BACKGROUND
uncoupling protein  <dig>  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  <cit> .
the physiological function of ucp <dig> is subject to an ongoing debate  <cit> .
regulation of ucp <dig> expression suggests a role in lipid metabolism.
skeletal muscle ucp <dig> transcription is increased in response to food deprivation, a robust mechanism consistently observable in man, rodents and even fish  <cit> .
further physiological conditions positively regulating ucp <dig> include cold exposure  <cit> , acute exercise  <cit>  and streptozotocin-induced diabetes  <cit> .
increased levels of circulating free fatty acids  are common to all these physiological states; infusion experiments imply that these are the primary cause for ucp <dig> upregulation  <cit> .
therefore it has been suggested, though not proven experimentally, that ucp <dig> is a fatty acid anion carrier  <cit> .
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 ucp <dig> in the defense against radical oxygen species , mitigating their generation by mild uncoupling  <cit> .
this possible function is corroborated by the finding that a product of ros induced lipid peroxidation, 4-hydroxy-2-nonenal, specifically induces uncoupling by ucp <dig> and that even a small reduction of membrane potential markedly decreases ros production  <cit> .
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  <cit> .
this mechanism would reduce ros production and at the same time reduce the level of non-esterified fatty acids in the matrix susceptible to peroxidation, thereby preventing deleterious effects in states of high lipid oxidation.
ucp <dig> is predominantly expressed in skeletal muscle  and brown adipose tissue , both tissues with exceptionally high lipid oxidation capacities.
the principal molecular constituents of ucp <dig> gene regulation have recently been identified by several copious studies.
heterodimers of peroxisome proliferator activated receptor α  and the retinoic x receptor α  bind to a response element  within the proximal promoter region and activate transcription depending on the presence of myogenic differentiation antigen  <dig> .
myod binds to a series of non-canonical e-boxes directly adjacent to the transcriptional start site .
induction is enhanced by the coactivator p <dig> protein , which acetylates myod and possibly surrounding histones  <cit> .
furthermore, the ppre in the ucp <dig> promoter is multifunctional, i.e.
can alternatively be targeted by heterodimers of the thyroid hormone receptor  and rxrα stimulating expression in the presence of myod  <cit> .
the ligands of pparα  and trs  along with the requirement of myod indicate that this mechanism is involved in the acute response of ucp <dig> expression in skm to physiological stimuli.
however, to our knowledge neither bat-specific nor differentiation specific regulation has been characterized in detail to date.
notably several studies have demonstrated interaction of ppars, myod and/or p <dig> with chicken ovalbumin upstream promoter transcription factor ii   <cit> .
this nuclear receptor is involved in organogenesis and other developmental processes  <cit>  including skm development  <cit> , but also co-regulates a number of metabolic genes  <cit> .
in this study we analyzed the upstream region of ucp <dig> of the djungarian hamster phodopus sungorus in silico 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 ucp <dig> gene in rodents.
RESULTS
structure of the hamster ucp <dig> gene
we successfully cloned the genomic ucp <dig> locus of the hamster.
primers to amplify fragments of the upstream region  were deduced from conserved segments of corresponding rodent genomic sequences.
resulting pcr products were sequenced and served as a framework to select primers for gap closure.
we extended this contig to the putative 5' adjacent gene.
ucp <dig> introns were amplified with exonic primers based on the known hamster cdna  and on the exon  <dig> sequence obtained as described above.
the resulting genomic contig of  <dig> bp included  <dig> bp of ucp <dig> 5' gene flanking region as well as all ucp <dig> exons and introns; the sequence of the terminal exon extends  <dig> bp 3' of the stop codon .
we identified two novel splice variants of the ucp <dig> transcript in bat .
one transcript is characterized by partial mobilization of the second intron, presumably resulting in a premature stop , the other lacks the second exon, possibly leading to alternative usage of the next in frame start codon wich is located in exon <dig> .
in our 5' race analysis, the tss was variable within a range of  <dig> bp .
to determine a reference point for relative sequence positions we arbitrarily chose position  <dig> of , represented by two clones obtained in bat and skm cdna race experiments, respectively.
of six additional sequenced clones, one contained a start site at - <dig> , two at - <dig> , one at - <dig> , one at + <dig>  and at + <dig> .
thus the tss did not show a distinct tissue specificity.
analysis of the ucp <dig> promoter
we compared rodent and human proximal promoter sequences to classify promoter elements by their conservation .
of the elements recently characterized by solanes and coworkers  – mediating transcriptional activation by pparα, trs and myod – the ppre/tre was located within a region that is identical in the compared species.
the  <dig> bp long myod-binding triple e-box only differs in three nucleotides of the first repeat and is otherwise identical.
in contrast, two tata-like boxes identified in the human promoter  <cit>  are absent in the known rodent sequences.
by in silico analysis of the hamster ucp <dig> promoter we detected several putative nuclear receptor binding sites, among them  <dig> elements predicted to possibly bind coup-tfii.
sequencing of hamster coup-tfii and qpcr
to derive homologous primers for qpcr we cloned and sequenced a  <dig> bp fragment of coup-tfii  comprising coding regions of exon  <dig> and  <dig>  hamster sequences were 98% identical with the murine orthologue.
the deduced amino acid sequences of hamster and mouse are identical.
in a first experiment we determined mrna expression levels of coup-tfii and ucp <dig> by qpcr analysis in a panel of nine tissues of hamsters housed under standard laboratory conditions and fed ad libitum .
ucp <dig> levels were highest in skm and bat, much lower in heart and white adipose tissue and virtually absent in all other tested tissues.
