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FTO mediates cell-autonomous effects on adipogenesis and adipocyte lipid content by regulating gene expression via 6mA DNA modifications

2018; Elsevier BV; Volume: 59; Issue: 8 Linguagem: Inglês

10.1194/jlr.m085555

ISSN

1539-7262

Autores

Jayne F. Martin Carli, Charles A. LeDuc, Yiying Zhang, George Stratigopoulos, Rudolph L. Leibel,

Tópico(s)

Cancer-related molecular mechanisms research

Resumo

SNPs in the first intron of α-ketoglutarate-dependent dioxygenase (FTO) convey effects on adiposity by mechanisms that remain unclear, but appear to include modulation of expression of FTO itself, as well as other genes in cis. FTO expression is lower in fibroblasts and iPSC-derived neurons of individuals segregating for FTO obesity risk alleles. We employed in vitro adipogenesis models to investigate the molecular mechanisms by which Fto affects adipocyte development and function. Fto expression was upregulated during adipogenesis, and was required for the maintenance of CEBPB and Cebpd/CEBPD expression in murine and human adipocytes in vitro. Fto knockdown decreased the number of 3T3-L1 cells that differentiated into adipocytes as well as the amount of lipid per mature adipocyte. This effect on adipocyte programming was conveyed, in part, by modulation of CCAAT enhancer binding protein (C/ebp)β-regulated transcription. We found that Fto also affected Cebpd transcription by demethylating DNA N6-methyldeoxyadenosine in the Cebpd promoter. Fto is permissive for adipogenesis and promotes maintenance of lipid content in mature adipocytes by enabling C/ebpβ-driven transcription and expression of Cebpd. These findings are consistent with the loss of fat mass in mice segregating for a dominant-negative Fto allele. SNPs in the first intron of α-ketoglutarate-dependent dioxygenase (FTO) convey effects on adiposity by mechanisms that remain unclear, but appear to include modulation of expression of FTO itself, as well as other genes in cis. FTO expression is lower in fibroblasts and iPSC-derived neurons of individuals segregating for FTO obesity risk alleles. We employed in vitro adipogenesis models to investigate the molecular mechanisms by which Fto affects adipocyte development and function. Fto expression was upregulated during adipogenesis, and was required for the maintenance of CEBPB and Cebpd/CEBPD expression in murine and human adipocytes in vitro. Fto knockdown decreased the number of 3T3-L1 cells that differentiated into adipocytes as well as the amount of lipid per mature adipocyte. This effect on adipocyte programming was conveyed, in part, by modulation of CCAAT enhancer binding protein (C/ebp)β-regulated transcription. We found that Fto also affected Cebpd transcription by demethylating DNA N6-methyldeoxyadenosine in the Cebpd promoter. Fto is permissive for adipogenesis and promotes maintenance of lipid content in mature adipocytes by enabling C/ebpβ-driven transcription and expression of Cebpd. These findings are consistent with the loss of fat mass in mice segregating for a dominant-negative Fto allele. SNPs in a region of strong linkage disequilibrium within the first intron of the α-ketoglutarate-dependent dioxygenase (FTO) gene are strongly associated with adiposity; each minor allele is associated with an increase in BMI of ∼0.36 kg/m2 (or body weight of ∼1.2 kg) in adults (1.Frayling T.M. Timpson N.J. Weedon M.N. Zeggini E. Freathy R.M. Lindgren C.M. Perry J.R. Elliott K.S. Lango H. Rayner N.W. et al.A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity.Science. 2007; 316: 889-894Crossref PubMed Scopus (3335) Google Scholar, 2.Dina C. Meyre D. Gallina S. Durand E. Korner A. Jacobson P. Carlsson L.M. Kiess W. Vatin V. Lecoeur C. et al.Variation in FTO contributes to childhood obesity and severe adult obesity.Nat. Genet. 