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. Author manuscript; available in PMC: 2011 Oct 22.
Published in final edited form as: Biochem Biophys Res Commun. 2010 Sep 19;401(3):390–395. doi: 10.1016/j.bbrc.2010.09.064

The obesity-associated Fto gene is a transcriptional coactivator

Qiong Wu 1,*, Rudel A Saunders 1,*, Maria Szkudlarek-Mikho 1, Ivana de la Serna 2, Khew-Voon Chin 1,2,3,§
PMCID: PMC2963669  NIHMSID: NIHMS238493  PMID: 20858458

Abstract

The fat mass and obesity associated, FTO, gene has been shown to be associated with obesity in human in several genome-wide association scans. In vitro studies suggest that Fto may function as a single-stranded DNA demethylase. In addition, homologous recombination-targeted knockout of Fto in mice resulted in growth retardation, loss of white adipose tissue, and increase energy metabolism and systemic sympathetic activation. Despite these intense investigations, the exact function of Fto remains unclear. We show here that Fto is a transcriptional coactivator that enhances the transactivation potential of the CCAAT/enhancer binding proteins (C/EBPs) from unmethylated as well as methylation-inhibited gene promoters. Fto also exhibits nuclease activity. We showed further that Fto enhances the binding C/EBP to unmethylated and methylated DNA. The coactivator role of FTO in modulating the transcriptional regulation of adipogenesis by C/EBPs is consistent with the temporal progressive loss of adipose tissue in the Fto-deficient mice, thus suggesting a role for Fto in the epigenetic regulation of the development and maintenance of fat tissue. How FTO reactivates transcription from methyl-repressed gene needs to be further investigated.

Keywords: Obesity, adipogenesis, transcriptional coactivator, Fto, CCAAT/enhancer binding protein, DNA methylation

Introduction

The pandemic rise in the incidence of obesity and its associated health problems has significant impact on the cost of global health care [1]. In the United States, approximately two-thirds of the adults are overweight, with one third of these considered obese. Presumably, the epidemic increase in obesity is due to sedentary lifestyle coupled with overconsumption of energy-rich foods, which create a chronic energy imbalance that leads to weight gain in the form of body fat [2; 3]. As adiposity increases, the risk of developing comorbidities such as diabetes, hypertension, and cardiovascular disease is also significantly elevated [4; 5; 6], thus prompting the search for the underlying genetic and environmental causes for obesity. The alarming rise in the incidence of obesity worldwide in the last two decades has prompted the search for genetic and environmental causes that contribute to this global health crisis [7]. Several genome-wide association scans have identified the association of FTO with body mass index [8; 9; 10; 11; 12]. FTO is a member of the family of non-heme Fe(II)- and 2-oxoglutarate-dependent dioxygenases [13; 14], that include the DNA demethylase AlkB (E. coli) and ABH (mammalian AlkB homolog) genes, which are involved in the repair of DNA alkylation damage [15; 16; 17]. FTO demethylates 3-methylthymine in vitro on single-stranded oligonucleotides [13; 18], and homologous recombination knockout of Fto in mice causes a near-complete loss of adipose tissue and increased energy expenditure [19]. The underlying link between the putative demethylase function of FTO and energy homeostasis is not apparent. The striking loss of fat tissue raises the question whether adipogenesis is impaired in the Fto−/ − mice and what the role of Fto in this might be.

Some members of the C/EBP family of transcription factors including C/EBPα , β , and δ , and the peroxisome proliferator-activated receptor γ (PPARγ ), are considered the master transcriptional regulators of adipogenesis [20; 21]. Though transcriptional regulation of adipogenesis has been intensely investigated, the role of Fto as a demethylase and epigenetic regulator in this process has not been reported, and epigenetic regulation of adipogenesis through either global or gene-specific DNA methylation and demethylation is underexplored. Methyl modification at CpG dinucleotide suppresses transcription by modulating DNA-protein interactions and altering the accessibility of transcription factors to the methylated control regions of genes [22; 23]. Recent studies suggest that cyclical DNA methylation and demethylation of gene promoters regulates transcription [24; 25]. Active DNA methylation and demethylation is important for resetting the epigenetic state of the genome for response to continuous gene environmental interactions resulting from dietary or environmental perturbants exposure. Aberrant methylation is associated with human diseases including developmental abnormalities and cancers. The enzymology of DNA methylation including the family of DNA methyltransferases (DNMTs) is well established [26]. In contrast, proteins that demethylate 5-methylcytosine are not well defined and the mechanisms that revert methylation remained controversial [27]. We examined here the ability of Fto to reactivate methylation-inhibited promoter reporter gene and modulate transcription factor binding to DNA. Our results showed that FTO is a transcriptional coactivator and enhances transcription through methyl-inhibited DNA. The implications of these findings are discussed.