coup-tfii was well detectable in all analyzed tissues with highest amounts in kidney and skm.
the individual variation for both ucp <dig> and coup-tfii was maximal in skm.
ucp <dig> mrna levels in bat were about 50% of skm expression, interestingly the same is true for coup-tfii.
to explore a possible coregulation of ucp <dig> and coup-tfii we exposed hamsters to cold and food deprivation, physiological challenges known to upregulate ucp <dig> expression  <cit>  .
in skm food deprivation led to a significant induction of ucp <dig> and coup-tfii.
in bat of cold exposed hamsters ucp <dig> was not systematically upregulated but displayed an increased individual variation.
a similar degree of variation without significant cold-induced regulation was observed for coup-tfii.
to test for a possible coregulation we measured ucp <dig> and coup-tfii transcript abundancies and compared respective levels of control and challenged animals .
under control conditions no correlation was observed.
however, we detected a highly significant correlation  between ucp <dig> and coup-tfii levels in challenged animals.
luciferase reporter gene assays with cotransfected transcription factors
we tested coup-tfii in cotransfection reporter gene assays with regard to its potential in coactivating a ucp <dig> promoter fragment of  <dig> bp cloned into the luciferase reporter gene vector pgl3basic .
to evaluate the influence of coup-tfii we compared cotransfections with or without expression vectors for pparα, myod, rxrα and/or the histone acetyltransferase p <dig>  factors known to regulate ucp <dig> expression  <cit>  .
without coup-tfii none of the tested factors upregulated -2244ucp3luc more than 5-fold.
cotransfection of several factors exhibited a synergistic effect, which was augmented by p <dig>  e.g.
adding rxrα to myod hardly increased reporter gene activity, but in the presence of p <dig> notably induced expression.
the strongest induction  was achieved by a cotransfection of pparα, myod and p <dig> in the presence of the synthetic pparα agonist wy <dig> .
cotransfection of coup-tfii systematically increased reporter gene expression in all combinations tested.
together with a single other factor it was particularly effective with myod or p <dig>  attaining an additional 3– <dig> fold induction.
in combination with multiple factors coup-tfii cotransfection consistently led to an approximate doubling of induction.
the maximal induction in comparison to the basal construct activity was achieved in combination with rxrα, myod and p <dig> , which is two fold higher than the maximal level observed in any experiment without coup-tfii .
the dimension of induction by coup-tfii is illustrated by comparison with the pparα activation through the ligand wy <dig>  on a background of pparα, myod and p <dig>  here transactivation by coup-tfii and the ligand wy <dig> , respectively, is in the same order of magnitude.
to exclude that overexpression of any nuclear receptor or coup-tf family member unspecifically leads to an induction of the ucp <dig> promoter we utilized an expression vector for coup-tfi, which is a close coup-tfii relative sharing  <dig> % of identical amino acid sequence, as a control.
replacing coup-tfii by coup-tfi in cotransfections with transcription factor combinations did not lead to activation of the ucp <dig> promoter, but conversely downregulated reporter gene activity , possibly due to competition for rxrα.
we therefore conclude that coup-tfii specifically transactivated the ucp <dig> promoter in synergy with other known transcription factors and coactivators.
to locate the responsible cis element, we studied the potential of coup-tfii to activate several reporter gene deletion constructs .
coup-tfii transfection alone in this assay increased luciferase activity on -2244ucp3luc 16-fold while all other constructs are much less sensitive.
there is, however, a certain unspecific effect on the empty pgl <dig> basic vector, that can also be seen for all constructs used.
this handicap was absent when we cotransfected the deletion constructs with myod, rxrα and p <dig>  the combination exhibiting the strongest effect on -2244ucp3luc.
the coup-tfii effect is limited to -2244ucp3luc, locating the crucial region between nucleotides - <dig> to - <dig>  which are exclusively found on this construct.
we identified several possible binding sites for coup-tfii in this fragment: - <dig> , - <dig> , - <dig> and - <dig> , - <dig>  and - <dig> .
electrophoretic mobility shifts assays
we tested five oligonucleotide probes corresponding to the putative response elements a-e  for binding of coup-tfii in electrophoretic mobility shift assays.
elements a, b, d and e did not display any specific interaction .
for element c we were able to show a strong complex formation in the presence of coup-tfii, that was specifically supershifted by the respective antibody.
binding of proteins to this probe was effectively blocked by competition with the unlabelled oligonucleotide.
the antibody was specific for coup-tfii and did not bind the close relative coup-tfi.
virtually all coup-tfii was detected in the nuclear fraction .
nuclear extracts from skeletal muscle tissue of food deprived  and control  hamsters were able to form a bandshift on element c of comparable size.
in two out of three starvation samples the complex was stronger than in control conditions .
the two elements present in oligonucleotide c form a functional repeat as is typical for binding sites of nuclear receptor dimers.
this inverse repeat is conserved in the rat ucp <dig> promoter, and also found slightly more upstream in the murine promoter albeit in reverse order .
mutational analyses of the coup-tfii binding element
we further analysed the two repeat elements on oligonucleotide c  by disrupting the 5' half , the 3' half  or both  on bandshift assay probes .
we chose to change two bases of the 3' half-site into adenine and two bases of the inverse 5' half-site into thymine correspondingly.
these probes were employed either labelled with nuclear extracts of mock or coup-tfii transfected cells or unlabelled as competitors for the interaction of coup-tfii with element c .
all mutated probes displayed a reduced complex formation with coup-tfii .
while probes cmut5' and cmut5'3' did not show any significant complex formation, probe cmut3' still formed a complex that was weaker in intensity and slightly smaller than the wt probe.