2007; 39: 724-726Crossref PubMed Scopus (1240) Google Scholar, 3.Scuteri A. Sanna S. Chen W.M. Uda M. Albai G. Strait J. Najjar S. Nagaraja R. Orru M. Usala G. et al.Genome-wide association scan shows genetic variants in the FTO gene are associated with obesity-related traits.PLoS Genet. 2007; 3: e115Crossref PubMed Scopus (1303) Google Scholar). The increased body weight in individuals segregating for the FTO risk allele, rs9939609, is attributable primarily to increased food intake (with preference for higher caloric density) rather than decreased energy expenditure (4.Speakman J.R. Rance K.A. Johnstone A.M. Polymorphisms of the FTO gene are associated with variation in energy intake, but not energy expenditure.Obesity (Silver Spring). 2008; 16: 1961-1965Crossref PubMed Scopus (260) Google Scholar, 5.Wardle J. Carnell S. Haworth C.M. Farooqi I.S. O'Rahilly S. Plomin R. Obesity associated genetic variation in FTO is associated with diminished satiety.J. Clin. Endocrinol. Metab. 2008; 93: 3640-3643Crossref PubMed Scopus (388) Google Scholar, 6.Tanofsky-Kraff M. Han J.C. Anandalingam K. Shomaker L.B. Columbo K.M. Wolkoff L.E. Kozlosky M. Elliott C. Ranzenhofer L.M. Roza C.A. et al.The FTO gene rs9939609 obesity-risk allele and loss of control over eating.Am. J. Clin. Nutr. 2009; 90: 1483-1488Crossref PubMed Scopus (189) Google Scholar, 7.Cecil J.E. Tavendale R. Watt P. Hetherington M.M. Palmer C.N. An obesity-associated FTO gene variant and increased energy intake in children.N. Engl. J. Med. 2008; 359: 2558-2566Crossref PubMed Scopus (530) Google Scholar). The mechanism(s) by which this association is conveyed is still unclear. The statistical strength of this association with adiposity and the large number of SNPs within the obesity-associated locus in the first intron of FTO suggest that there may be multiple causal variants contributing to the observed effect. For example, we have demonstrated that two SNPs in the obesity-associated locus (rs8050136 and rs1421085) differentially bind the transcription factor, cut-like homeobox 1 (CUX1). The protective alleles of these SNPs preferentially bind the activating isoform of CUX1, p110, proteolytically processed from the p200 isoform by cathepsin L, increasing expression of FTO and neighboring RPGRIP1-like (RPGRIP1L) (8.Stratigopoulos G. Padilla S.L. LeDuc C.A. Watson E. Hattersley A.T. McCarthy M.I. Zeltser L.M. Chung W.K. Leibel R.L. Regulation of Fto/Ftm gene expression in mice and humans.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2008; 294: R1185-R1196Crossref PubMed Scopus (247) Google Scholar, 9.Stratigopoulos G. Burnett L.C. Rausch R. Gill R. Penn D.B. Skowronski A.A. LeDuc C.A. Lanzano A.J. Zhang P. Storm D.R. et al.Hypomorphism of Fto and Rpgrip1l causes obesity in mice.J. Clin. Invest. 2016; 126: 1897-1910Crossref PubMed Scopus (58) Google Scholar, 10.Stratigopoulos G. LeDuc C.A. Cremona M.L. Chung W.K. Leibel R.L. Cut-like homeobox 1 (CUX1) regulates expression of the fat mass and obesity-associated and retinitis pigmentosa GTPase regulator-interacting protein-1-like (RPGRIP1L) genes and coordinates leptin receptor signaling.J. Biol. Chem. 2011; 286: 2155-2170Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Mice congenitally null for Fto exhibit a complex phenotype that includes postnatal lethality (11.Fischer J. Koch L. Emmerling C. Vierkotten J. Peters T. Bruning J.C. Ruther U. Inactivation of the Fto gene protects from obesity.Nature. 