The long non-coding RNA Paupar promotes KAP1-dependent chromatin changes and regulates olfactory bulb neurogenesis.

Many long non-coding RNAs (lncRNAs) are expressed during central nervous system (CNS) development, yet their in vivo roles and mechanisms of action remain poorly understood. Paupar, a CNS-expressed lncRNA, controls neuroblastoma cell growth by binding and modulating the activity of transcriptional regulatory elements in a genome-wide manner. We show here that the Paupar lncRNA directly binds KAP1, an essential epigenetic regulatory protein, and thereby regulates the expression of shared target genes important for proliferation and neuronal differentiation. Paupar promotes KAP1 chromatin occupancy and H3K9me3 deposition at a subset of distal targets, through the formation of a ribonucleoprotein complex containing Paupar, KAP1 and the PAX6 transcription factor. Paupar-KAP1 genome-wide co-occupancy reveals a fourfold enrichment of overlap between Paupar and KAP1 bound sequences, the majority of which also appear to associate with PAX6. Furthermore, both Paupar and Kap1 loss-of-function in vivo disrupt olfactory bulb neurogenesis. These observations provide important conceptual insights into the trans-acting modes of lncRNA-mediated epigenetic regulation and the mechanisms of KAP1 genomic recruitment, and identify Paupar and Kap1 as regulators of neurogenesis in vivo.

The long non-coding RNA Paupar regulates the expression of both local and distal genes.

Although some long noncoding RNAs (lncRNAs) have been shown to regulate gene expression in cis, it remains unclear whether lncRNAs can directly regulate transcription in trans by interacting with chromatin genome-wide independently of their sites of synthesis. Here, we describe the genomically local and more distal functions of Paupar, a vertebrate-conserved and central nervous system-expressed lncRNA transcribed from a locus upstream of the gene encoding the PAX6 transcription factor. Knockdown of Paupar disrupts the normal cell cycle profile of neuroblastoma cells and induces neural differentiation. Paupar acts in a transcript-dependent manner both locally, to regulate Pax6, as well as distally by binding and regulating genes on multiple chromosomes, in part through physical association with PAX6 protein. Paupar binding sites are enriched near promoters and can function as transcriptional regulatory elements whose activity is modulated by Paupar transcript levels. Our findings demonstrate that a lncRNA can function in trans at transcriptional regulatory elements distinct from its site of synthesis to control large-scale transcriptional programmes.

The mutant Moonwalker TRPC3 channel links calcium signaling to lipid metabolism in the developing cerebellum.

The Moonwalker (Mwk) mouse is a model of dominantly inherited cerebellar ataxia caused by a gain-of-function mutation in the transient receptor potential (TRP) channel TRPC3. Here, we report impairments in dendritic growth and synapse formation early on during Purkinje cell development in the Mwk cerebellum that are accompanied by alterations in calcium signaling. To elucidate the molecular effector pathways that regulate Purkinje cell dendritic arborization downstream of mutant TRPC3, we employed transcriptomic analysis of developing Purkinje cells isolated by laser-capture microdissection. We identified significant gene and protein expression changes in molecules involved in lipid metabolism. Consistently, lipid homeostasis in the Mwk cerebellum was found to be disturbed, and treatment of organotypic cerebellar slices with ceramide significantly improved dendritic outgrowth of Mwk Purkinje cells. These findings provide the first mechanistic insights into the TRPC3-dependent mechanisms, by which activated calcium signaling is coupled to lipid metabolism and the regulation of Purkinje cell development in the Mwk cerebellum.

Neuron-specific antioxidant OXR1 extends survival of a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a devastating neurodegenerative disorder characterized by the progressive loss of spinal motor neurons. While the aetiological mechanisms underlying the disease remain poorly understood, oxidative stress is a central component of amyotrophic lateral sclerosis and contributes to motor neuron injury. Recently, oxidation resistance 1 (OXR1) has emerged as a critical regulator of neuronal survival in response to oxidative stress, and is upregulated in the spinal cord of patients with amyotrophic lateral sclerosis. Here, we tested the hypothesis that OXR1 is a key neuroprotective factor during amyotrophic lateral sclerosis pathogenesis by crossing a new transgenic mouse line that overexpresses OXR1 in neurons with the SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Interestingly, we report that overexpression of OXR1 significantly extends survival, improves motor deficits, and delays pathology in the spinal cord and in muscles of SOD1(G93A) mice. Furthermore, we find that overexpression of OXR1 in neurons significantly delays non-cell-autonomous neuroinflammatory response, classic complement system activation, and STAT3 activation through transcriptomic analysis of spinal cords of SOD1(G93A) mice. Taken together, these data identify OXR1 as the first neuron-specific antioxidant modulator of pathogenesis and disease progression in SOD1-mediated amyotrophic lateral sclerosis, and suggest that OXR1 may serve as a novel target for future therapeutic strategies.