in competition assays the mutated competitor oligonucleotides were less efficient in competing coup-tfii complex formation on the cwt probe than the unlabelled cwt oligonucleotide itself .
again cmut5' and cmut5'3' generated a similar pattern of no binding of coup-tfii to these mutated elements.
mutation cmut3' did not entirely loose its ability to compete complex formation, which is in line with its demonstrated retained capability of interaction with coup-tfii.
we introduced the same mutations cmut5' and cmut3' into our reporter gene vector -2244ucp3luc generating -2244cmut5'luc and -2244cmut3'luc .
disruption of the 3'half-site on -2244cmut3'luc did not lower responsiveness to coup-tfii alone or on a background of myod, rxrα and p <dig>  but did even proportionally increase luciferase activity by a small amount.
conversely, -2244cmut5'luc was devoid of any luciferase activity in all conditions tested and displayed a pattern resembling our empty control vector pgl <dig> basic.
discussion
a prominent hypothesis considers uncoupling protein  <dig>  to be a crucial component of lipid metabolism with implications for the regulation of body weight and composition  <cit> .
this role is further substantiated by the identification of polymorphisms/alleles in the human ucp <dig> gene that are associated with an elevated body mass index  <cit> .
a more detailed analysis of the machinery regulating ucp <dig> transcription is therefore of importance for identifying regulatory networks controlling energy partitioning.
our comparison of previously characterized ucp <dig> promoter elements in rat, mouse and human with the hamster sequence, shows full conservation of the binding sites for myod, pparα/rxrα and tr/rxrα heterodimers.
the sequence alignment furthermore demonstrates that two tata-like boxes present in the human promoter  <cit>  are absent in rodent sequences including the hamster.
this might prove to be crucial considering, that in the study of riquet and coworkers  the activities of human constructs were investigated in murine tissue.
the tss of the human ucp <dig> gene is quite variable and displays a distinct tissue specificity, whereas in mouse the tss is located at a single site  <cit> .
our result of variable tss in p. sungorus demonstrates that the constancy in mouse does not reflect a common trait of rodent species.
however, in the hamster the tss did not show a distinct tissue specificity as found in humans.
by in silico analysis we identified coup-tfii as a candidate transcription factor for the regulation of ucp <dig> expression.
coup-tfii is a  <dig> kd nuclear orphan receptor and member of the coup-tf family.
the amino acid sequence of the ligand binding and the dna binding domain is conserved across species to a very high extent  indicating an important role for these domains.
coup-tfii has mainly been described as a crucial factor in developmental processes  <cit> .
by qpcr we measured ucp <dig> and coup-tfii transcript levels in a panel of nine different tissues.
we confirmed that bat and skm are the major sites of ucp <dig> mrna expression.
coup-tfii was detected in both tissues, but in line with previous data on human adult tissue distribution  <cit>  is rather ubiquitously expressed with highest levels in kidney, liver and heart.
contrary to reports that coup-tfii is expressed in preadipocytes and myoblasts and downregulated during differentiation  <cit> , we were able to detect considerable amounts of coup-tfii transcript in bat and skm tissue.
apparantely cell culture systems are devoid of the appropriate physiological stimuli promoting coup-tfii mrna expression in tissues.
this is supported by the absence of coup-tfii in human embryonal kidney cells as shown in our western blot experiment  despite its presence in human kidney tissue in vivo  <cit> .
it has been demonstrated that coup-tfii plays an important role in the regulation of several genes encoding key metabolic enzymes  <cit> , which are certainly regulated in terminally differentiated cells.
ucp <dig> gene expression was upregulated in skm of food deprived hamsters.
in line with the function of coup-tfii in metabolic regulation we also observed a significant increase of mrna expression in response to this challenge.
probably owing to the short duration of cold exposure in our study we did not observe a significant cold induced increase in ucp <dig> mrna expression in bat as published previously  <cit> .
however, coup-tfii and ucp <dig> mrna expression in bat displayed a similar cold-induced increase in variation.
the resemblance of expression levels in skm and bat and the response to physiological stimuli culminates in a highly significant correlation of coup-tfii and ucp <dig> mrna abundancy under challenged conditions .
the absence of such a correlation in the control group suggests that coup-tfii requires additional factors in order to enhance ucp <dig> mrna expression which must be recruited and/or activated beforehand.
we could support this model in reporter gene assays, in which coup-tfii strongly coactivated ucp <dig> promotor activity in synergy with pparα, myod, rxrα and/or p <dig>  while the effect of coup-tfii was much lower alone.
these constituents of the well described basal transcription factor complex in our experiments affected ucp <dig> expression as described previously  <cit> , i.e.
strong activation by pparα/rxrα and p <dig>  dependent on agonist stimulation and presence of myod.
coup-tfii specifically enhanced expression in synergy with these factors.
in contrast to our data, coup-tfii has been shown to negatively interact with myod and p <dig> in skm, reducing their potential to activate e-box driven reporter gene constructs  <cit> .
this discrepancy may be explained by a differential recruitment of the multiple coup-tf family cofactors in different physiological environments .
the specific complex of transcription factors, to which coup-tfii is recruited, may determine the final function as described for interaction with the glucocorticoid receptor  <cit> .
in general, coup-tf proteins display a conflicting pattern of positive or negative interaction with nuclear receptors like ppars or the estrogen receptor depending on the target gene .
there are genes where transcription is increased by ppars and decreased by coup-tfs  as well as genes for which the situation is opposite  or at which both ppars and coup-tfs act synergistically .
even a complete reversal of the effect of coup-tfii on a single target has been reported  <cit> ; transcription of the phosphoenolpyruvate carboxykinase gene is induced or repressed by coup-tfii in a tissue specific manner.
interestingly, specific pparγ agonists upregulate coup-tfii in the heart  <cit>  and also ucp <dig> gene expression in bat and skm  <cit> , although to our knowledge no direct interaction of pparγ with the ucp <dig> promoter has been verified so far.
this effect might therefore be due to pparγ mediated transactivation of coup-tfii, which in turn enhances ucp <dig> expression.
we were able to confine the sequence element mediating the coup-tfii effect to - <dig> to - <dig> by utilizing reporter gene deletion constructs.