2009; 458: 894-898Crossref PubMed Scopus (709) Google Scholar, 12.Gao X. Shin Y.H. Li M. Wang F. Tong Q. Zhang P. The fat mass and obesity associated gene FTO functions in the brain to regulate postnatal growth in mice.PLoS One. 2010; 5: e14005Crossref PubMed Scopus (162) Google Scholar, 13.McMurray F. Church C.D. Larder R. Nicholson G. Wells S. Teboul L. Tung Y.C. Rimmington D. Bosch F. Jimenez V. et al.Adult onset global loss of the fto gene alters body composition and metabolism in the mouse.PLoS Genet. 2013; 9: e1003166Crossref PubMed Scopus (106) Google Scholar). Mice that survive have impaired linear growth; in adulthood, these mice have reduced adipose tissue mass compared to controls and are protected from diet-induced obesity (11.Fischer J. Koch L. Emmerling C. Vierkotten J. Peters T. Bruning J.C. Ruther U. Inactivation of the Fto gene protects from obesity.Nature. 2009; 458: 894-898Crossref PubMed Scopus (709) Google Scholar, 14.Ronkainen J. Huusko T.J. Soininen R. Mondini E. Cinti F. Makela K.A. Kovalainen M. Herzig K.H. Jarvelin M.R. Sebert S. et al.Fat mass- and obesity-associated gene Fto affects the dietary response in mouse white adipose tissue.Sci. Rep. 2015; 5: 9233Crossref PubMed Scopus (42) Google Scholar). Conversely, Fto-overexpressing mice display increased body weight and adiposity (15.Church C. Moir L. McMurray F. Girard C. Banks G.T. Teboul L. Wells S. Bruning J.C. Nolan P.M. Ashcroft F.M. et al.Overexpression of Fto leads to increased food intake and results in obesity.Nat. Genet. 2010; 42: 1086-1092Crossref PubMed Scopus (525) Google Scholar). A separate mouse model of ENU-mutagenized Fto, which exhibits partial loss-of-function, is spared the postnatal lethality and impaired skeletal growth seen in constitutive Fto knockout models (16.Church C. Lee S. Bagg E.A. McTaggart J.S. Deacon R. Gerken T. Lee A. Moir L. Mecinovic J. Quwailid M.M. et al.A mouse model for the metabolic effects of the human fat mass and obesity associated FTO gene.PLoS Genet. 2009; 5: e1000599Crossref PubMed Scopus (248) Google Scholar). These mice, free of the confounds of impaired growth, exhibit decreased adiposity when fed both chow and high-fat diet as adults. Adult mice that are null for Fto have smaller adipocytes (11.Fischer J. Koch L. Emmerling C. Vierkotten J. Peters T. Bruning J.C. Ruther U. Inactivation of the Fto gene protects from obesity.Nature. 2009; 458: 894-898Crossref PubMed Scopus (709) Google Scholar) and Fto-overexpressing mice have larger adipocytes than controls (15.Church C. Moir L. McMurray F. Girard C. Banks G.T. Teboul L. Wells S. Bruning J.C. Nolan P.M. Ashcroft F.M. et al.Overexpression of Fto leads to increased food intake and results in obesity.Nat. Genet. 2010; 42: 1086-1092Crossref PubMed Scopus (525) Google Scholar). These phenotypes suggest a role for Fto in enabling facultative adipose tissue expansion, possibly in the context of diet-induced obesity. In support of these inferences, 24-week-old mice segregating for the partial loss-of-function Fto mutation exhibit decreased adiposity, but increased circulating concentrations of fasting glucose, triglyceride, cholesterol, and HDL levels, consistent with a lipodystrophic metabolic profile (16.Church C. Lee S. Bagg E.A. McTaggart J.S. Deacon R. Gerken T. Lee A. Moir L. Mecinovic J. Quwailid M.M. et al.A mouse model for the metabolic effects of the human fat mass and obesity associated FTO gene.PLoS Genet. 2009; 5: e1000599Crossref PubMed Scopus (248) Google Scholar, 17.Unger R.H. Scherer P.E. Gluttony, sloth and the metabolic syndrome: a roadmap to lipotoxicity.Trends Endocrinol. Metab. 