization (Figure 6C). A majority of OVOL2 and C/EBPα co-localized in the nuclei when these two proteins were co-expressed in 3T3-L1 pre-adipocytes (Figure 6C). Further mapping experiments revealed the C-terminal zinc finger domains of OVOL2, and C-terminal basic leucine zipper (bZIP) domains of C/EBPα were required for their interaction (Figures 6D–6F). Compared to WT OVOL2, the mutant OVOL2^boh protein (C120Y) had a reduced interaction with C/EBPα (Figure 6G). Collectively, these data demonstrate that C/EBPα interacts with OVOL2, and that this interaction is disrupted by theboh mutation.

OVOL2 suppresses adipogenesis through inhibition of the transcriptional activity of C/EBPα

The C/EBPα binding ability of OVOL2 suggested a potential regulatory role of OVOL2 towards C/EBPα as a transcription factor. In a luciferase reporter system with 8 copies (8x) of C/EBPα binding motifs, HA tagged WT OVOL2-A (HA-Ovol2-A) significantly reduced 8xC/EBPα luciferase activity, while HA-OVOL2-B had no such inhibition (Figure 6H). C/EBPα is one of the major transcriptional activators during adipogenesis. We used the mouse 3T3-L1 cell line as a well-established cultured adipocyte system to study adipogenesisin vitro. To better test the roles of two different OVOL2 isoforms (OVOL2-A, OVOL2-B) and OVOL2^boh mutants, we generatedOvol2 KO 3T3-L1 cells by CRISPR/Cas9 targeting and then re-introduced CRISPR resistant (CR) transgenes encoding WT OVOL2 or OVOL2^boh versions of both isoforms. Although both WT andboh mutant OVOL2 proteins were expressed at similar levels (Figure 6K), when expressed throughout the differentiation period of 3T3-L1 cells (days 0–10), WT OVOL2-A dramatically inhibited adipogenesis as revealed by decreased Oil Red O staining and expression of mature adipocyte markers, while theboh mutant OVOL2-A had no such effect (Figures 6I–6K). Compared with OVOL2-A, OVOL2-B had a weak inhibitory effect (Figures 6I–6K). Notably, human OVOL2-A and OVOL2-B showed a similar trend of inhibition when overexpressed in primary human subcutaneous adipocytes, as indicated by decreased adipogenic gene expression (Figures S6D–S6F). Since OVOL2 interacted with the C-terminal portion of C/EBPα containing the bZIP domain (Figures 6D–6F), we hypothesized that OVOL2 binding occupies the DNA binding region of C/EBPα and thus inhibits the function of C/EBPα as an active transcription factor. Electrophoretic mobility shift assay (EMSA) showed that purified His-OVOL2 protein inhibited the binding of His-C/EBPα protein to a biotin-labeled DNA probe containing a classic C/EBPα binding motif (Figure 6L). Moreover, chromatin immunoprecipitation (ChIP) assay revealed that WT OVOL2 but not OVOL2^boh inhibited the binding of C/EBPα to the promoter region of C/EBPα target genesFabp4 andGlut4 in 3T3-L1 cells (Figures 6M and6N). InOvol2^boh/− mice where there was no inhibition by OVOL2, the expression levels ofFabp4 andGlut4 were significantly increased in iWAT but not in iBAT (Figure 6O). Taken together, these findings indicate that OVOL2 blocks adipogenesis through direct inhibition of the transcriptional effects of C/EBPα.

Adipocyte-specific transgenic expression of OVOL2 reduces high-fat diet-induced obesity