-2244ucp3luc was the only construct being induced by coup-tfii on a background of myod, rxrα and p <dig> and was exclusively activated high above an unspecific effect by coup-tfii alone.
of five candidate elements a-e within this region tested by electrophoretic mobility shift assays we confirmed specific binding of coup-tfii to element c. notably, element c did not only show complex formation with overexpressed coup-tfii but also with nuclear extracts of hamster skm .
extracts of all six animals tested were able to form a complex of comparable size and food deprivation of hamsters led to an increase in band intensity.
the element is located at - <dig> to - <dig> and constitutes a functional repeat structure.
by mutational analyses we were able to confine coup-tfii binding to the 5' half-site of this element.
in reporter gene assays disruption of this site surprisingly led to a complete loss of activity and responsiveness to any treatment tested.
while this fact certainly underlines the importance of this element, its indifference to myod, rxrα and p <dig> treatment is in conflict with the retained effect of this treatment on -664ucp3luc and -243ucp3luc, that are also devoid of this element.
it seems that the element exhibits a function beyond direct transactivation by coup-tfii, that depends on the presence of the surrounding region.
these characteristics point towards a model of coup-tfii deactivating or displacing a so far unknown repressor of ucp <dig> transcription.
this repressor could be expected to bind in proximity to the 5' half-site of elementc, possibly even to the 3' half-site of this same element.
in the light of this hypothetical model several formerly negligible seeming facts gain relevancy: mutated construct -2244cmut3'luc displayed an overall increase of luciferase activity as compared to -2244ucp3luc .
deletion construct -664ucp3luc had a higher basal activity and could be activated by myod, rxrα and p <dig> to a higher extent than -2244ucp3luc .
the complex size of bandshift probe cmut5' was slightly changed .
this model alone, however, does not account for the activation of -2244ucp3luc by myod, rxrα, p <dig> and pparα in the absence of cotransfected coup-tfii.
therefore there would have to be an endogenous coup-tfii expression in hek <dig> cells that we can either not detect with our western blots or is induced by transfection of factors like myod and p <dig>  at present we cannot decide wether repressor displacement/deactivation itself or subsequent direct coactivation is the dominant means of coup-tfii mediated transcriptional induction.
the conservation of the inverse repeat structure and its coup-tfii binding site across rodent species suggests an important role for this element; its preservation in the murine promoter despite an obvious event of rearrangement during evolution supports this role.
the upstream location of the binding element and its synergy with pparα resembles a comparable situation, where coup-tfii directly coactivates the lipoprotein lipase gene in cooperation with ppar/rxr heterodimers as described previously  <cit> .
notably lipoprotein lipase is a crucial determinant of fatty acid uptake from circulating triglycerides.
an explanation of the link between increased lipid utilization and the recruitment of coup-tfii in bat and skm is hampered by the lack of knowledge about a specific ligand and the posttranslational mechanism of activation.
it is beyond the scope of this study to clarify whether coup-tfii is directly regulated in response to ffa levels and thus possibly conveys ffa-dependent ucp <dig> transcription.
our qpcr data indicate that de novo synthesis of coup-tfii is the dominant means of target gene upregulation rather than ligand binding or posttranslational modification.
so far, transcription of coup-tfii has been linked to map kinase pathway activity  <cit>  and the presence of ets family transcription factors  <cit> .
further research is required to clarify whether these mechanisms represent a link between lipid utilization and coup-tfii upregulation.
on the functional level beyond its role in organismic development coup-tfii has been assumed to be an integral part of the glucose response complex, where it functions as an inhibitor of glucose dependent activators  <cit> .
this hypothesis is based on the ability of coup-tfii to inhibit upstream stimulatory factor -dependent transactivation of a glucose response element in the l-type pyruvate kinase gene  and a similar finding regarding the atpa gene encoding the alpha subunit of the f1f <dig> atp synthase complex  <cit> .
recently, bardoux and coworkers , after discovering a role for coup-tfii in insulin secretion and sensitivity, assigned coup-tfii as an important regulator of glucose homeostasis  <cit> .
our finding of coup-tfii upregulation in states of augmented lipid oxidation confirms these models and extends them to a new aspect.
the inhibition of glucose induced gene transcription may occur in conjunction with a positive regulation of lipid metabolism genes like ucp <dig> and lipoprotein lipase.
CONCLUSIONS
coup-tfii is a strong activator of ucp <dig> gene transcription by binding to an upstream element in the ucp <dig> promoter as shown by luciferase assays and electrophoretic mobility shift assays.
coup-tfii mrna expression correlates with ucp <dig> mrna levels in tissues of hamsters subjected to physiological challenges inducing lipid oxidation.
transcriptional upregulation of the coup-tfii gene in response to these challenges seems to regulate the ucp <dig> gene in brown adipose and skeletal muscle tissue determining the final appropriate rate of ucp <dig> mrna synthesis.
the mechanism of coup-tfii inducing ucp <dig> transcription seems to involve displacement/deactivation of an unknown repressor.
these findings add a crucial component to the complex transcriptional machinery controlling expression of ucp <dig>  at the same time they further manifest a function of coup-tfii in the regulation of lipid metabolism genes.