2010; 21: 345-352Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 18.Virtue S. Vidal-Puig A. Adipose tissue expandability, lipotoxicity and the metabolic syndrome–an allostatic perspective.Biochim. Biophys. Acta. 2010; 1801: 338-349Crossref PubMed Scopus (688) Google Scholar). Humans who are homozygous for null mutations of FTO display severe brain (microcephaly, hydrocephalus, and lissencephaly) and cardiac (ventricular and atrial septal defects, patent ductus arteriosus) malformations that result in growth retardation and death within the first few years of life (19.Boissel S. Reish O. Proulx K. Kawagoe-Takaki H. Sedgwick B. Yeo G.S. Meyre D. Golzio C. Molinari F. Kadhom N. et al.Loss-of-function mutation in the dioxygenase-encoding FTO gene causes severe growth retardation and multiple malformations.Am. J. Hum. Genet. 2009; 85: 106-111Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 20.Daoud H. Zhang D. McMurray F. Yu A. Luco S.M. Vanstone J. Jarinova O. Carson N. Wickens J. Shishodia S. et al.Identification of a pathogenic FTO mutation by next-generation sequencing in a newborn with growth retardation and developmental delay.J. Med. Genet. 2016; 53: 200-207Crossref PubMed Scopus (40) Google Scholar, 21.Rohena L. Lawson M. Guzman E. Ganapathi M. Cho M.T. Haverfield E. Anyane-Yeboa K. FTO variant associated with malformation syndrome.Am. J. Med. Genet. A. 2016; 170A: 1023-1028Crossref PubMed Scopus (11) Google Scholar). Therefore, effects on adiposity per se cannot be accurately assessed in these individuals. Individuals heterozygous for a variety of FTO mutations have no obvious phenotypic characteristics (22.Meyre D. Proulx K. Kawagoe-Takaki H. Vatin V. Gutierrez-Aguilar R. Lyon D. Ma M. Choquet H. Horber F. Van Hul W. et al.Prevalence of loss-of-function FTO mutations in lean and obese individuals.Diabetes. 2010; 59: 311-318Crossref PubMed Scopus (82) Google Scholar, 23.Deliard S. Panossian S. Mentch F.D. Kim C.E. Hou C. Frackelton E.C. Bradfield J.P. Glessner J.T. Zhang H. Wang K. et al.The missense variation landscape of FTO, MC4R, and TMEM18 in obese children of African ancestry.Obesity (Silver Spring). 2013; 21: 159-163Crossref PubMed Scopus (21) Google Scholar, 24.Zheng Z. Hong L. Huang X. Yang P. Li J. Ding Y. Yao R.E. Geng J. Shen Y. Shen Y. et al.Screening for coding variants in FTO and SH2B1 genes in Chinese patients with obesity.PLoS One. 2013; 8: e67039Crossref PubMed Scopus (15) Google Scholar). The number of these individuals with variants predicted or proven to have deleterious effects on FTO function is extremely small, however, limiting the strength of inferences regarding the protein's effects on metabolic homeostasis. FTO is a member of the ALKB homolog family of non-heme dioxygenases [Fe(II)-dependent dioxygenase and FTO] (25.Sanchez-Pulido L. Andrade-Navarro M.A. The FTO (fat mass and obesity associated) gene codes for a novel member of the non-heme dioxygenase superfamily.BMC Biochem. 2007; 8: 23Crossref PubMed Scopus (158) Google Scholar, 26.Gerken T. Girard C.A. Tung Y.C. Webby C.J. Saudek V. Hewitson K.S. Yeo G.S. McDonough M.A. Cunliffe S. McNeill L.A. et al.The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.Science. 2007; 318: 1469-1472Crossref PubMed Scopus (1167) Google Scholar) and exhibits in vitro demethylase activity directed at single-stranded N6-methyladenosine (m6A) RNA and N6-methyldeoxyadenosine (6mA) DNA (27.Jia G. Fu Y. Zhao X. Dai Q. Zheng G. Yang Y. Yi C. Lindahl T. Pan T. Yang Y.G. et al.N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.Nat. Chem. Biol. 2011; 7: 885-887Crossref PubMed Scopus (2188) Google Scholar). m6A is an abundant mRNA modification that is thought to play a role in the regulation of mRNA stability, splicing, and/or translation. These modifications are increased around stop codons and 3′UTRs, as well as along long exons (28.Dominissini D. Moshitch-Moshkovitz S. Schwartz S. Salmon-Divon M. Ungar L. Osenberg S. Cesarkas K. Jacob-Hirsch J. Amariglio N. Kupiec M. et al.Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.Nature. 2012; 485: 201-206Crossref PubMed Scopus (2653) Google Scholar, 29.Meyer K.D. Saletore Y. Zumbo P. Elemento O. Mason C.E. Jaffrey S.R. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons.Cell. 2012; 149: 1635-1646Abstract Full Text Full Text PDF PubMed Scopus (2318) Google Scholar, 30.Fu Y. Dominissini D. Rechavi G. He C. Gene expression regulation mediated through reversible m(6)A RNA methylation.Nat. Rev. Genet. 2014; 15: 293-306Crossref PubMed Scopus (1017) Google Scholar). 6mA is a prevalent DNA modification in prokaryotes, where it plays a part in defense mechanisms designated as "restriction-modification systems," which detect and degrade foreign (unmethylated) DNA (31.Wion D. Casadesus J. N6-methyl-adenine: an epigenetic signal for DNA-protein interactions.Nat. Rev. Microbiol. 2006; 4: 183-192Crossref PubMed Scopus (381) Google Scholar). The 6mA also regulates DNA repair and replication as well as transcription in prokaryotes (32.Løbner-Olesen A. Skovgaard O. Marinus M.G. Dam methylation: coordinating cellular processes.Curr. Opin. Microbiol. 2005; 8: 154-160Crossref PubMed Scopus (193) Google Scholar). Recently, 6mA has been detected in lower quantities in eukaryotic organisms (33.Greer E.L. Blanco M.A. Gu L. Sendinc E. Liu J. Aristizabal-Corrales D. Hsu C.H. Aravind L. He C. Shi Y. DNA methylation on N6-adenine in C. elegans.Cell. 2015; 161: 868-878Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 34.Koziol M.J. Bradshaw C.R. Allen G.E. Costa A.S.H. Frezza C. Gurdon J.B. Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications.Nat. Struct. Mol. Biol. 2016; 23: 24-30Crossref PubMed Scopus (163) Google Scholar), suggesting an epigenetic role in mammalian cells. Although expressed most highly in the brain, Fto is also expressed in adipose tissue (12.Gao X. Shin Y.H. Li M. Wang F. Tong Q. Zhang P. The fat mass and obesity associated gene FTO functions in the brain to regulate postnatal growth in mice.PLoS One. 2010; 5: e14005Crossref PubMed Scopus (162) Google Scholar, 26.Gerken T. Girard C.A. Tung Y.C. Webby C.J. Saudek V. Hewitson K.S. Yeo G.S. McDonough M.A. Cunliffe S. McNeill L.A. et al.The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.Science. 2007; 318: 1469-1472Crossref PubMed Scopus (1167) Google Scholar) where its functions remain unclear. While eQTL studies examining the expression of FTO in individuals segregating for obesity risk or protective alleles have not shown different levels of expression, sample sizes were small and, in some cases, whole adipose tissue was examined, potentially masking effects in specific cell types such as adipocytes or preadipocytes (35.Grunnet L.G. Nilsson E. Ling C. Hansen T. Pedersen O. Groop L. Vaag A. Poulsen P. Regulation and function of FTO mRNA expression in human skeletal muscle and subcutaneous adipose tissue.Diabetes. 2009; 58: 2402-2408Crossref PubMed Scopus (77) Google Scholar, 36.Klöting N. Schleinitz D. Ruschke K. Berndt J. Fasshauer M. Tönjes A. Schön M.R. Kovacs P. Stumvoll M. Blüher M. Inverse relationship between obesity and FTO gene expression in visceral adipose tissue in humans.Diabetologia. 2008; 51: 641-647Crossref PubMed Scopus (84) Google Scholar, 37.Zabena C. Gonzalez-Sanchez J.L. Martinez-Larrad M.T. Torres-Garcia A. Alvarez-Fernandez-Represa J. Corbaton-Anchuelo A. Perez-Barba M. Serrano-Rios M. The FTO obesity gene. Genotyping and gene expression analysis in morbidly obese patients.Obes. Surg. 2009; 19: 87-95Crossref PubMed Scopus (92) Google Scholar, 38.Claussnitzer M. Dankel S.N. Kim K.H. Quon G. Meuleman W. Haugen C. Glunk V. Sousa I.S. Beaudry J.L. Puviindran V. et al.FTO obesity variant circuitry and adipocyte browning in humans.N. Engl. J. Med. 2015; 373: 895-907Crossref PubMed Scopus (799) Google Scholar). In mice, Fto expression is decreased in mesenteric adipose tissue upon fasting (8.Stratigopoulos G. Padilla S.L. LeDuc C.A. Watson E. Hattersley A.T. McCarthy M.I. Zeltser L.M. Chung W.K. Leibel R.L. Regulation of Fto/Ftm gene expression in mice and humans.