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

Abstract

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.

Figure 1

Alignment of the human and three rodent proximal Ucp3 promoter regions. Positions of identical bases are marked (*). The PPRE/TRE (box) as well as the MyoD binding triple E-box (dashed box) are highly conserved. Two TATA-like boxes (bold) can only be found in the human promoter. Transcriptional start sites of P. sungorus were identified within a wide range (black background), the arbitrarily chosen reference TSS is indicated (#). Rn = R. norvegicus, Mm = M. musculus, Ps = P. sungorus, Hs = H. sapiens

Figure 2

Expression level of Coup-TFII and Ucp3 mRNA in tissues of P. sungorus. Coup-TFII mRNA is expressed in all examined tissue types as measured by qPCR. Ucp3 mRNA is found together with Coup-TFII mRNA in BAT and SKM. Both display highest variability in SKM (SKM/BAT n = 9, other tissues n = 3). Black dots represent individual results, bars are mean values. WAT = white adipose tissue, SKM = skeletal muscle, BAT = brown adipose tissue.

Figure 3

Response of Coup-TFII and Ucp3 mRNA expression to physiological challenges inducing lipid utilization. (A) Expression of Coup-TFII and Ucp3 mRNA in BAT of cold exposed and SKM of food deprived hamsters as compared to control conditions. Shown are mean values with standard error, * = p < 0,05. (B) Relationship of Ucp3 and Coup-TFII mRNA levels in SKM and BAT of hamsters housed under standard conditions with ad libitum access to food; r² = 0.211, p = not significant (SKM n = 9, BAT n = 9). (C)Correlation of Ucp3 and Coup-TFII mRNA abundancies in SKM of food deprived and BAT of cold exposed animals; r² = 0.834, p < 0.001 (SKM n = 9, BAT n = 9).

Figure 4

Coup-TFII transactivates the Ucp3 promoter in cotransfection reporter gene assays. (A) Activity of reporter gene construct -2244UCP3luc in cotransfection experiments with various transcription factors (fold change relative to basal -2244UCP3luc level). Coup-TFII coactivates the Ucp3 promoter in all combinations. It is especially effective together with MyoD, RXRα and p300. Coup-TFI does not activate reporter gene expression demonstrating a specific effect of Coup-TFII. (B) Identification of the region mediating Coup-TFII transctivation by analysis of deletion reporter gene constructs. Shown in the upper panel is the basal activity and induction by Coup-TFII of all constructs, in the lower panel the experiment was repeated on a background of MyoD, RXRα and p300 (note the different axis scaling). The shaded area highlights the region that is exclusively found on -2244UCP3luc and is responsible for the Coup-TFII effect in both setups.

Figure 5

Coup-TFII binds to a conserved element of the Ucp3 promoter. (A) Electrophoretic mobility shift assay with candidate probes A-E and nuclear extracts of HEK293 cells. Overexpression of Coup-TFII leads to formation of a specific complex on probeC only, that can be supershifted with a Coup-TFII antibody. All complexes are subject to competition with 100 fold molar excess of unlabelled probe C. (B) Western Blot of protein fractions of HEK293 cells with a Coup-TFII-antibody. Coup-TFII is specifically detected in the nuclear fraction and absent in untransfected cells. M = Marker, - = mock transfected, CI = Coup-TFI transfected, CII = Coup-TFII transfected. (C) Alignment of the human and rodent promoter regions of the Ucp3 gene (Ps: -821 to -857). The positions of identical bases are marked (*). The putatively Coup-TFII binding elements (box, dashed box) located on probe C are conserved in all rodent species. In the murine promoter they are found in reverse order slightly more upstream. The human promoter does not feature comparable sequences in the compared region. Rn = R. norvegicus, Mm = M. musculus, Ps = P. sungorus, Hs = H. sapiens (D) Electrophoretic mobility shift assay with skeletal muscle nuclear extracts from control and food deprived hamsters. The complex shown is of a size comparable to the Coup-TFII complex above. Starvation induces complex formation in comparison to control conditions.

Figure 6

Disruption of the 5' half-site leads to loss of Coup-TFII binding. (A) Electrophoretic mobility shift assay with element C and mutated derivatives (compare (D)). Bands were quantified, background corrected and analyzed. Probe = labelled oligonucleotide used; comp. = unlabelled competitor oligonucleotide used; CII = nuclear extract of Coup-TFII transfected HEK293 cells; mock = nuclear extract of mock transfected HEK293 cells. (B) Quantification of complex intensity on labelled probes Cwt, Cmut5', Cmut3' and Cmut5'3'. Probe Cmut3' retained a lowered ability to bind Coup-TFII while Cmut5'and Cmut5'3'were unable to do so. (C) Accordingly unlabelled competitor probe Cmut3' was still able to partially compete for Coup-TFII binding to the labelled wt probe, while Cmut5' and Cmut5'3' were not. (D) Overview of the probes used in electrophoretic mobility shift assays. Probe Cwt represents element C, the mutated derivatives are depicted below.

Figure 7

Disruption of the 5' half-site leads to complete loss of activity. Reporter gene assays with mutated luciferase constructs. In the upper panel basal luciferase activity and induction by Coup-TFII is shown, in the lower panel the experiment was repeated on a background of MyoD, RXRα and p300. Disruption of the 3' half site on -2244Cmut3'luc did not lower activity in any condition tested. Disrupting the 5' half-site on -2244Cmut5'luc led to a complete loss of basal activity and responsiveness to Coup-TFII and MyoD, RXRα and p300.