Materials and Methods

Fto cDNA and C/EBP response-element reporter constructs

Full-length cDNA of murine Fto was obtained from American Type Culture Collection (ATCC), PCR-amplified and subcloned into either pGEX-6P-2 vector (GE Healthcare) or the expression vector pcDNA3-FLAG (Addgene), to generate either a glutathione-S-transferase (GST)-FTO fusion protein or an N-terminal FLAG tag-FTO hybrid, respectively. The GST-FTO hybrid was batched purified using GST beads and FTO was recovered after cleaving from the fusion protein using PreScission Protease (GE Healthcare). Oligonucleotides containing three tandem repeats of the C/EBP-response elements were ligated into pGL3-Luc reporter vector and sequence-verified to produce the reporter plasmid.

Plasmid methylation

HhaI restriction site is present in each of the C/EBP response element, seven in the coding region within the luciferase gene, and 19 more scattered in the remaining portion of the vector. CEBPRE plasmid was methylated in vitro using HhaI methyltransferase according to manufacturer’s specification (New England Biolab), and extent of methylation assessed by resistance to HhaI endonuclease restriction. CEBPRE reporter demethylation was performed in the presence of 1 μg recombinant FTO in a reaction mixture of 50 mM TRIS-HCl, pH 7.5, 1 mM 2-oxoglutarate, 2 mM ascorbate, 75 μM ferrous ammonium sulfate [(NH4)2Fe(SO4)2], 50 μM BSA, 5 mM DTT, 1 mM MgCl2, and 150 mM NaCl, in 100 μl total volume, at 37° C for 2 hr. Reaction was terminated with 10 mM EDTA (final concentration) and plasmid recovered using the Plasmid Mini Kit (Qiagen). Isolated DNA was subjected to restriction analysis using HhaI endonuclease on 1% agarose gel. Alternatively, plasmid was methylated with radiolabelled S-adenosyl-L-[methyl-3H]methionine, and demethylated by FTO as described above. Methylation was monitored by scintillation counting of eluted plasmid DNA and flow through from column.

Chromatin Immunoprecipitations (ChIPs)

ChIPs were performed as before [28]. In brief, after formaldehyde crosslinking, nuclei isolated in a buffer containing 50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% TritonX, and protease inhibitors, were washed, lysed, immunoprecipitated, followed by five washes in RIPA buffer (50 mM HEPES, 500 mM LiCl, 0.1 mM EDTA, 1.0% NP-40 and 0.7% Na-Deoxycholate) and once with TE containing 50 mM NaCl. Eluted immune complexes were subjected to real-time PCR analysis.

Cell culture and reporter assay

HEK293 cells (ATCC) were cultured at 37°C in 5% CO2 with DMEM (Invitrogen) supplemented with 10% (v/v) fetal calf serum (Invitrogen) and antibiotics accordingly. OP9 stromal preadipocytes (a gift of Dr. Perry Bickel, University of Texas Health Science Centre, Houston) were cultured and differentiated with 15% KnockOut SR (Invitrogen) as described [29]. Transfection was performed in HEK293 and OP9 cells typically with 25 ng of reporter plasmids mixed with 10 ng of expression constructs for Fto, C/EBPs, and CBP using the LTX transfection reagent (Invitrogen). Renilla luciferase reporter was used as internal control and relative luciferase activities were determined 24 h following transfection and normalized to the Renilla luciferase activity.

Statistical analyses

Single factor ANOVA or Student’s t-tests were used for all statistical analysis using PRISM (GraphPad Software, Inc.). P values of less than or equal to 0.05 were considered statistically significant.