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2008; 294: R1185-R1196Crossref PubMed Scopus (247) Google Scholar) and increased in subcutaneous and visceral adipose tissue upon acute exposure to high-fat diets (39.Wang C.Y. Shie S.S. Wen M.S. Hung K.C. Hsieh I.C. Yeh T.S. Wu D. Loss of FTO in adipose tissue decreases Angptl4 translation and alters triglyceride metabolism.Sci. Signal. 2015; 8: ra127Crossref PubMed Scopus (26) Google Scholar), indicating that nutritional status might confound efforts to attribute expression levels of FTO to obesity-risk genotypes. We assessed the possibility that Fto may be involved in adipocyte differentiation by developmentally timed reduction of Fto gene expression in 3T3-L1 preadipocytes. We also analyzed the effect of FTO knockdown on adipogenesis in human adipose tissue-derived preadipocytes. We found that Fto expression was required for adipogenesis; and by using RNA-sequencing (RNA-Seq), we identified CCAAT enhancer binding protein (C/ebp)β activity and Cebpd expression as primary targets of Fto. C/ebpβ and C/ebpδ are transcription factors that initiate adipogenesis; C/ebpβ function is required to maintain lipid homeostasis in adipocytes (40.Rosen E.D. MacDougald O.A. Adipocyte differentiation from the inside out.Nat. Rev. Mol. Cell Biol. 2006; 7: 885-896Crossref PubMed Scopus (1938) Google Scholar). We did not find evidence that these transcripts are differentially methylated or exhibit altered stability or translational efficiency following Fto knockdown, effects that have been proposed for m6A (41.Meyer K.D. Jaffrey S.R. The dynamic epitranscriptome: N6-methyladenosine and gene expression control.Nat. Rev. Mol. Cell Biol. 2014; 15: 313-326Crossref PubMed Scopus (618) Google Scholar) and which could be affected by m6A demethylation by Fto (27.Jia G. Fu Y. Zhao X. Dai Q. Zheng G. Yang Y. Yi C. Lindahl T. Pan T. Yang Y.G. et al.N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.Nat. Chem. Biol. 2011; 7: 885-887Crossref PubMed Scopus (2188) Google Scholar). We did find evidence that, in addition to its coactivation activity with C/ebpβ (42.Wu Q. Saunders R.A. Szkudlarek-Mikho M. Serna Ide L. Chin K.V. The obesity-associated Fto gene is a transcriptional coactivator.Biochem. Biophys. Res. Commun. 2010; 401: 390-395Crossref PubMed Scopus (78) Google Scholar), Fto demethylated 6mA DNA at the promoter of Cebpd. By these mechanisms, the downstream targets of C/ebpβ and Cebpd, Pparg and Cebpa (40.Rosen E.D. MacDougald O.A. Adipocyte differentiation from the inside out.Nat. Rev. Mol. Cell Biol. 2006; 7: 885-896Crossref PubMed Scopus (1938) Google Scholar), are activated. The 3T3-L1 preadipocytes were purchased from ATCC and maintained in sub-confluent cultures. Growth medium consisted of DMEM (with 25 mM glucose, GlutaMAX™, and sodium pyruvate) supplemented with 10% newborn calf serum (both Thermo Fisher). Differentiation was achieved by treating cells 2 days after they reached confluence with differentiation medium containing DMEM plus insulin (from bovine pancreas, 1 μg/ml), dexamethasone (0.25 μM), and isobutylmethylxanthine (IBMX; 0.5 mM; all Sigma-Aldrich) supplemented with 10% FBS (Thermo Fisher). Two days later, differentiation medium was replaced with DMEM containing 1 μg/ml insulin and 10% FBS. Insulin medium was replaced every 2–3 days; cells were filled with lipid and considered to be mature adipocytes by days 8–12. De-identified human subcutaneous adipose stromal cells (ASCs) were generously provided by the Boston Nutrition Obesity Research Center and were cultured and differentiated as previously described (43.Lee M.J. Fried S.K. Optimal protocol for the differentiation and metabolic analysis of human adipose stromal cells.Methods Enzymol. 