Expression profiling of mouse subplate reveals a dynamic gene network and disease association with autism and schizophrenia.

The subplate zone is a highly dynamic transient sector of the developing cerebral cortex that contains some of the earliest generated neurons and the first functional synapses of the cerebral cortex. Subplate cells have important functions in early establishment and maturation of thalamocortical connections, as well as in the development of inhibitory cortical circuits in sensory areas. So far no role has been identified for cells in the subplate in the mature brain and disease association of the subplate-specific genes has not been analyzed systematically. Here we present gene expression evidence for distinct roles of the mouse subplate across development as well as unique molecular markers to extend the repertoire of subplate labels. Performing systematic comparisons between different ages (embryonic days 15 and 18, postnatal day 8, and adult), we reveal the dynamic and constant features of the markers labeling subplate cells during embryonic and early postnatal development and in the adult. This can be visualized using the online database of subplate gene expression at https://molnar.dpag.ox.ac.uk/subplate/. We also identify embryonic similarities in gene expression between the ventricular zones, intermediate zone, and subplate, and distinct postnatal similarities between subplate, layer 5, and layers 2/3. The genes expressed in a subplate-specific manner at some point during development show a statistically significant enrichment for association with autism spectrum disorders and schizophrenia. Our report emphasizes the importance of the study of transient features of the developing brain to better understand neurodevelopmental disorders.

Gene expression analysis of the embryonic subplate.

The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of thalamocortical axons. At later developmental stages, they are predominantly involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. We aimed to further characterize the embryonic murine subplate population by establishing a gene expression profile at embryonic day (E) 15.5 using laser capture microdissection and microarrays. The microarray identified over 300 transcripts with higher expression in the subplate compared with the cortical plate at this stage. Using quantitative reverse transcription-polymerase chain reaction, in situ hybridization (ISH), and immunohistochemistry (IHC), we have confirmed specific expression in the E15.5 subplate for 13 selected genes, which have not been previously associated with this compartment (Abca8a, Cdh10, Cdh18, Csmd3, Gabra5, Kcnt2, Ogfrl1, Pls3, Rcan2, Sv2b, Slc8a2, Unc5c, and Zdhhc2). In the reeler mutant, the expression of the majority of these genes (9 of 13) was shifted in accordance with the altered position of subplate. These genes belong to several functional groups and likely contribute to synapse formation and axonal growth and guidance in subplate cells.

Pre-symptomatic development of lower motor neuron connectivity in a mouse model of severe spinal muscular atrophy.

The childhood motor neuron disease spinal muscular atrophy (SMA) results from reduced expression of the survival motor neuron (SMN) gene. Previous studies using in vitro model systems and lower organisms have suggested that low levels of Smn protein disrupt prenatal developmental processes in lower motor neurons, influencing neuronal outgrowth, axon branching and neuromuscular connectivity. The extent to which these developmental pathways contribute to selective vulnerability and pathology in the mammalian neuromuscular system in vivo remains unclear. Here, we have investigated the pre-symptomatic development of neuromuscular connectivity in differentially vulnerable motor neuron populations in Smn(-/-);SMN2 mice, a model of severe SMA. We show that reduced Smn levels have no detectable effect on morphological correlates of pre-symptomatic development in either vulnerable or stable motor units, indicating that abnormal pre-symptomatic developmental processes are unlikely to be a prerequisite for subsequent pathological changes to occur in vivo. Microarray analyses of spinal cord from two different severe SMA mouse models demonstrated that only minimal changes in gene expression were present in pre-symptomatic mice. In stark contrast, microarray analysis of late-symptomatic spinal cord revealed widespread changes in gene expression, implicating extracellular matrix integrity, growth factor signalling and myelination pathways in SMA pathogenesis. Taken together, these data suggest that reduced Smn levels induce SMA pathology by instigating rapidly progressive neurodegenerative pathways in lower motor neurons around the time of disease onset rather than by modulating pre-symptomatic neurodevelopmental pathways.

