Introduction
Obesity is becoming more widespread with global projections of more than 1.12 billion obese individuals by 2030 (Kelly, Yang, Chen, Reynolds, & He, 2008). Excess fat accumulation in obese individuals occurs when energy intake exceeds energy expenditure, where their response to such imbalance is partially governed by genetic predisposition (Herrera, Keildson, & Lindgren, 2011). Twin studies have estimated heritability to account for 40-75% of the phenotypic variance of obesity in children and adults, demonstrating how the occurrence of obesity depends on the interplay of both environmental and genetic factors (Rhee, Phelan, & McCaffery, 2012). In attempts to track down common genetic variants influencing obesity, the Fat mass and obesity associated (FTO) gene was identified by genome-wide association studies (Herrera et al., 2011). Not only were genetic variants of the FTO gene associated with human adiposity and metabolic disorders, they were also linked to cancer (Zhao, Yang, Sun, Zhao, & Yang, 2014). However, the exact molecular mechanism of the effects of the FTO gene on obesity still remains largely unknown (Zhao et al., 2014).
Background
FTO expression is highest in areas of the hypothalamus associated with feeding, and have been shown to be linked with sensitivity to satiety in children (Wardle, Llewllyn, Sanderson, & Plomin, 2008). This suggests that the FTO gene may be influencing regulatory drivers underlying food intake (Wardle et al., 2008). The existence of single nucleotide polymorphisms (SNPs) within the gene may be responsible for an increased risk of obesity in humans since carriers of the FTO rs9939609 risk allele altered food intake, with 505 kJ and 1,231 kJ more in A allele and AA allele carriers than TT homozygotes, respectively (Zhao et al., 2014). Evidence has shown that primary transcripts containing an at-risk A risk allele at SNP rn9939609 are more abundant in blood and fibroblast RNA samples of individuals than those with the T allele (Church et al., 2011). This suggests that increased expression of FTO is correlated with obesity, which was tested in mouse models with additional copies of the Fto gene, resulting in increased fat mass and obesity via hyperphagia (Chuch et al., 2011).
The FTO protein is a Fe(II) and 2-oxoglutarate-dependent demethylase of single stranded DNA and RNA, which mainly targets 6-methyladenosine (m6A), 3-methyluracil (m3U) and 3-methylthymidine (m3T) (Merkestein et al., 2014). However, m3T is rare in mammalian genomic DNA, and m3U are deeply buried within folded rRNA, they are unlikely to be the physiological substrates of FTO (Jia et al., 2013). On the other hand, m6A is the most prevalent modification in eukaryotic mRNA and can affect RNA processing, RNA transport, and translation efficiency (Merkestein et al., 2014). Previous studies have shown that m6A levels in the brain are low during embryogenesis but drastically increase by adulthood, indicating that m6A plays a role in neuronal maturation and normal brain functions (McGuinness & McGuinness, 2014). Hess et al. examined the FTO protein’s control over m6A methylation and its critical role in regulating gene transcription and expression of key components in dopamine (DA) signaling in the midbrain (2013). Since FTO only targets a unique subset of mRNAs involved in neuronal function, they measured methylation on Kcnj6, Grin1 and Drd3, of which are all key regulators of DA activity (Hess et al., 2013). In the absence of FTO, mRNAs showed increased methylation and increased expression, whereas protein expression of the gene products was significantly attenuated. However, mRNA and protein expression levels of other hypermethylated mRNAs remained unchanged when FTO was absent, indicating that the effect of m6A on mRNA and protein expression is governed by specific rules or through indirect complex mechanisms (Hess et al., 2013). The studied concluded that FTO influences the translation of specific proteins in the D2R-D3R-GIRK signaling cascade, where the absence of FTO resulted in reduced protein expression which ultimately attenuated D2R and D3R signaling (Hess et al., 2013). Since dopaminergic (DA) signaling governs the control of many complex behaviors including learning, reward behavior, motor functions and feeding, FTO’s enzymatic role in regulating the neuronal pathway of the brain is of specific interest as it could provide insight on FTO’s mechanism in controlling obesity- related feeding behavior.
Increasing amounts of evidence suggest that epigenetic changes that occur on genomic DNA and histone proteins, regulating gene activity and expression patterns during development and beyond, are sensitive to extrinsic factors, such as diet and nutrients (Almén et al., 2012). Studies have shown that long-term administration of a diet lacking in methyl donors, such as folic acid, methionine and choline, caused global DNA hypermethylation in the brain (Pogribny et al., 2008). B-vitamin folate, methionine, betaine and choline are all essential for transfer reactions, which regulates the transfer of methyl groups of methylation reactions (Ishii et al., 2014). Thus, diet-induced epigenetic rearrangement during brain maturation could alter future development and behavior and even influence the onset of various diseases including obesity. Since m6A methylation states on mRNA are dynamically regulated by FTO and methyltransferase activities in response to internal and external cellular cues, is methyl donor supplementation in the diet during a developmental period sufficient to alter m6A methylation levels on mRNA and protein expression of Kcnj6, Grin1 and Drd3 in the brain? Also, does the altered diet induce similar effects in individuals overexpressing FTO?