2014; 538: 49-65Crossref PubMed Scopus (55) Google Scholar). Briefly, cells were maintained in αMEM supplemented with 10% FBS (all Thermo Fisher) to avoid confluence. To differentiate, cells were allowed to reach confluence and 2 days later were treated with a chemically defined serum-free medium [DMEM/F12 containing HEPES and antibiotic-antimycotic (Thermo Fisher), NaHCO3, d-biotin, pantothenate, dexamethasone, human insulin, rosiglitazone, IBMX, T3, and transferrin (Sigma-Aldrich)]. Differentiation medium was replaced every 2–3 days and switched to maintenance medium after 3–7 days (differentiation medium without rosiglitazone, IBMX, T3, and transferrin). Cells were filled with lipid and assayed between 10 and 14 days after initial treatment with differentiation factors. Transfections were performed prior to differentiation by incubation for 24 h of preconfluent cells with a mix of three Stealth siRNAs targeted against Fto/FTO (designed to span multiple exons) or three nontargeted controls with Lipofectamine 2000 (40 nM siRNA) or Lipofectamine 3000 (75 nM siRNA). Fto/protein tyrosine phosphatase, receptor type, V (Ptprv) and Fto/spondin 2 (Spon2) double knockdowns were performed by adding a single Stealth siRNA against Ptprv or Spon2 to the mix of three anti-Fto siRNAs (all predesigned by Thermo Fisher). Knockdown of Fto in mature adipocytes was achieved by electroporation (44.Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing.Proc. Natl. Acad. Sci. USA. 2003; 100: 7569-7574Crossref PubMed Scopus (306) Google Scholar, 45.Okada S. Mori M. Pessin J.E. Introduction of DNA into 3T3–L1 adipocytes by electroporation.Methods Mol. Med. 2003; 83: 93-96PubMed Google Scholar, 46.Kilpeläinen T.O. Carli J.F.M. Skowronski A.A. Sun Q. Kriebel J. Feitosa M.F. Hedman Å.K. Drong A.W. Hayes J.E. Zhao J. et al.Genome-wide meta-analysis uncovers novel loci influencing circulating leptin levels.Nat. Commun. 2016; 7: 10494Crossref PubMed Scopus (110) Google Scholar) using the same siRNAs after 3T3-L1 cells had been differentiated. Total RNA was isolated using the RNeasy lipid tissue mini kit and columns were treated with DNase (both Qiagen). cDNA was reverse transcribed with the Transcriptor First Strand cDNA synthesis kit (Roche) using both OligoDT and random hexamer primers. Quantitative real-time PCR (qPCR) was performed on a LightCycler 480 using SYBR Green I Master (Roche). Analysis was performed by the LightCycler 480 software (Roche) using the second derivative maximum calculation based on a standard curve. qPCR primers were designed using Primer 3 (http://bioinfo.ut.ee/primer3/) to span exon-exon junctions and are provided in supplemental Tables S1 and S2. Analysis of mRNA in Fto/FTO knockdown experiments in vitro was normalized to Rplp0/RPLP0 unless otherwise indicated. Sample integrity for RNA utilized for RNA-Seq was assessed with an Agilent 2100 bioanalyzer. All samples had RIN numbers greater than 8.0. mRNA was isolated using poly-A pulldown and reverse transcribed to generate cDNA (47.Dominissini D. Moshitch-Moshkovitz S. Salmon-Divon M. Amariglio N. Rechavi G. Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing.Nat. Protoc. 