A mouse model for the metabolic effects of the human fat mass and obesity associated FTO gene.

Human FTO gene variants are associated with body mass index and type 2 diabetes. Because the obesity-associated SNPs are intronic, it is unclear whether changes in FTO expression or splicing are the cause of obesity or if regulatory elements within intron 1 influence upstream or downstream genes. We tested the idea that FTO itself is involved in obesity. We show that a dominant point mutation in the mouse Fto gene results in reduced fat mass, increased energy expenditure, and unchanged physical activity. Exposure to a high-fat diet enhances lean mass and lowers fat mass relative to control mice. Biochemical studies suggest the mutation occurs in a structurally novel domain and modifies FTO function, possibly by altering its dimerisation state. Gene expression profiling revealed increased expression of some fat and carbohydrate metabolism genes and an improved inflammatory profile in white adipose tissue of mutant mice. These data provide direct functional evidence that FTO is a causal gene underlying obesity. Compared to the reported mouse FTO knockout, our model more accurately reflects the effect of human FTO variants; we observe a heterozygous as well as homozygous phenotype, a smaller difference in weight and adiposity, and our mice do not show perinatal lethality or an age-related reduction in size and length. Our model suggests that a search for human coding mutations in FTO may be informative and that inhibition of FTO activity is a possible target for the treatment of morbid obesity.

Alternative splicing events are a late feature of pathology in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy is a severe motor neuron disease caused by inactivating mutations in the SMN1 gene leading to reduced levels of full-length functional SMN protein. SMN is a critical mediator of spliceosomal protein assembly, and complete loss or drastic reduction in protein leads to loss of cell viability. However, the reason for selective motor neuron degeneration when SMN is reduced to levels which are tolerated by all other cell types is not currently understood. Widespread splicing abnormalities have recently been reported at end-stage in a mouse model of SMA, leading to the proposition that disruption of efficient splicing is the primary mechanism of motor neuron death. However, it remains unclear whether splicing abnormalities are present during early stages of the disease, which would be a requirement for a direct role in disease pathogenesis. We performed exon-array analysis of RNA from SMN deficient mouse spinal cord at 3 time points, pre-symptomatic (P1), early symptomatic (P7), and late-symptomatic (P13). Compared to littermate control mice, SMA mice showed a time-dependent increase in the number of exons showing differential expression, with minimal differences between genotypes at P1 and P7, but substantial variation in late-symptomatic (P13) mice. Gene ontology analysis revealed differences in pathways associated with neuronal development as well as cellular injury. Validation of selected targets by RT-PCR confirmed the array findings and was in keeping with a shift between physiologically occurring mRNA isoforms. We conclude that the majority of splicing changes occur late in SMA and may represent a secondary effect of cell injury, though we cannot rule out significant early changes in a small number of transcripts crucial to motor neuron survival.

Cross-hierarchical plasticity of corticofugal projections to dLGN after neonatal monocular enucleation.

Perception is the result of interactions between the sensory periphery, thalamus, and cerebral cortex. Inputs from the retina project to the first-order dorsal lateral geniculate nucleus (dLGN), which projects to the primary visual cortex (V1). In return, the cortex innervates the thalamus. While layer 6 projections innervate all thalamic nuclei, cortical layer 5 neurons selectively project to the higher order lateral posterior nucleus (LP) and not to dLGN. It has been demonstrated that a subpopulation of layer 5 (Rbp4-Cre+) projections rewires to dLGN after monocular or binocular enucleation in young postnatal mice. However, the exact cortical regional origin of these projections was not fully determined, and it remained unclear whether these changes persisted into adulthood. In this study, we report gene expression changes observed in the dLGN after monocular enucleation at birth using microarray, qPCR at P6, and in situ hybridization at P8. We report that genes that are normally enriched in dLGN, but not LP during development are preferentially downregulated in dLGN following monocular enucleation. Comparisons with developmental gene expression patters in dLGN suggest more immature and delayed gene expression in enucleated dLGN. Combined tracing and immuno-histochemical analysis revealed that the induced layer 5 fibers that innervate enucleated dLGN originate from putative primary visual cortex and they retain increased VGluT1+ synapse formation into adulthood. Our results indicate a new form of plasticity when layer 5 driver input takes over the innervation of an originally first-order thalamic nucleus after early sensory deficit.

