ChIP-seq Data from C2C12 cells demonstrate MyoD binding to the region deleted in Δ4b.

<p>(A) Publicly available ChIP-seq data for MyoD and Myogenin in C2C12 cells were mapped to the first intron of UCP3. Interestingly, only one of these experiments demonstrates MyoD binding to the promoter of UCP3. Furthermore, ChIP-seq data for the co-activator p300 and RNA polymerase 2 were mapped. Screenshot taken from the ENCODE browser. ChIP-seq data for BAT was not available. (B) Alignment of the three intronic binding elements in hamster (Psu), rat (Rno) and mouse (Mmu). Intronic sequences were obtained from ENSEMBL (<a href="http://www.ensembl.org" target="_blank">www.ensembl.org</a>). Putative binding elements are marked by boxes. The fourth row of sequence resembles the <i>Phodopus</i> reporter gene construct carrying the deletion Δ4b. Shown are 25 bp of 36 bp deleted in Δ4b, but not in Δ4a. Numbers in brackets denote bases left out for the sake of clarity.</p

A Novel SP1/SP3 Dependent Intronic Enhancer Governing Transcription of the UCP3 Gene in Brown Adipocytes

<div><p>Uncoupling protein (UCP) 3 is a mitochondrial inner membrane protein implicated in lipid handling and metabolism of reactive oxygen species. Its transcription is mainly regulated by peroxisome proliferator-activated receptors (PPAR), a family of nuclear hormone receptors. Employing bandshift assays, RNA interference and reporter gene assays we examine an intronic region in the UCP3 gene harboring a <i>cis</i>-element essential for expression in brown adipocytes. We demonstrate binding of SP1 and SP3 to this element which is adjacent to a direct repeat 1 element mediating activation of UCP3 expression by PPARγ agonists. Transactivation mediated by these elements is interdependent and indispensable for UCP3 expression. Systematic deletion uncovered a third binding element, a putative NF1 site, in close proximity to the SP1/3 and PPARγ binding elements. Data mining demonstrated binding of MyoD and Myogenin to this third element in C2C12 cells, and, furthermore, revealed recruitment of p300. Taken together, this intronic region is the main enhancer driving UCP3 expression with SP1/3 and PPARγ as the core factors required for expression.</p></div

The IVS1+1505G element binds SP1 and SP3 in EMSA.

<p>EMSA bands were obtained incubating either the Cy5 labeled probes IVS1+1505G (A, lane 1) or SP1/SP3 consensus (B, lane 1) with nuclear extracts from HIB1b cells followed by native PAGE. Non-labeled competitors IVS1+1505G, IVS1+1504A and SP1/3 consensus were added to the binding reaction along with labeled probe where indicated. Different spacing between the complexes and a non-SP complex formed with the IVS1+1505G probe (arrows in (A), competition with SP1/3 consensus) hint to different complex compositions. (C) Supershift experiments by addition of antibodies against SP1, SP3, PPARγ and RXRα to test the identity of the proteins binding to the IVS1+1505G element. A representative experiment of 3 independent repetitions is shown in C.</p

A downstream SP element is a common feature of many mammalian UCP3 genes.

<p>The UCP3 genes of mouse (<i>Mus</i>), rat (<i>Rattus</i>), pig (<i>Sus</i>) and human (<i>Homo</i>) were analyzed for SP/DR modules downstream of their promoter using the Genomatix software package. For all species one (mouse, rat, human) or two (pig, E1&E2) modules were predicted roughly 1500 bp downstream of the transcriptional start site. Oligonucleotides resembling the predicted SP site were annealed and used as cold competitor in EMSA against a Cy5 labeled <i>Phodopus</i> IVS1+1505G probe. As a negative control, IVS1+1504A, a probe lacking a crucial C of the GC-Box, does not compete at all. Shown is one representative EMSA out of at least 4 independent experiments.</p

PPAR agonist-mediated UCP3 expression depends on combined presence of the intronic SPx/DR1 double element.

