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Targeting SP1 and/or SP3 via RNAi decreases reportergene activity of the IVS1+1505G reporter gene construct. 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 mM) and rosiglitazone (rosi, 10 mM). (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 6 s.d. Stars denote a significant difference from both control vectors for the respective agonist (one way ANOVA for miRNA, HolmSidak method, Log transformed data). doi:10.1371/journal.pone.0083426.g003
Source publication
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 exam...
Contexts in source publication
Context 1
... of either SP1 or SP3 led to 40% (SP1 vs Ctrl U: p,.01; SP1 vs. Ctrl Z: p,.001) and 47% (SP3 vs Ctrl U: p,.01; SP3 vs. Ctrl Z: p,.001 vs Ctrl U/Z) reduction in IVS1+1505G construct activity, respectively (Fig. 3A). Knockdown of both SP1 and SP3 reduced activity by 61% (SP1+SP3 vs Ctrl U: p,.001; SP1+SP3 vs Ctrl Z p,.001). All three knockdown conditions were significantly different from either control after adjusting for multiple testing. Conversely, even the double knockdown did not have a statistically significant effect on the mutant ...
Context 2
... vectors were delivered by the Nucleofection transfection method. The results reproduce the effects described above and are shown in Figure 3C. ...
Context 3
... effect was independent of the presence of the promoter DR1 element. Repetition of the experiment in immortalized primary brown preadipocytes replicates these findings ( Figure S3). Strikingly, none of the two DR1 elements can confer PPAR ligand dependent activation without the presence of the intronic SP1/3 element (''G''). ...
Context 4
... is a representative experiment of more than 4 independent blots. (TIF) Figure S3 Interdependence of SP1/3 binding and PPARc agonist activity in SV40-LTA immortalized primary brown preadipocytes. Five of the reporter constructs used for the experiments shown in Figure 5 were transfected into immortalized preadipocytes and stimulated for 24 h with Wy14643 and Rosiglitazone. ...
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Background
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Methods
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Background:
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Methods:
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Citations
... sp3a encodes a transcription factor belonging to Sp1 related family genes which can have bi-functional roles in stimulating or repressing the transcription of numerous target genes (Majello et al. 1997). In humans, it has been shown that Sp3 (encoded by an mammalian orthologue of sp3a) and Sp1 can have synergistic or opposite regulatory effects on transcription, while binding to the same regulatory element upstream of genes playing a role in lipid metabolism and the pathogenesis of obesity in adipose tissues (Barth et al. 2003;Hoffmann et al. 2013). Interestingly, it has already been demonstrated in mammals that leptin signal can enhance the regulatory effects of Sp1 and Sp3 on the transcription of their target genes (Lin et al. 2006;García-Ruiz et al. 2012). ...
The signal mediated by leptin hormone and its receptor is a major regulator of body weight, food intake and metabolism. In mammals and many teleost fish species, leptin has an anorexigenic role and inhibits food intake by influencing the appetite centres in the hypothalamus. However, the regulatory connections between leptin and downstream genes mediating its appetite-regulating effects are still not fully explored in teleost fish. In this study, we used a loss of function leptin receptor zebrafish mutant and real-time quantitative PCR to assess brain expression patterns of several previously identified anorexigenic genes downstream of leptin signal under different feeding conditions (normal feeding, 7-day fasting, 2 and 6-h refeeding). These downstream factors include members of cart genes, crhb and gnrh2 , as well as selected genes co-expressed with them based on a zebrafish co-expression database. Here, we found a potential gene expression network (GRN) comprising the abovementioned genes by a stepwise approach of identifying co-expression modules and predicting their upstream regulators. Among the transcription factors (TFs) predicted as potential upstream regulators of this GRN, we found expression pattern of sp3a to be correlated with transcriptional changes of the downstream gene network. Interestingly, the expression and transcriptional activity of Sp3 orthologous gene in mammals have already been implicated to be under the influence of leptin signal. These findings suggest a potentially conserved regulatory connection between leptin and sp3a , which is predicted to act as a transcriptional driver of a downstream gene network in the zebrafish brain.
... Nevertheless, there are numerous studies demonstrating instances in which transcription factor binding to intron regions can regulate the expression of a gene. A previous study reported that SP1/SP3 transcription factors enhance the expression of the UCP3 gene because they bind to an intron of the UCP3 gene 46 . Another report shows that GATA-2, a transcription factor that has important roles in various organs during embryogenesis, is regulated by Gata2 endothelium enhancer in the fourth intron region, and Gata2 modulation by this enhancer is restricted to the endocardial, lymphatic, and vascular endothelium 47 . ...
Nurr1, a transcription factor belonging to the orphan nuclear receptor, has an essential role in the generation and maintenance of dopaminergic neurons and is important in the pathogenesis of Parkinson’ disease (PD). In addition, Nurr1 has a non-neuronal function, and it is especially well known that Nurr1 has an anti-inflammatory function in the Parkinson’s disease model. However, the molecular mechanisms of Nurr1 have not been elucidated. In this study, we describe a novel mechanism of Nurr1 function. To provide new insights into the molecular mechanisms of Nurr1 in the inflammatory response, we performed Chromatin immunoprecipitation sequencing (ChIP-Seq) on LPS-induced inflammation in BV2 cells and finally identified the RasGRP1 gene as a novel target of Nurr1. Here, we show that Nurr1 directly binds to the RasGRP1 intron to regulate its expression. Moreover, we also identified that RasGRP1 regulates the Ras-Raf-MEK-ERK signaling cascade in LPS-induced inflammation signaling. Finally, we conclude that RasGRP1 is a novel regulator of Nurr1’s mediated inflammation signaling.