Results

Reactivation of methyl-inhibited reporter gene expression by Fto

To investigate Fto demethylase activity, we first examined its effect on the expression of a methylated cytomegalovirus (CMV) early promoter-driven luciferase reporter. The unmethylated reporter showed constitutive luciferase activity and methylation by HhaI DNA methyltransferase inhibited HhaI endonuclease restriction and luciferase expression (Fig. S1). Coexpression with Fto, but not the vector, reactivated the methyl-repressed reporter expression, thus suggesting that Fto may epigenetically regulate gene expression. In light of the progressive and gradual loss of white adipose tissue in Fto knockout mice [19], it is conceivable that Fto-deficiency could disrupt adipogenesis. The C/EBPs and PPARγ transcription factors are the master regulators of adipogenesis, hence we considered the effect of Fto demethylase on their transactivation potential. Since PPARγ response elements, NNNAGGTCANAGGTCA [30; 31], do not contain CpG dinucleotide, therefore, we focused on the transactivation of the C/EBP response element (CEBPRE)-driven promoter reporter, which contained three CEBPREs in tandem, each with a CpG site for methylation (Fig. 1A). The vector contained additional CpG sites. Methylation of CEBPRE-reporter inhibited HhaI cleavage (Fig. 1B) and C/EBPβ transactivation from the promoter (Fig. 1C). Expression of Fto alone had no effect on either the unmethylated or the methylated reporters. Cotransfecting C/EBPβ with Fto synergistically activated transcription from the unmethylated promoter. Albeit attenuated, Fto enhanced C/EBPβ transactivation from the methyl-inhibited promoter (Fig. 1C). Moreover, reactivation of C/EBPβ transactivation was markedly reduced with an Fto deletion mutant (Δ 274–317), which lacked the Fe(II)- and carboxylate-binding residues (Fig. 2A and B). We further compared Fto to the transcriptional coactivator CREB binding protein (CBP), a histone acetyltransferase [32], and found that overexpression of CBP enhanced the transcriptional activity of C/EBPβ from an unmethylated-reporter, but failed to reactivate transcription from the methylation-inhibited reporter (Fig. 2C). These results were recapitulated with C/EBPα and δ (Fig. S2).

Figure 1. Fto is a transcriptional coactivator.

Figure 1

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(A) Nucleotide sequence of the minimal promoter of CEBPRE reporter that includes a TATA binding site (boxed) and three C/EBP response elements (underlined) in tandem with each containing a CpG dinucleotide (asterisk) targeted for methylation by the HhaI methyltransferase (consensus site: GCGC). (B) Methylation status of the CEBPRE reporter plasmid assessed by HhaI endonuclease restriction analysis shows that in vitro methylated reporter was resistant to endonuclease cleavage. (C) Fto and C/EBPβ were ectopically expressed in OP9 preadipocytes by transfection and show that Fto synergistically coactivated C/EBPβ-mediated transactivation from the unmethylated CEBPRE luciferase reporter, and reactivated C/EBPβ transactivation from the methyl-inhibited reporter. All luciferase assays were performed in triplicates and statistics were conducted as student t-test. Results are means ± s.e.m., normalized to Renilla luciferase activity.

Figure 2. Transactivation of CEBPRE reporter by mutant Fto and CBP.

Figure 2

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(A) OP9 preadipocytes were transfected with expression vector for FLAG-tagged Fto and Fto(Δ 274–317). Immunoblot with anti-FLAG antibody shows the expression of FTO and FTO(Δ 274–317). (B) OP9 preadipocytes ectopically expressing Fto and Fto(Δ 274–317) show reactivation of CEBPRE reporter by Fto and attenuated reactivation by Fto(Δ 274–317). (C) OP9 preadipocytes co-expressing either Fto or CBP with C/EBPβ shows CBP failed to reactivate methylation-inhibited CEBPRE reporter compared to Fto. All studies were performed in triplicates. Results are means ± s.e.m., normalized to Renilla luciferase activity.

