Chicken ovalbumin upstream promoter transcription factor II regulates uncoupling protein 3 gene transcription in Phodopus sungorus

BMC Molecular Biology Chicken ovalbumin upstream promoter transcription factor II regulates uncoupling protein 3 gene transcription in Phodopus sungorus Tobias Fromme 1 Kathrin Reichwald 0 Matthias Platzer mplatzer@fli- 0 Xing-Sheng Li 1 Martin Klingenspor 1 0 Genome Analysis, Leibniz- Institute for Age Research - Fritz Lipmann Institute , D-07745 Jena , Germany 1 Department of Animal Physiology, Faculty of Biology, Philipps-University , D-35043 Marburg , Germany Background: Ucp3 is an integral protein of the inner mitochondrial membrane with a role in lipid metabolism preventing deleterious effects of fatty acids in states of high lipid oxidation. Ucp3 is expressed in brown adipose tissue and skeletal muscle and controlled by a transcription factor complex including PPARalpha, MyoD and the histone acetyltransferase p300. Several studies have demonstrated interaction of these factors with chicken ovalbumin upstream promoter transcription factor II (Coup-TFII). This nuclear receptor is involved in organogenesis and other developmental processes including skeletal muscle development, but also co-regulates a number of metabolic genes. In this study we in silico analyzed the upstream region of Ucp3 of the Djungarian hamster Phodopus sungorus and identified several putative response elements for Coup-TFII. We therefore investigated whether Coup-TFII is a further player in the transcriptional control of the Ucp3 gene in rodents. Results: By quantitative PCR we demonstrated a positive correlation of Coup-TFII and Ucp3 mRNA expression in skeletal muscle and brown adipose tissue in response to food deprivation and cold exposure, respectively. In reporter gene assays Coup-TFII enhanced transactivation of the Ucp3 promoter conveyed by MyoD, PPARalpha, RXRalpha and/or p300. Using deletions and mutated constructs, we identified a Coup-TFII enhancer element 816-840 bp upstream of the transcriptional start site. Binding of Coup-TFII to this upstream enhancer was confirmed in electrophoretic mobility shift and supershift assays. Conclusion: Transcriptional regulation of the Coup-TFII gene in response to starvation and cold exposure seems to be the regulatory mechanism of Ucp3 mRNA expression in brown adipose and skeletal muscle tissue determining the final appropriate rate of transcript synthesis. These findings add a crucial component to the complex transcriptional machinery controlling expression of Ucp3. Given the substantial evidence for a function of Ucp3 in lipid metabolism, Coup-TFII may not only be a negative regulator of glucose responsive genes but also transactivate genes involved in lipid metabolism. - Background Uncoupling protein 3 (Ucp3) is a member of the family of uncoupling proteins, which are located in the inner mitochondrial membrane and uncouple the respiratory chain from ATP synthesis by dissipating the proton motive force [1,2]. The physiological function of Ucp3 is subject to an ongoing debate [3]. Regulation of Ucp3 expression suggests a role in lipid metabolism. Skeletal muscle Ucp3 transcription is increased in response to food deprivation, a robust mechanism consistently observable in man, rodents and even fish [4]. Further physiological conditions positively regulating Ucp3 include cold exposure [5,6], acute exercise [7] and streptozotocin-induced diabetes [8]. Increased levels of circulating free fatty acids (FFA) are common to all these physiological states; infusion experiments imply that these are the primary cause for Ucp3 upregulation [9]. Therefore it has been suggested, though not proven experimentally, that Ucp3 is a fatty acid anion carrier [10]. The biochemical properties of the protein as measured in mitochondrial proton leak assays by parallel recording of membrane potential and oxygen consumption infer a role for Ucp3 in the defense against radical oxygen species (ROS), mitigating their generation by mild uncoupling [11]. This possible function is corroborated by the finding that a product of ROS induced lipid peroxidation, 4hydroxy-2-nonenal, specifically induces uncoupling by Ucp3 and that even a small reduction of membrane potential markedly decreases ROS production [12]. A controversial hypothesis which takes into account both physiological and biochemical data emphasizes that the export of fatty acids or hydroperoxy fatty acids and subsequent protonated re-influx of a certain fraction into the matrix would result in a net proton import detectable as mild uncoupling [13,14]. This mechanism would reduce ROS production and at the same time reduce the level of nonesterified fatty acids in the matrix susceptible to peroxidation, thereby preventing deleterious effects in states of high lipid oxidation. Ucp3 is predominantly expressed in skeletal muscle (SKM) and brown adipose tissue (BAT), both tissues with exceptionally high lipid oxidation capacities. The principal molecular constituents of Ucp3 gene regulation have recently been identified by several copious studies. Heterodimers of peroxisome proliferator activated receptor (PPAR) and the retinoic X receptor (RXR) bind to a response element (PPRE) within the proximal promoter region and activate transcription depending on the presence of myogenic differentiation antigen 1 (MyoD). MyoD binds to a series of non-canonical E-boxes directly adjacent to the transcriptional start site (TSS). Induction is enhanced by the coactivator p300 protein (p300), which acetylates MyoD and possibly surrounding histones [15]. Furthermore, the PPRE in the Ucp3 promoter is multifunctional, i.e. can alternatively be targeted by heterodimers of the thyroid hormone receptor (TR) and RXR stimulating expression in the presence of MyoD [16]. The ligands of PPAR (fatty acids) and TRs (T3) along with the requirement of MyoD indicate that this mechanism is involved in the acute response of Ucp3 expression in SKM to physiological stimuli. However, to our knowledge neither BATspecific nor differentiation specific regulation has been characterized in detail to date. Notably several studies have demonstrated interaction of PPARs, MyoD and/or p300 with chicken ovalbumin upstream promoter transcription factor II (Coup-TFII, official gene name: Nr2f2) [17-20]. This nuclear receptor is involved in organogenesis and other developmental processes [21] including SKM development [22], but also co-regulates a number of metabolic genes [18,23-25]. In this study we analyzed the upstream region of Ucp3 of the Djungarian hamster Phodopus sungorus in silico and identified several putative response elements for CoupTFII. We therefore investigated whether Coup-TFII is a further player in the transcriptional control of the Ucp3 gene in rodents. Results Structure of the hamster Ucp3 gene We successfully cloned the genomic Ucp3 locus of the hamster. Primers to amplify fragments of the upstream region (approx. -3500 to +50) were deduced from conserved segments of corresponding rodent genomic sequences. Resulting PCR products w (...truncated)