Transcriptome sequencing, microarray, and proteomic analyses reveal cellular and metabolic impact of hepatitis C virus infection in vitro.

UNLABELLED: Hepatitis C virus (HCV) is a major cause of liver disease but the full impact of HCV infection on the hepatocyte is poorly understood. RNA sequencing (RNA-Seq) is a novel method to analyze the full transcriptional activity of a cell or tissue, thus allowing new insight into the impact of HCV infection. We conducted the first full-genome RNA-Seq analysis in a host cell to analyze infected and noninfected cells, and compared this to microarray and proteomic analyses. The combined power of the triple approach revealed that HCV infection affects a number of previously unreported canonical pathways and biological functions, including pregnane X receptor/retinoic acid receptor activation as a potential host antiviral response, and integrin-linked kinase signaling as an entry factor. This approach also identified several mechanisms implicated in HCV pathogenesis, including an increase in reactive oxygen species. HCV infection had a broad effect on cellular metabolism, leading to increases in cellular cholesterol and free fatty acid levels, associated with a profound and specific decrease in cellular glucose levels. CONCLUSION: RNA-Seq technology, especially when combined with established methods, demonstrated that HCV infection has potentially wide-ranging effects on cellular gene and protein expression. This in vitro study indicates a substantial metabolic impact of HCV infection and highlights new mechanisms of virus-host interaction which may be highly relevant to pathogenesis in vivo.

AF4 is a critical regulator of the IGF-1 signaling pathway during Purkinje cell development.

Deregulation of the insulin-like growth factor 1 (IGF-1) signaling pathway is a recurrent finding in mouse models and human patients with cerebellar ataxia and thus represents a common pathological cascade in neuronal cell death that may be targeted for therapy. We have previously identified a point mutation in AF4, a transcription cofactor of RNA polymerase II elongation and chromatin remodeling, that causes progressive and highly specific Purkinje cell (PC) death in the ataxic mouse mutant robotic, leading to the accumulation of AF4 in PCs. Here we confirm that the spatiotemporal pattern of PC degeneration in the robotic cerebellum correlates with the specific profile of AF4 upregulation. To identify the underlying molecular pathways, we performed microarray gene expression analysis of PCs obtained by laser capture microdissection (LCM) at the onset of degeneration. Igf-1 was significantly downregulated in robotic PCs compared with wild-type controls before and throughout the degenerative process. Consistently, we observed a decrease in the activation of downstream signaling molecules including type 1 IGF receptor (IGF-1R) and the extracellular signal-regulated kinase (ERK) 1 and ERK2. Chromatin immunoprecipitation confirmed that Igf-1 is a direct and the first validated target of the AF4 transcriptional regulatory complex, and treatment of presymptomatic robotic mice with IGF-1 indeed markedly delayed the progression of PC death. This study demonstrates that small changes in the levels of a single transcriptional cofactor can deleteriously affect normal cerebellum function and opens new avenues of research for the manipulation of the IGF-1 pathway in the treatment of cerebellar ataxia in humans.

Novel markers reveal subpopulations of subplate neurons in the murine cerebral cortex.

The subplate lays the foundation of the developing cerebral cortex, and abnormalities have been suggested to contribute to various brain developmental disorders. The causal relationship between cellular pathologies and cognitive disorders remains unclear, and therefore, a better understanding of the role of subplate cells in cortical development is essential. Only by determining the molecular taxonomy of this diverse class of neurons can we identify the subpopulations that may contribute differentially to cortical development. We identified novel markers for murine subplate cells by comparing gene expression of subplate and layer 6 of primary visual and somatosensory cortical areas of postnatal day (P)8 old mice using a microarray-based approach. We examined the utility of these markers in well-characterized mutants (reeler, scrambler, and p35-KO) where the subplate is displaced in relation to the cortical plate. In situ hybridization or immunohistochemistry confirmed subplate-selective expression of complexin 3, connective tissue growth factor, nuclear receptor-related 1/Nr4a2, and monooxygenase Dbh-like 1 while transmembrane protein 163 also had additional expression in layer 5, and DOPA decarboxylase was also present in the white matter. Localization of marker-positive cells in the reeler and p35-KO cortices suggests different subpopulations of subplate cells. These new markers open up possibilities for further identification of subplate subpopulations in research and in neuropathological diagnosis.