Demethylation of 5-methylcytosine and DNA cleavage by Fto

These observations are intriguing given that FTO does not demethylate 5-methylcytosine in vitro [13]. The mechanism of 5-methylcytosine demethylation is unclear and base excision repair via the excision of methylated base by DNA glycosylases [33], or direct reversal of the methyl group by dioxygenases and photolyases has been suggested [17]. To determine if FTO demethylase targets 5-methylcytosine in intact cells, we methylated the CEBPRE reporter with radiolabelled coenzyme S-adenosyl methionine (SAM) and assessed the ability of FTO to demethylate the reporter by monitoring changes in the radiolabelled reporter plasmid retained on affinity purification cartridge. If FTO removes 5-methylcytosine by direct oxidative demethylation, plasmid DNA would remain intact and be retained on the cartridge. In contrast, base excision by FTO would result in DNA cleavage, producing DNA fragments due to the presence of multiple methylated sites on the plasmid that would elute from the cartridge into the flow through. Our results showed that 3[H]-SAM-methylated CEBPRE reporter was resistant to HhaI (Fig. S3). Incubation with purified FTO caused a dose-dependent decrease in radiolabelled methylated plasmid with a concomitant increase in radioactivity in the flow-through from the purification column, whereas treatment with GST did not affect the incorporated radioactivity levels either in the eluted DNA or cartridge flow through (Fig. 3A). Further, treatment with FTO resulted in DNA cleavage but not with GST (Fig. 3B), thus suggesting an excision of 5-methylcytosine by FTO. It is also intriguing that both unmethylated and methylated reporters were susceptible to cleavage following exposure to FTO (Fig. S3).

Figure 3. FTO is a demethylase and endonuclease.

Figure 3

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(A) CEBPRE reporter plasmids were methylated and then incubated with purified FTO, followed by extraction with DNA affinity column as described in Experimental. Decrease in eluted [3H]methylated-DNA and increase in [3H]-count in flow through occur in an FTO dose-dependent manner. All studies were performed in triplicates and results are means ± s.e.m. (B) Purified FTO, but not GST, exhibits endonucleolytic activity and caused cleavage of methylated CEBPRE reporter.

Recruitment of Fto coactivator to target gene promoter

To characterize the transcriptional coactivation role of Fto, electrophoretic mobility shift assay (EMSA) using nuclear extracts from Fto and C/EBPβ transfected HEK293 cells showed that FTO did not bind to methylated-CEBPRE oligonucleotides, but enhanced C/EBPβ binding to the methylated-DNA. The DNA-protein complex was supershifted by an anti-C/EBPβ , but not anti-FTO antibody (Fig. 4A). ChIP assay confirmed that FTO increased the association of C/EBPβ with both unmodified and methyl-modified CEBPRE (Fig. 4B). Since C/EBPα and PPARγ positively regulate each other’s expression in adipogenesis [34], we further demonstrated the association of Fto with C/EBPβ at the PPARγ gene promoter in OP9 preadipocytes undergoing differentiation, compared to an undifferentiated and vector transfected controls (Fig. 4C), thus suggesting that Fto is recruited to both unmethylated and methylated promoters and enhances C/EBPs binding to DNA.

Figure 4. FTO binding to unmethylated and methylated DNA.

Figure 4

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(A) EMSA using nuclear extracts from HEK293 cells transfected with either C/EBPβ or FLAG-Fto to monitor protein-DNA interaction using the CEBPRE oligonucleotides (Figure 1A) shows FTO enhanced C/EBPβ binding to response elements that was supershifted with anti-C/EBPβ but not Fto antibody. ChIP analysis of: (B) HEK293 cells transfected with either unmethylated or methylated CEBPRE reporter plasmid, and in the presence or the absence of C/EBPβ and Fto as indicated; or (C) PPARγ gene promoter in C/EBPα and Fto-transfected OP9 preadipocytes undergoing differentiation at either 24 or 30 hrs compared to undifferentiated or vector transfected controls. ChIP analysis was performed in triplicates for C/EBPα and β , and FTO; and immunoprecipitates were normalized to β-actin; and results are means ± s.e.m.

Discussion

We show here that Fto is a transcriptional coactivator that facilitates transcription from unmethylated and also methyl-inhibited gene. In contrast, the ubiquitous transcriptional coactivator, CBP, fails to potentiate C/EBPs-mediated transactivation from the methyl-inhibited promoter, thus revealing the unique dual role of Fto to regulate non-epigenetic and epigenetic transcription.