<p>In the IVS1+1505G and A reporter gene constructs either one or both of the two putative DR1 elements were mutated. The 8 constructs were transfected into HIB1b cells and exposed to Wy14643 and Rosiglitazone (in combination, 10 µM each) or DMSO in differentiation medium for 24 hours. Black boxes represent the first 2 exons of UCP3. Crossed circles represent mutation of the respective elements indicated above. Circles with “G” or “A” indicate the allele at the IVS1+1505 position in intron 1. GLuc: Gaussia Luciferase. N = 3–4 for Wy/Rosi and N = 2–3 for DMSO. Bars represent mean ± s.d. # marks constructs that respond to PPAR agonist stimulation compared to vehicle. (two way ANOVA for Vector and Agonist, Holm-Sidak Method).</p

Mithramycin suppresses PPAR agonist mediated activation of the IVS1+1505G reporter gene construct.

<p>HIB1b cells were transiently transfected with the reporter gene vectors IVS1+1505G, IVS1+1505A or a 3xPPRE consensus element and subsequently stimulated by the PPAR agonists Wy14643 and Rosiglitazone (in combination, 10 µM each) or DMSO for 24 hours in presence or absence of different concentrations of Mithramycin. Mithramycin concentrations used were 25 ng/ml, 100 ng/ml and 400 ng/ml or no Mithramycin (DMSO/vehicle). Bars represent mean ± s.d. (one way ANOVA for Mithramycin concentration, Holm-Sidak method, Log transformed data).</p

Targeting SP1 and/or SP3 via RNAi decreases reportergene activity of the IVS1+1505G reporter gene construct.

<p>miRNA-expressing HIB1b cells were transiently transfected with IVS1+1505G or A, induced and differentiated. During the last 24 hours of differentiation cells were stimulated by a combination of Wy14643 (Wy, 10 µM) and rosiglitazone (rosi, 10 µM). (A) Each cell line expresses two miRNAs targeting either twice SP1, twice SP3, each SP1&SP3 once, UCP1 (Ctrl. U, no transcript detectable in HIB1b cells) or LacZ/shBle (Ctrl. Z, two bacterial genes) The experiment was repeated 8 and 7 times for IVS1+1505G and IVS1+1505A, respectively, each time in triplicates using cells from 2 independent rounds of infection and selection. scram: scrambled shRNA sequence C) Replication of the miRNA experiment using transient transfection of shRNAs with independent sequences. The experiment was carried out 3 times in duplicates. Bars represent mean ± s.d. Stars denote a significant difference from both control vectors for the respective agonist (one way ANOVA for miRNA, Holm-Sidak method, Log transformed data).</p

Stepwise deletion of the first intron reveals additional regulatory elements.

<p>Using PCR-mediated deletion, several 300–400 bp deletion covering most of the first intron in the IVS1+1505G (“G”) reporter gene construct were generated. All constructs were transfected into HIB1b brown adipocytes and exposed to a combination of Wy14643 (Wy, 10 µM) and rosiglitazone (rosi, 10 µM) or vehicle. Black boxes represent the first 2 exons of UCP3. Crossed circles represent mutation of the elements indicated above. GLuc: Gaussia Luciferase. n = 4 to 5 for Wy/Rosi and n = 3 for DMSO. Bars represent mean values ± s.d. Stars denote a significant difference from the IVS1+1505G vector in the presence or absence of agonists, respectively (two way ANOVA for construct and agonist, Holm-Sidak Method).</p

Regulation of UCP3 expression: Refined model.

<p>SP1 and SP3 bind to the intronic GC-box and recruit, in presence of the respective agonists, PPARγ and RXRα to the intronic DR1 element. This complex then recruits p300 to open the chromatin and enables initiation of transcription. Factors binding to the nearby NF1 site, (at least in muscle: MyoD and MyoG) join the complex and further increase the activating potency. The three intronic elements then, in cooperation with promoter elements and an upstream regulatory inverted repeat, regulate the expression of UCP3.</p

S4 Table -

Results of Levene’s test of equality of variance of the reported assay-specific Ct/Cq values per MPX positive sample for A) MPXV detection and B) OPXV detection. When equality of variance was not found (labeled in red colour), one collective (marked with *) was identified and excluded before rerun the test to be able to show the equality of the variances for the other collectives (labeled in green colour). (XLSX)</p