Hypothesis and Prediction
Since RNA methylation involves the transfer of a methyl group similar to DNA methylation, methyl-donor supplementation in the diet during development will likely alter RNA methylation states as well. Thus, supplementation with folate, methionine, betaine and choline will be sufficient in inducing changes in m6A methylation levels on the mRNA transcripts of Kcnj6, Grin1 and Drd3. Since Hess et al. found that FTO-deficient mice displayed hypermethylation of mRNAS of Kcnj6, Grin1 and Drd3 in the brain, FTO overexpression will likely cause hypomethylation (2013). However, FTO-overexpressing mice supplemented with methyl donors should show lower levels of hypomethylation compared to mice provided a diet without supplementation. Increased levels of available methyl groups could drive greater activity of methyltransferases for increased methylation, which would thus counteract the demethylation activity by FTO.
Experimental Design
Mouse Model
The FTO-overexpression will be induced in mice by globally expressing either one (FTO-3) or two (FTO-4) additional copies of the Fto gene, as done in a study by Church et al. (2010). Wild-type C57BL/6J littermates (FTO-2) will be used as the control, expressing only 2 copies of the Fto gene. An increase in Fto mRNA expression in the brain can be confirmed later by qRT-PCR. The FTO-2, FTO-3 and FTO-4 mice populations will be kept in separate cages in a temperature and humidity controlled room on 12∶12 light-dark cycle with free access to water and food. From each of the 3 genetically different groups, half (n=5) will be fed chow highly supplemented with methyl donors (MS) while the other half supplemented with the mice chow. The MS chow will contain a 2-fold increase in folic acid, vitamin B12, choline and betaine levels relative to the control chow (Wolff, Kodell, Moore, & Cooney, 1998). Mice will be given their assigned diet starting from birth until 5 weeks of age. Previous studies have shown that FTO-4 mice begin increasing in body weight starting at 5 weeks, so mice will be killed by cervical dislocation at this point during development (Merkestein et al., 2014). This control for any methylation changes occurring as a secondary consequence of increased body weight or of other varying external factors (Merkestein et al., 2014). The brain will then be immediately dissected and kept on dry ice (-80°C) until RNA extraction.
MeRIP- Seq
Full-length RNA fragments in the brain will be digested to approximately 100nt long fragments following the recommendations of the sequencing platform (Illumina) (Meter et al., 2012). Immunoprecipitation of total mouse brain methylated RNA will be performed using a commercial rabbit antibody to m6A (Meyer et al., 2012). MeRIP-Seq high-throughput sequencing will be performed on replicates of midbrain RNA isolated from FTO-4, FTO-3 and FTO-2 (WT) mice. Genomic alignment will be done using the Burrows-Wheeler Aligner (BWA) and only peaks that reached significance in all replicates for each sample will be used for final peak analysis (Hess et al., 2013).
Gene Ontology (GO) Analysis
Peaks will be identified for all samples and the genome coordinates for FTO-4 peaks and FTO-3 peaks will be intersected with FTO-2 peaks to compare and identify any differences in m6A methylation. Specifically, UCSC Genome Browser plots of MeRIP-Seq reads for the Kcnj6, Grin1 and Drd3 genes will be analyzed, since they had been previously identified by GO analysis to regulate the DA signaling pathway (Hess et al., 2013). mRNA levels will also be determined for the three genes by quantitative PCR of all mice samples to compare their relative levels using box plots as done by Hess et al. (2013). GIRK2, GRIN1 and DRD3 protein expression levels in the midbrain tissues will also be compared across the FTO-4, FTO-3, and FTO-2 mice using SDS-PAGE and Western blot analysis, utilizing β-actin and calnexin as loading controls (Hess et al., 2013).
Discussion and Results
If my hypothesis is correct, mice given the MS diet will show greater levels of m6A hypermethylation of Kcnj6, Grin1 and Drd3 mRNAs relative to the un-supplemented groups. However, such hypermethylation will be to a lesser extent in FTO-overexpressing mice (FTO-4 and FTO-3) compared to FTO control mice (FTO-2) due to FTO demethylation activity. Thus, FTO-4 and FTO-3 mice will also exhibit greater GIRK2, GRIN1 and DRD3 protein expression than FTO-2 from the lesser degree of hypermethylation in both MS and control diet groups. These results would suggest that FTO functions to demethylate the mRNA of genes involved in neuronal DA pathways while increasing protein expression to enhance DA signaling activity. This could explain individuals with the risk allele and mice overexpressing FTO have the tendency to increase feeding behavior resultant from increased DA- associated activity in reward centres of the brain. If methyl donor supplementation increases methylation of DA-related genes, such a result can be useful in the development of nutrient supplementation therapies for decreasing the risk of obesity. However, if methyl supplementation is not sufficient to induce hypermethylation of Kcnj6, Grin1 and Drd3 mRNA, it will not have an affect on protein expression and thus DA reward pathways.
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