The cleavage of DNA following incubation with FTO is curious because previous in vitro study under similar condition did not result in the degradation of oligonucleotides [13]. Moreover, the GST protein serving as control, purified in parallel with FTO under similar experimental conditions did not exhibit DNA cleavage. AlkB, ABH2 and ABH3 have been shown to directly reverse alkylation damage by oxidative demethylation of single-stranded DNA [16]. It is possible that the differences in the DNA templates used (single-stranded oligonucleotides versus double-stranded plasmid DNA) as well as substrate specificity (1-alkyladenine and 3-alkylcytosine versus 5-methylcytosine) may account for the differences in the observed activity of FTO in our study. Nevertheless, the DNA cleavage observed in our study is reminiscent of XPF and XPG, which are involved in nucleotide excision repair, serving to excise the modified or damaged nucleotides [35], and resulting in the appearance of small nucleolytic DNA fragments, thus further raises the possibility that 5-methylcytosine is excised from DNA in a step analogous to either nucleotide excision or base excision repair.

The precise mechanism of DNA demethylation is still controversial [36] and the notion that FTO demethylates CpG by excision is provocative. That FTO increases C/EBPs binding to and potentiates transcription from both unmethylated and methylated DNA also seems counterintuitive to its role as a demethylase. Such a mechanism is not inconceivable given that nucleotide excision repair factors including XPB and XPD are also part of the basal transcription initiation machinery [37; 38]; and moreover, transcription coupled repair of oxidative damage requires an XPG function distinct from its nucleotide excision repair endonuclease activity [39].

More likely, it is conceivable that the partially purified FTO may contain impurities including nucleases or the association and co-purification of DNA glycosylases with FTO that caused the observed DNA cleavage. Hence, further investigation with homogeneously purified FTO or its enzymatically inactive mutants will yield insights into the precise mechanisms of FTO as a transcriptional coactivator. Therefore, we speculate that Fto may serve as a transcriptional coactivator and also in recruiting DNA demethylase, a mechanism that is similar to XPB and XPD in DNA repair and transcription initiation. We envision that during epigenetic reactivation of gene expression, within the transcription initiation complex, Fto functions to recruit associated factors that recognize 5-methylcytosine, which then excises the methyl-modified base from the promoter. Following excision, cellular DNA polymerase and ligase presumably would fill in and seal the gap resulting the excision, thereby restoring the DNA for further cyclical epigenetic regulation.

In addition to the striking loss of adipose tissue, Fto−/ − mice also exhibit an increase in energy expenditure that may be a result of elevated sympathetic activity. Therefore, it is possible that Fto may have a critical role in the central control of energy homeostasis but not adipogenesis. It has been shown that C/EBPβ and δ are expressed in the hypothalamus and paraventricular nucleus in the brain [40; 41], which seemed to overlap with the localization of Fto [13]. Therefore, the notion that coactivation of C/EBPs by Fto that results in the dual regulation of energy homeostasis and adipogenesis cannot be ruled out. It is also unclear how the present finding relates to the obesity-associated single nucleotide polymorphisms [11].

It is possible that these intronic genetic variants may influence the tissue-specific expression of FTO and differentially modulate satiety and adipogenesis in the brain and adipose tissues, respectively. Taken together, our results point to an epigenetic role for Fto as a transcriptional coactivator in regulating adipogenesis, and its absence causes decreased adiposity, which is consistent with the disappearance of white adipose tissue in Fto-deficient mice.

Conclusions

Our results revealed that Fto is a transcriptional coactivator for the C/EBP family of transcriptional regulator, thus providing the first evidence that Fto may play a role in the epigenetic regulation of adiposity. Hence targeting the Fto pathway may be a novel mechanistic approach for the development of anti-obesity pharmacotherapeutics.

Supplementary Material

01

Acknowledgments

This research was supported by the American Cancer Society, Ohio Division, to IDLS; and the NIH [CA102204] to KVC. IDLS is also a recipient of NIH K22-ES12981 award.

Footnotes

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