The elevated fat weight of OVOL2 deficient mice (Ovol2^boh/boh orOvol2^boh/−) was due to both adipocyte hypertrophy and hyperplasia (Figures S1D–S1G andS2M–S2P), and we sought to determine the effect ofOvol2 overexpression using a tetracycline responsive element (TRE)-drivenOvol2 transgene (TRE-Ovol2) (Figure 7A). An adiponectin promoter-driven reverse tetracycline-dependent transcriptional activator (Apn-rtTA) (Sun et al., 2012) was also used (Figure 7A). In the presence of doxycycline (Dox), rtTA binds TRE and activatesOvol2 expression (Figure 7A). We tested fourTRE-Ovol2 founder lines withApn-rtTA drivers after 4 weeks of high fat diet and Dox (HFD-Dox) feeding, and selected TG line #38015 because it showed the highest inducibleTRE-Ovol2-derived mRNA (Figure S7A) and protein levels (Figure S7B) in iBAT, eWAT, and iWAT, but not in liver. 6-week-old mice expressingTRE-Ovol2 andApn-rtTA transgenes were fed HFD-Dox for 12 weeks (Figure 7A) to induce adipocyte specificOvol2 expression (Figure 7B). Before HFD-Dox feeding, the body weights ofTRE-Ovol2; Apn-rtTA mice andApn-rtTA littermates were similar. However, after 12 weeks HFD-Dox feeding, the body weights ofTRE-Ovol2; Apn-rtTA mice averaged 22% lower than those ofApn-rtTA littermates (Figures 7C and7D). MRS showed thatTRE-Ovol2; Apn-rtTA mice had reduced ratios of fat to lean weight (Figure 7E) compared withApn-rtTA littermates, which resulted from a 40% average reduction in absolute fat weight (Figure 7F) but no change in lean weight (Figure 7G). Necropsy revealed that adipose tissue beds (eWAT, iWAT, and iBAT) ofTRE-Ovol2; Apn-rtTA mice were decreased in size relative to those inApn-rtTA littermates (Figures 7H andS7C–S7E). H&E staining revealed slightly reduced size of adipocytes from iBAT and iWAT, but not from eWAT (Figures 7I–7N andS7F–S7G). Plotting of adipocyte volume to adipose tissue weight showed fewer adipocytes from iWAT and eWAT inTRE-Ovol2; Apn-rttA mice (Figures S7H andS7I). In addition to the decreased obesity, adipocyte-specific OVOL2 expression reduced both fasting glucose (Figure 7O) and insulin levels (Figure 7P). Presumably due to decreased adiposity,TRE-Ovol2; Apn-rtTA mice had less leptin in the serum (Figure 7Q). Adipocyte-specific OVOL2 overexpression also reduced fasting cholesterol levels (Figure 7R), while no significant differences in the fasting triglyceride levels were observed (Figure 7S). HFD-Dox induced large, pallid fatty livers inApn-rtTA control mice whereasTRE-Ovol2; Apn-rtTA mice had smaller, reddish livers, with decreased lipid storage based on H&E and Oil Red O staining (Figures S7J–S7N).TRE-Ovol2; Apn-rtTA mice had increased energy expenditure at 23 °C (Figures 7T and7U) with no change in RER and physical activities (Figures 7V andS7O–S7R). When the housing temperature decreased from 30 °C to 6 °C within a 12 h period,TRE-Ovol2; Apn-rtTA mice increased energy expenditure to a greater extent compared withApn-rtTA control mice (Figures 7W and7X). Moreover, both UCP1 protein level detected by immunohistochemical staining (Figure 7Y) andUcp1 mRNA level (Figure 7Z) were increased in BAT fromTRE-Ovol2; Apn-rtTA mice. These findings indicate that OVOL2 overexpression in adipocytes limits high fat diet-induced obesity and adiposity in mice.

Limitations of study

This study demonstrates that OVOL2 deficient mice are cold intolerant with a defect of brown adipose tissue contributing to reduced nonshivering thermogenesis. We cannot exclude the possibility that muscle mediated shivering thermogenesis is also affected by OVOL2 deficiency. Our study reveals dual roles of OVOL2 in brown and white adipose tissue development and function. The contribution of these two adipose tissues toboh obesity is limited by the whole-body KO or mutant mice used in our study and remains incompletely understood. To corroborate and extend these findings and to rule out thermogenic mechanisms outside of brown adipose tissues, future works will involve tissue/cell type specificOvol2 deletion. Although we found C/EBPα as an OVOL2 interacting protein in white adipocytes, we do not fully understand the role of OVOL2 in brown adipose tissues. The mechanism of OVOL2 in promoting brown adipocyte development and/or function is an area we are actively investigating.

Resource Availability

Experimental Model and Subject Details

Method Details

Quantification and Statistical Analysis

Data are presented as mean ± SD in all graphs depicting error bars. The statistical significance of differences between experimental groups was determined by Student’st test or one-way ANOVA with Tukey’s multiple comparison test using GraphPad Prism 9. The quantification of size and number of adipocytes was adapted from (Parlee et al., 2014). Image J 1.53k was used to analyze adipose histology images. A linear correlation with a two-tailed comparison of slope and intercept was calculated and compared between different mouse groups. No statistical method was used to determine whether the data met assumptions of the statistical approach.

KEY RESOURCES TABLE.

Highlights.

A viable hypomorphic allele ofOvol2 (Ovol2^boh, p.C120Y) causes obesity in mice

Obesity inOvol2^boh/boh develops with no hyperphagia and reduced energy expenditure

OVOL2 deficient mice are cold intolerant with defective brown/beige adipose tissues

OVOL2 inhibits transcriptional activity of C/EBPα and suppresses white adipogenesis

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Supplementary Materials

Data Availability Statement

• All data generated in this study are available in the main text and supplementary information. Original western blot images and analyzed data are available asData S1.

• This paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.