High quality RNA from multiple brain regions simultaneously acquired by laser capture microdissection.

BACKGROUND: Laser capture microdissection enables the isolation of single cells or small cell groups from histological sections under direct microscopic observation. Combined with quantitative PCR or microarray, it is a very powerful approach for studying gene expression profiles in discrete cell populations. The major challenge for such studies is to obtain good quality RNA from small amounts of starting material. RESULTS: We have developed a simple, flexible, and low-cost method for simultaneously producing RNA from discrete cell groups in embryonic day 15 mouse brain. In particular, we have optimized the following key steps in the procedure: staining, cryosectioning, storage of sections and harvesting of microdissected cells. We obtained the best results when staining 20 mum-thick sections with 1% cresyl violet in 70% ethanol and harvesting the microdissected tissue in RNA stabilization solution. In addition, we introduced three stop-points in the protocol which makes the tedious process of laser capture microdissection more flexible, without compromising RNA quality. CONCLUSION: Using this optimized method, we have consistently obtained RNA of high quality from all four simultaneously microdissected cell groups. RNA integrity numbers were all above 8, and long cDNA fragments (> 1.2 kb) were successfully amplified by reverse transcription PCR from all four samples. We conclude that RNAs isolated by this method are well suited for downstream quantitative PCR or microarray studies.

Genes involved in the formation of the earliest cortical circuits.

Building the brain is like erecting a house of cards. The early connections provide the foundation of the adult structure, and disruption of these may be the source of many developmental flaws. Cerebral cortical developmental disorders (including schizophrenia and autism) and perinatal injuries involve cortical neurons with early connectivity. The major hindrance of progress in understanding the early neural circuits during cortical development and disease has been the lack of reliable markers for specific cell populations. Due to the advance of powerful approaches in gene expression analysis and the utility of models with reporter gene expressions in specific cortical cell types, our knowledge of the early cortical circuits is rapidly increasing. With focus on the sub-plate, layer VI and layer V projection neurons, we shall illustrate the progress made in the understanding of their neurochemical properties, physiological characteristics and their integration into the early intracortical and extracortical circuitry. This field benefited from recent developments in mouse genetics in generating models with subtype specific gene expression patterns, powerful cell dissection and separation methods combined with microarray analysis. The emergence of cortical cell type specific biomarkers will not only help neuropathological diagnosis, but will also eventually reveal the causal relations in the pathogenesis of various cortical developmental disorders.

Genotype-Dependent Effects of COMT Inhibition on Cognitive Function in a Highly Specific, Novel Mouse Model of Altered COMT Activity.

Catechol-O-methyltransferase (COMT) modulates dopamine levels in the prefrontal cortex. The human gene contains a polymorphism (Val158Met) that alters enzyme activity and influences PFC function. It has also been linked with cognition and anxiety, but the findings are mixed. We therefore developed a novel mouse model of altered COMT activity. The human Met allele was introduced into the native mouse COMT gene to produce COMT-Met mice, which were compared with their wild-type littermates. The model proved highly specific: COMT-Met mice had reductions in COMT abundance and activity, compared with wild-type mice, explicitly in the absence of off-target changes in the expression of other genes. Despite robust alterations in dopamine metabolism, we found only subtle changes on certain cognitive tasks under baseline conditions (eg, increased spatial novelty preference in COMT-Met mice vs wild-type mice). However, genotype differences emerged after administration of the COMT inhibitor tolcapone: performance of wild-type mice, but not COMT-Met mice, was improved on the 5-choice serial reaction time task after tolcapone administration. There were no changes in anxiety-related behaviors in the tests that we used. Our findings are convergent with human studies of the Val158Met polymorphism, and suggest that COMT's effects are most prominent when the dopamine system is challenged. Finally, they demonstrate the importance of considering COMT genotype when examining the therapeutic potential of COMT inhibitors.

Hyperglycaemia induces metabolic dysfunction and glycogen accumulation in pancreatic ß-cells.