2013; 8: 176-189Crossref PubMed Scopus (385) Google Scholar). cDNA was sequenced using single-ended sequencing on a HiSeq2000 according to the manufacturer's recommendations (Illumina) at the Columbia Genome Center, 1 × 100 bp read length, 30 M read count. The pass filter reads were mapped to mouse reference genome mm9 using TopHat (version 2.0.11; https://ccb.jhu.edu/software/tophat/index.shtml). TopHat infers novel exon-exon junctions and combines them with junctions from known mRNA sequences as the reference annotation. For each read, up to three mismatches and 10 multiple hits were allowed during mapping. FPKM values indicating relative abundance were estimated using Cufflinks software (version 2.2.1; http://cole-trapnell-lab.github.io/cufflinks/). Only transcripts with FPKM values that were verified as acceptable in at least three of four replicates with average FPKM values of all replicates in both conditions (siCtr and siFto) >1 were considered. Transcripts with average FPKM values below 1 were excluded from data analyses, but included in adjustments for multiple testings. The m6A RNA immunoprecipitation (m6A RIP) was performed based on previously published protocols (29.Meyer K.D. Saletore Y. Zumbo P. Elemento O. Mason C.E. Jaffrey S.R. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons.Cell. 2012; 149: 1635-1646Abstract Full Text Full Text PDF PubMed Scopus (2318) Google Scholar, 47.Dominissini D. Moshitch-Moshkovitz S. Salmon-Divon M. Amariglio N. Rechavi G. Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing.Nat. Protoc. 2013; 8: 176-189Crossref PubMed Scopus (385) Google Scholar). Briefly, RNA was isolated (as described above, including DNase treatment) from 2 day postconfluent 3T3-L1 preadipocytes following Fto knockdown and a 5 μg aliquot was treated with RiboMinus (Thermo Fisher) to decrease rRNA content. Heat-denatured RNA (300 ng) was pulled down with rabbit anti-m6A antibody (Synaptic Systems) or rabbit IgG negative control coupled to Protein A/G Dynabeads in the presence of an RNase inhibitor, SUPERase·In (all Thermo Fisher). Eluted RNA was isolated by QIAzol:chloroform extraction and column purification (as described above, without DNase treatment). The resulting RNA and a reserved aliquot containing 10% of the input were reverse transcribed and analyzed by qPCR. No transcript was detectable in the IgG negative control condition. Levels of m6A enriched mRNA transcripts were compared with transcript levels in the input control. Transcript stability was measured following treatment with actinomycin D (ActD). Cells were treated with 1 μg/ml ActD (stock: 1 mg/ml in DMSO; Thermo Fisher) for the indicated time periods prior to isolation of RNA for analysis by qPCR. Transcript levels were normalized by the amount of 18S transcript present, which remained constant throughout all 8 h of ActD treatment. The 6mA DNA immunoprecipitation was performed using the MAGnify chromatin immunoprecipitation (ChIP) system (Thermo Fisher). One hundred and fifty thousand cells per replicate were collected from 2 day postconfluent 3T3-L1 preadipocytes that had been transfected with siRNA against Fto prior to reaching confluence. Sonicated DNA was pulled down with the same rabbit anti-6mA antibody (Synaptic Systems) used in the m6A RIP experiment or a rabbit IgG negative control (Thermo Fisher). Eluted DNA and a reserved aliquot containing 10% of the input were analyzed by qPCR. No template was detectable after pulldown in the IgG negative control condition. Levels of 6mA-enriched DNA content were compared with DNA content of the input control. Primers spanning the Cebpd promoter (493 bases upstream to 200 bases downstream of the transcriptional start site) as well as Gapdh (intron 2) are listed in supplemental Table S1. Rpl30 (intron 2)

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