 uncoupling protein 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. the physiological function of ucp is subject to an ongoing debate. regulation of ucp expression suggests a role in lipid metabolism. skeletal muscle ucp transcription is increased in response to food deprivation, a robust mechanism consistently observable in man, rodents and even fish. further physiological conditions positively regulating ucp include cold exposure, acute exercise and streptozotocin-induced diabetes. increased levels of circulating free fatty acids are common to all these physiological states; infusion experiments imply that these are the primary cause for ucp upregulation. therefore it has been suggested, though not proven experimentally, that ucp is a fatty acid anion carrier. 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 ucp in the defense against radical oxygen species, mitigating their generation by mild uncoupling. this possible function is corroborated by the finding that a product of ros induced lipid peroxidation, 4-hydroxy-2-nonenal, specifically induces uncoupling by ucp and that even a small reduction of membrane potential markedly decreases ros production. 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. this mechanism would reduce ros production and at the same time reduce the level of non-esterified fatty acids in the matrix susceptible to peroxidation, thereby preventing deleterious effects in states of high lipid oxidation. ucp is predominantly expressed in skeletal muscle and brown adipose tissue, both tissues with exceptionally high lipid oxidation capacities. the principal molecular constituents of ucp gene regulation have recently been identified by several copious studies. heterodimers of peroxisome proliferator activated receptor α and the retinoic x receptor α bind to a response element within the proximal promoter region and activate transcription depending on the presence of myogenic differentiation antigen. myod binds to a series of non-canonical e-boxes directly adjacent to the transcriptional start site. induction is enhanced by the coactivator p protein, which acetylates myod and possibly surrounding histones. furthermore, the ppre in the ucp promoter is multifunctional, i.e. can alternatively be targeted by heterodimers of the thyroid hormone receptor and rxrα stimulating expression in the presence of myod. the ligands of pparα and trs along with the requirement of myod indicate that this mechanism is involved in the acute response of ucp expression in skm to physiological stimuli. however, to our knowledge neither bat-specific nor differentiation specific regulation has been characterized in detail to date. notably several studies have demonstrated interaction of ppars, myod and/or p with chicken ovalbumin upstream promoter transcription factor ii. this nuclear receptor is involved in organogenesis and other developmental processes including skm development, but also co-regulates a number of metabolic genes. in this study we analyzed the upstream region of ucp of the djungarian hamster phodopus sungorus in silico 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 ucp gene in rodents.  structure of the hamster ucp gene we successfully cloned the genomic ucp locus of the hamster. primers to amplify fragments of the upstream region were deduced from conserved segments of corresponding rodent genomic sequences. resulting pcr products were sequenced and served as a framework to select primers for gap closure. we extended this contig to the putative 5' adjacent gene. ucp introns were amplified with exonic primers based on the known hamster cdna and on the exon sequence obtained as described above. the resulting genomic contig of bp included bp of ucp 5' gene flanking region as well as all ucp exons and introns; the sequence of the terminal exon extends bp 3' of the stop codon. we identified two novel splice variants of the ucp transcript in bat. one transcript is characterized by partial mobilization of the second intron, presumably resulting in a premature stop, the other lacks the second exon, possibly leading to alternative usage of the next in frame start codon wich is located in exon. in our 5' race analysis, the tss was variable within a range of bp. to determine a reference point for relative sequence positions we arbitrarily chose position of, represented by two clones obtained in bat and skm cdna race experiments, respectively. of six additional sequenced clones, one contained a start site at -, two at -, one at -, one at + and at +. thus the tss did not show a distinct tissue specificity. analysis of the ucp promoter we compared rodent and human proximal promoter sequences to classify promoter elements by their conservation. of the elements recently characterized by solanes and coworkers – mediating transcriptional activation by pparα, trs and myod – the ppre/tre was located within a region that is identical in the compared species. the bp long myod-binding triple e-box only differs in three nucleotides of the first repeat and is otherwise identical. in contrast, two tata-like boxes identified in the human promoter are absent in the known rodent sequences. by in silico analysis of the hamster ucp promoter we detected several putative nuclear receptor binding sites, among them elements predicted to possibly bind coup-tfii. sequencing of hamster coup-tfii and qpcr to derive homologous primers for qpcr we cloned and sequenced a bp fragment of coup-tfii comprising coding regions of exon and hamster sequences were 98% identical with the murine orthologue. the deduced amino acid sequences of hamster and mouse are identical. in a first experiment we determined mrna expression levels of coup-tfii and ucp by qpcr analysis in a panel of nine tissues of hamsters housed under standard laboratory conditions and fed ad libitum. ucp levels were highest in skm and bat, much lower in heart and white adipose tissue and virtually absent in all other tested tissues. coup-tfii was well detectable in all analyzed tissues with highest amounts in kidney and skm. the individual variation for both ucp and coup-tfii was maximal in skm. ucp mrna levels in bat were about 50% of skm expression, interestingly the same is true for coup-tfii. to explore a possible coregulation of ucp and coup-tfii we exposed hamsters to cold and food deprivation, physiological challenges known to upregulate ucp expression. in skm food deprivation led to a significant induction of ucp and coup-tfii. in bat of cold exposed hamsters ucp was not systematically upregulated but displayed an increased individual variation. a similar degree of variation without significant cold-induced regulation was observed for coup-tfii. to test for a possible coregulation we measured ucp and coup-tfii transcript abundancies and compared respective levels of control and challenged animals. under control conditions no correlation was observed. however, we detected a highly