Insulin secretion from pancreatic ß-cells is impaired in all forms of diabetes. The resultant hyperglycaemia has deleterious effects on many tissues, including ß-cells. Here we show that chronic hyperglycaemia impairs glucose metabolism and alters expression of metabolic genes in pancreatic islets. In a mouse model of human neonatal diabetes, hyperglycaemia results in marked glycogen accumulation, and increased apoptosis in ß-cells. Sulphonylurea therapy rapidly normalizes blood glucose levels, dissipates glycogen stores, increases autophagy and restores ß-cell metabolism. Insulin therapy has the same effect but with slower kinetics. Similar changes are observed in mice expressing an activating glucokinase mutation, in in vitro models of hyperglycaemia, and in islets from type-2 diabetic patients. Altered ß-cell metabolism may underlie both the progressive impairment of insulin secretion and reduced ß-cell mass in diabetes.

Increased Expression of the Diabetes Gene SOX4 Reduces Insulin Secretion by Impaired Fusion Pore Expansion.

The transcription factor Sox4 has been proposed to underlie the increased type 2 diabetes risk linked to an intronic single nucleotide polymorphism in CDKAL1 In a mouse model expressing a mutant form of Sox4, glucose-induced insulin secretion is reduced by 40% despite normal intracellular Ca(2+) signaling and depolarization-evoked exocytosis. This paradox is explained by a fourfold increase in kiss-and-run exocytosis (as determined by single-granule exocytosis measurements) in which the fusion pore connecting the granule lumen to the exterior expands to a diameter of only 2 nm, which does not allow the exit of insulin. Microarray analysis indicated that this correlated with an increased expression of the exocytosis-regulating protein Stxbp6. In a large collection of human islet preparations (n = 63), STXBP6 expression and glucose-induced insulin secretion correlated positively and negatively with SOX4 expression, respectively. Overexpression of SOX4 in the human insulin-secreting cell EndoC-ßH2 interfered with granule emptying and inhibited hormone release, the latter effect reversed by silencing STXBP6 These data suggest that increased SOX4 expression inhibits insulin secretion and increased diabetes risk by the upregulation of STXBP6 and an increase in kiss-and-run exocytosis at the expense of full fusion. We propose that pharmacological interventions promoting fusion pore expansion may be effective in diabetes therapy.

Kinetic Studies of the Reactions of Pentacyanoferrate(II) Complexes with Peroxydisulfate.

Reactions of Fe(CN)(5)L(3-) (L = 4-aminopyridine (4-ampy), pyridine (py), 4,4'-bipyridine (4,4'-bpy), and pyrazine (pz)) with peroxydisulfate, Fe(CN)(5)L(3-) + S(2)O(8)(2-) right harpoon over left harpoon Fe(CN)(5)L(2-) + SO(4)(-) + SO(4)(2-), have been found to follow an outer-sphere electron transfer mechanism. The specific rate constants of oxidation are 1.45 +/- 0.01, (9.00 +/- 0.02) x 10(-2), (5.60 +/- 0.01) x 10(-2), and (2.89 +/- 0.01) x 10(-2) M(-1) s(-1), for L = 4-ampy, py, 4,4'-bpy, and pz, respectively, at &mgr; = 0.50 M LiClO(4), T = 25 degrees C, pH = 4.4-8.8. The rate constants of oxidation for the corresponding Ru(NH(3))(5)L(2+) complexes were also measured and were found to be faster than those of Fe(CN)(5)L(3-) complexes by a factor of approximately 10(2) even after the corrections for the differences in reduction potentials and in the charges of the complexes. The difference in reactivity may arise from the hydrogen bonding between peroxydisulfate and the ammonia ligands of Ru(NH(3))(5)L(2+) and nonadiabaticity observed in the Fe(CN)(5)L(3-) complexes.

Changes in gene expression associated with FTO overexpression in mice.

Single nucleotide polymorphisms in the first intron of the fat-mass-and-obesity-related gene FTO are associated with increased body weight and adiposity. Increased expression of FTO is likely underlying this obesity phenotype, as mice with two additional copies of Fto (FTO-4 mice) exhibit increased adiposity and are hyperphagic. FTO is a demethylase of single stranded DNA and RNA, and one of its targets is the m6A modification in RNA, which might play a role in the regulation of gene expression. In this study, we aimed to examine the changes in gene expression that occur in FTO-4 mice in order to gain more insight into the underlying mechanisms by which FTO influences body weight and adiposity. Our results indicate an upregulation of anabolic pathways and a downregulation of catabolic pathways in FTO-4 mice. Interestingly, although genes involved in methylation were differentially regulated in skeletal muscle of FTO-4 mice, no effect of FTO overexpression on m6A methylation of total mRNA was detected.