significant correlation between ucp and coup-tfii levels in challenged animals. luciferase reporter gene assays with cotransfected transcription factors we tested coup-tfii in cotransfection reporter gene assays with regard to its potential in coactivating a ucp promoter fragment of bp cloned into the luciferase reporter gene vector pgl3basic. to evaluate the influence of coup-tfii we compared cotransfections with or without expression vectors for pparα, myod, rxrα and/or the histone acetyltransferase p factors known to regulate ucp expression. without coup-tfii none of the tested factors upregulated -2244ucp3luc more than 5-fold. cotransfection of several factors exhibited a synergistic effect, which was augmented by p e.g. adding rxrα to myod hardly increased reporter gene activity, but in the presence of p notably induced expression. the strongest induction was achieved by a cotransfection of pparα, myod and p in the presence of the synthetic pparα agonist wy. cotransfection of coup-tfii systematically increased reporter gene expression in all combinations tested. together with a single other factor it was particularly effective with myod or p attaining an additional 3– fold induction. in combination with multiple factors coup-tfii cotransfection consistently led to an approximate doubling of induction. the maximal induction in comparison to the basal construct activity was achieved in combination with rxrα, myod and p, which is two fold higher than the maximal level observed in any experiment without coup-tfii. the dimension of induction by coup-tfii is illustrated by comparison with the pparα activation through the ligand wy on a background of pparα, myod and p here transactivation by coup-tfii and the ligand wy, respectively, is in the same order of magnitude. to exclude that overexpression of any nuclear receptor or coup-tf family member unspecifically leads to an induction of the ucp promoter we utilized an expression vector for coup-tfi, which is a close coup-tfii relative sharing % of identical amino acid sequence, as a control. replacing coup-tfii by coup-tfi in cotransfections with transcription factor combinations did not lead to activation of the ucp promoter, but conversely downregulated reporter gene activity, possibly due to competition for rxrα. we therefore conclude that coup-tfii specifically transactivated the ucp promoter in synergy with other known transcription factors and coactivators. to locate the responsible cis element, we studied the potential of coup-tfii to activate several reporter gene deletion constructs. coup-tfii transfection alone in this assay increased luciferase activity on -2244ucp3luc 16-fold while all other constructs are much less sensitive. there is, however, a certain unspecific effect on the empty pgl basic vector, that can also be seen for all constructs used. this handicap was absent when we cotransfected the deletion constructs with myod, rxrα and p the combination exhibiting the strongest effect on -2244ucp3luc. the coup-tfii effect is limited to -2244ucp3luc, locating the crucial region between nucleotides - to - which are exclusively found on this construct. we identified several possible binding sites for coup-tfii in this fragment: -, -, - and -, - and -. electrophoretic mobility shifts assays we tested five oligonucleotide probes corresponding to the putative response elements a-e for binding of coup-tfii in electrophoretic mobility shift assays. elements a, b, d and e did not display any specific interaction. for element c we were able to show a strong complex formation in the presence of coup-tfii, that was specifically supershifted by the respective antibody. binding of proteins to this probe was effectively blocked by competition with the unlabelled oligonucleotide. the antibody was specific for coup-tfii and did not bind the close relative coup-tfi. virtually all coup-tfii was detected in the nuclear fraction. nuclear extracts from skeletal muscle tissue of food deprived and control hamsters were able to form a bandshift on element c of comparable size. in two out of three starvation samples the complex was stronger than in control conditions. the two elements present in oligonucleotide c form a functional repeat as is typical for binding sites of nuclear receptor dimers. this inverse repeat is conserved in the rat ucp promoter, and also found slightly more upstream in the murine promoter albeit in reverse order. mutational analyses of the coup-tfii binding element we further analysed the two repeat elements on oligonucleotide c by disrupting the 5' half, the 3' half or both on bandshift assay probes. we chose to change two bases of the 3' half-site into adenine and two bases of the inverse 5' half-site into thymine correspondingly. these probes were employed either labelled with nuclear extracts of mock or coup-tfii transfected cells or unlabelled as competitors for the interaction of coup-tfii with element c. all mutated probes displayed a reduced complex formation with coup-tfii. while probes cmut5' and cmut5'3' did not show any significant complex formation, probe cmut3' still formed a complex that was weaker in intensity and slightly smaller than the wt probe. in competition assays the mutated competitor oligonucleotides were less efficient in competing coup-tfii complex formation on the cwt probe than the unlabelled cwt oligonucleotide itself. again cmut5' and cmut5'3' generated a similar pattern of no binding of coup-tfii to these mutated elements. mutation cmut3' did not entirely loose its ability to compete complex formation, which is in line with its demonstrated retained capability of interaction with coup-tfii. we introduced the same mutations cmut5' and cmut3' into our reporter gene vector -2244ucp3luc generating -2244cmut5'luc and -2244cmut3'luc. disruption of the 3'half-site on -2244cmut3'luc did not lower responsiveness to coup-tfii alone or on a background of myod, rxrα and p but did even proportionally increase luciferase activity by a small amount. conversely, -2244cmut5'luc was devoid of any luciferase activity in all conditions tested and displayed a pattern resembling our empty control vector pgl basic. discussion a prominent hypothesis considers uncoupling protein to be a crucial component of lipid metabolism with implications for the regulation of body weight and composition. this role is further substantiated by the identification of polymorphisms/alleles in the human ucp gene that are associated with an elevated body mass index. a more detailed analysis of the machinery regulating ucp transcription is therefore of importance for identifying regulatory networks controlling energy partitioning. our comparison of previously characterized ucp promoter elements in rat, mouse and human with the hamster sequence, shows full conservation of the binding sites for myod, pparα/rxrα and tr/rxrα heterodimers. the sequence alignment furthermore demonstrates that two tata-like boxes present in the human promoter are absent in rodent sequences including the hamster. this might prove to be crucial considering, that in the study of riquet and coworkers the activities of human constructs were investigated in murine tissue. the tss of the human ucp gene is quite variable and displays a distinct tissue specificity, whereas in mouse the tss is located at a single site. our result of variable tss in p. sungorus demonstrates that the constancy in mouse does not reflect a common trait of rodent species. however, in the hamster the tss did not show a distinct tissue specificity as found in humans. by in silico analysis we identified coup-tfii as a candidate transcription factor for the regulation of ucp expression. coup-tfii is a kd nuclear orphan receptor and member of the coup-tf family. the amino acid sequence of the ligand binding and the dna binding domain is conserved across species to a very high extent indicating an important role for these domains. coup-tfii has mainly been described as a crucial factor in developmental processes. by qpcr we measured ucp and coup-tfii transcript levels in a panel of nine different tissues. we confirmed that bat and skm are the major sites of ucp mrna expression. coup-tfii was detected in both tissues, but in line with previous data on human adult tissue distribution is rather ubiquitously expressed with highest levels in kidney, liver and heart. contrary to reports that coup-tfii is expressed in preadipocytes and myoblasts and downregulated during differentiation, we were able to detect considerable amounts of coup-tfii transcript in bat and skm tissue. apparantely cell culture systems are devoid of the appropriate physiological stimuli promoting coup-tfii mrna expression in tissues. this is supported by the absence of coup-tfii in human embryonal kidney cells as shown in our western blot experiment despite its presence in human kidney tissue in vivo. it has been demonstrated that coup-tfii plays an important role in the regulation of several genes encoding key metabolic enzymes, which are certainly regulated in terminally differentiated cells. ucp gene expression was upregulated in skm of food deprived hamsters. in line with the function of coup-tfii in metabolic regulation we also observed a significant increase of mrna expression in response to this challenge. probably owing to the short duration of cold exposure in our study we did not observe a significant cold induced increase in ucp mrna expression in bat as published previously. however, coup-tfii and ucp mrna expression in bat displayed a similar cold-induced increase in variation. the resemblance of expression levels in skm and bat and the response to physiological stimuli culminates in a highly significant correlation of coup-tfii and ucp mrna abundancy under challenged conditions. the absence of such a correlation in the control group suggests that coup-tfii requires additional factors in order to enhance ucp mrna expression which must be recruited and/or activated beforehand. we could support this model in reporter gene assays, in which coup-tfii strongly coactivated ucp promotor activity in synergy with pparα, myod, rxrα and/or p while the effect of coup-tfii was much lower alone. these constituents of the well described basal transcription factor complex in our experiments affected ucp expression as described previously, i.e. strong activation by pparα/rxrα and p dependent on agonist stimulation and presence of myod. coup-tfii specifically enhanced expression in synergy with these factors. in contrast to our data, coup-tfii has been shown to negatively interact with myod and p in skm, reducing their potential to activate e-box driven reporter gene constructs. this discrepancy may be explained by a differential recruitment of the multiple coup-tf family cofactors in different physiological environments. the specific complex of transcription factors, to which coup-tfii is recruited, may determine the final function as described for interaction with the glucocorticoid receptor. in general, coup-tf proteins display a conflicting pattern of positive or negative interaction with nuclear receptors like ppars or the estrogen receptor depending on the target gene. there are genes where transcription is increased by ppars and decreased by coup-tfs as well as genes for which the situation is opposite or at which both ppars and coup-tfs act synergistically. even a complete reversal of the effect of coup-tfii on a single target has been reported; transcription of the phosphoenolpyruvate carboxykinase gene is induced or repressed by coup-tfii in a tissue specific manner. interestingly, specific pparγ agonists upregulate coup-tfii in the heart and also ucp gene expression in bat and skm, although to our knowledge no direct interaction of pparγ with the ucp promoter has been verified so far. this effect might therefore be due to pparγ mediated transactivation of coup-tfii, which in turn enhances ucp expression. we were able to confine the sequence element mediating the coup-tfii effect to - to - by utilizing reporter gene deletion constructs. -2244ucp3luc was the only construct being induced by coup-tfii on a background of myod, rxrα and p and was exclusively activated high above an unspecific effect by coup-tfii alone. of five candidate elements a-e within this region tested by electrophoretic mobility shift assays we confirmed specific binding of coup-tfii to element c. notably, element c did not only show complex formation with overexpressed coup-tfii but also with nuclear extracts of hamster skm. extracts of all six animals tested were able to form a complex of comparable size and food deprivation of hamsters led to an increase in band intensity. the element is located at - to - and constitutes a functional repeat structure. by mutational analyses we were able to confine coup-tfii binding to the 5' half-site of this element. in reporter gene assays disruption of this site surprisingly led to a complete loss of activity and responsiveness to any treatment tested. while this fact certainly underlines the importance of this element, its indifference to myod, rxrα and p treatment is in conflict with the retained effect of this treatment on -664ucp3luc and -243ucp3luc, that are also devoid of this element. it seems that the element exhibits a function beyond direct transactivation by coup-tfii, that depends on the presence of the surrounding region. these characteristics point towards a model of coup-tfii deactivating or displacing a so far unknown repressor of ucp transcription. this repressor could be expected to bind in proximity to the 5' half-site of elementc, possibly even to the 3' half-site of this same element. in the light of this hypothetical model several formerly negligible seeming facts gain relevancy: mutated construct -2244cmut3'luc displayed an overall increase of luciferase activity as compared to -2244ucp3luc. deletion construct -664ucp3luc had a higher basal activity and could be activated by myod, rxrα and p to a higher extent than -2244ucp3luc. the complex size of bandshift probe cmut5' was slightly changed. this model alone, however, does not account for the activation of -2244ucp3luc by myod, rxrα, p and pparα in the absence of cotransfected coup-tfii. therefore there would have to be an endogenous coup-tfii expression in hek cells that we can either not detect with our western blots or is induced by transfection of factors like myod and p at present we cannot decide wether repressor displacement/deactivation itself or subsequent direct coactivation is the dominant means of coup-tfii mediated transcriptional induction. the conservation of the inverse repeat structure and its coup-tfii binding site across rodent species suggests an important role for this element; its preservation in the murine promoter despite an obvious event of rearrangement during evolution supports this role. the upstream location of the binding element and its synergy with pparα resembles a comparable situation, where coup-tfii directly coactivates the lipoprotein lipase gene in cooperation with ppar/rxr heterodimers as described previously. notably lipoprotein lipase is a crucial determinant of fatty acid uptake from circulating triglycerides. an explanation of the link between increased lipid utilization and the recruitment of coup-tfii in bat and skm is hampered by the lack of knowledge about a specific ligand and the posttranslational mechanism of activation. it is beyond the scope of this study to clarify whether coup-tfii is directly regulated in response to ffa levels and thus possibly conveys ffa-dependent ucp transcription. our qpcr data indicate that de novo synthesis of coup-tfii is the dominant means of target gene upregulation rather than ligand binding or posttranslational modification. so far, transcription of coup-tfii has been linked to map kinase pathway activity and the presence of ets family transcription factors. further research is required to clarify whether these mechanisms represent a link between lipid utilization and coup-tfii upregulation. on the functional level beyond its role in organismic development coup-tfii has been assumed to be an integral part of the glucose response complex, where it functions as an inhibitor of glucose dependent activators. this hypothesis is based on the ability of coup-tfii to inhibit upstream stimulatory factor -dependent transactivation of a glucose response element in the l-type pyruvate kinase gene and a similar finding regarding the atpa gene encoding the alpha subunit of the f1f atp synthase complex. recently, bardoux and coworkers, after discovering a role for coup-tfii in insulin secretion and sensitivity, assigned coup-tfii as an important regulator of glucose homeostasis. our finding of coup-tfii upregulation in states of augmented lipid oxidation confirms these models and extends them to a new aspect. the inhibition of glucose induced gene transcription may occur in conjunction with a positive regulation of lipid metabolism genes like ucp and lipoprotein lipase.  coup-tfii is a strong activator of ucp gene transcription by binding to an upstream element in the ucp promoter as shown by luciferase assays and electrophoretic mobility shift assays. coup-tfii mrna expression correlates with ucp mrna levels in tissues of hamsters subjected to physiological challenges inducing lipid oxidation. transcriptional upregulation of the coup-tfii gene in response to these challenges seems to regulate the ucp gene in brown adipose and skeletal muscle tissue determining the final appropriate rate of ucp mrna synthesis. the mechanism of coup-tfii inducing ucp transcription seems to involve displacement/deactivation of an unknown repressor. these findings add a crucial component to the complex transcriptional machinery controlling expression of ucp at the same time they further manifest a function of coup-tfii in the regulation of lipid metabolism genes. 
transcriptional regulation of the coup-tfii gene in response to starvation and cold exposure seems to be the regulatory mechanism of ucp mrna expression in brown adipose and skeletal muscle tissue determining the final appropriate rate of transcript synthesis. ucp 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. these findings add a crucial component to the complex transcriptional machinery controlling expression of ucp given the substantial evidence for a function of ucp in lipid metabolism, coup-tfii may not only be a negative regulator of glucose responsive genes but also transactivate genes involved in lipid metabolism. ucp is expressed in brown adipose tissue and skeletal muscle and controlled by a transcription factor complex including pparalpha, myod and the histone acetyltransferase p several studies have demonstrated interaction of these factors with chicken ovalbumin upstream promoter transcription factor ii. by quantitative pcr we demonstrated a positive correlation of coup-tfii and ucp mrna expression in skeletal muscle and brown adipose tissue in response to food deprivation and cold exposure, respectively. in this study we in silico analyzed the upstream region of ucp of the djungarian hamster phodopus sungorus and identified several putative response elements for 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. we therefore investigated whether coup-tfii is a further player in the transcriptional control of the ucp gene in rodents. binding of coup-tfii to this upstream enhancer was confirmed in electrophoretic mobility shift and supershift assays.
 ucp 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. ucp is expressed in brown adipose tissue and skeletal muscle and controlled by a transcription factor complex including pparalpha, myod and the histone acetyltransferase p several studies have demonstrated interaction of these factors with chicken ovalbumin upstream promoter transcription factor ii. 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 ucp 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 ucp gene in rodents.  by quantitative pcr we demonstrated a positive correlation of coup-tfii and ucp 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 ucp promoter conveyed by myod, pparalpha, rxralpha and/or p using deletions and mutated constructs, we identified a coup-tfii enhancer element 816– bp upstream of the transcriptional start site. binding of coup-tfii to this upstream enhancer was confirmed in electrophoretic mobility shift and supershift assays.  transcriptional regulation of the coup-tfii gene in response to starvation and cold exposure seems to be the regulatory mechanism of ucp 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 ucp given the substantial evidence for a function of ucp in lipid metabolism, coup-tfii may not only be a negative regulator of glucose responsive genes but also transactivate genes involved in lipid metabolism. 