... The existence of some alteration in the promoter region makes biological sense which confirms regulatory sequences should be variable. Since gene promoters are critical role players in gene regulation, so they ordinarily receive signals from different sources to down and up-regulate the level of transcription, which mainly determines gene expression (Rey et al., 2010;Hoffmann et al., 2013;Murata et al., 2013). For the reason, that transcription start sites and surrounding regulatory elements normally are often in the upstream region of gene sequence. ...
... The output of prediction represents that -40 T/A located in the probable consensus binding motifs in the promoter region and might combine with MyoD which qualified in this study. (Bailey et al., 1998;Solanes et al., 2000;Hoffmann et al., 2013). If this hypothesis becomes true so the significant changes in the mentioned traits will become more justified. ...
... If this hypothesis becomes true so the significant changes in the mentioned traits will become more justified. According to text mining further transcription factors have been found related to UCP3 such as Coup-TFII, MyoD, Sp1, PPAR-alpha, and the others (Lu and Sack, 2008;Hoffmann et al., 2013). As biology is mysterious and complicated we guess other regulators have been located out of our eyes. ...
The Avian Uncoupling Protein (avUCP) belongs to the mitochondrial anion transporter family. It has a pivotal homeostatic mechanism that associated with energy regulation and lipid metabolism. The avUCP considered as a candidate gene for chicken growth-related traits according to its predominant expression is in skeletal muscle. To address the genetic distance pattern of UCP3 between mammalian and avian species, sequence similarity analysis using the protein alignment of UCP3 identified the high amino acid identity between the species and complementarily detected two protein conserved regions which are known as the ADP/ATP transporter translocase and the Mitochondria-carrier. Likewise, for mutation detection, samples were genotyped, afterward, PCR-SSCP method implemented. In addition, association analysis was performed for investigating single nucleotide polymorphism within the UCP3 gene relating to the given economic traits. A detected polymorphic site, on the promoter region of UCP3 (-40 T/A substitution), has displayed significant influences on the Feed Conversion Rate (FCR), Residual Feed
Intake (RFI), Average Daily Gain (ADG), and Carcass Weight (CW%). In the case that, birds with genotype AA had better FCR, ADG, RFI as compared to the genotype BB and birds with genotype AA revealed a higher CW% as compared to the genotype BB. According to the obtained results from the in-silico survey, Myoblast determination protein (MyoD) was predicted as a best-matched transcription factor with a consensus sequence harboring the -40 T/A -novel SNP- in the promoter region of UCP3, where might be responsible for phenotypic variation between two genotypes. In conclusion, the result suggests important roles for UCP3 polymorphism in feed efficiency and growth traits which is better to be used in broiler chicken breeding programs.
... Nevertheless, PPARG binding differences were not really related to SNPs in Ucp1 PPARG binding sites. Therefore, the PPARG binding occupancy that varies between strains must be caused by the proximal presence of other TFs that bind to the Ucp1 locus, consistent with the notion that PPARG binding depends on local chromatin accessibility as mediated by other collaborating TFs (Grossman et al., 2017;Hoffmann et al., 2013;Simicevic et al., 2013). In an attempt to explore the SNPs at the Ucp1 locus that likely affect TF binding activity, we found a few TFs with altered predicted binding motif affinity. ...
Recruitment of brite/beige cells, known as browning of white adipose tissue (WAT), is an efficient way to turn an energy-storing organ into an energy-dissipating one and may therefore be of therapeutic value in combating obesity. However, a comprehensive understanding of the regulatory mechanisms mediating WAT browning is still lacking. Here, we exploit the large natural variation in WAT browning propensity between inbred mouse strains to gain an inclusive view of the core regulatory network coordinating this cellular process. Combining comparative transcriptomics, perturbation-based validations, and gene network analyses, we present a comprehensive gene regulatory network of inguinal WAT browning, revealing up to four distinct regulatory modules with key roles for uncovered transcriptional factors, while also providing deep insights into the genetic architecture of brite adipogenesis. The presented findings therefore greatly increase our understanding of the molecular drivers mediating the intriguing cellular heterogeneity and plasticity of adipose tissue. : Browning of white fat, which turns an energy-storing organ into an energy-dissipating one, holds promise for the development of novel therapeutics against obesity. By exploiting intrinsic mouse strain variation and applying a systems-genetics approach, Li et al. reveal a comprehensive gene regulatory network that controls this browning process. Keywords: UCP1, browning, thermogenesis, inbred mouse strains, molecular network, brite/beige adipogenesis, adipose tissue, obesity, transcription factor
... The SGBS human preadipocytes cell strain, primary human preadipocytes, Huh7 human hepatoma, C2C12 mouse myoblast, INS-l rat insulinoma, 293T human embryonic kidney and the HIB1B mouse brown adipocyte cell line were cultured, induced and differentiated as previously described (6,25). Ethical committee approval for primary human preadipocyte cells was obtained from the Faculty of Medicine of the Technical University of Munich, Germany. ...
... Protein concentration was quantitated using Bradford assay. HIB 1B nuclear extracts were prepared as described (25); in brief, cells were washed with PBS, lysed in pre-chilled homogenization buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 20 mM NaF, 0.5 mM DTT). Nuclei were collected by centrifugation, resuspended in low salt buffer (20 mM HEPES pH 7.9, 1.5 mM MgCl 2 , 20 mM KCl, 0.2 mM EDTA, 20 mM NaF, 25% v/v glycerol, 0.5 mM DTT), lysed by adding high salt buffer containing 1.2 M KCl followed by vigorous shaking, centrifuged, and the nuclear extract supernatant was recovered for further analysis. ...
Genome-wide association studies identified numerous disease risk loci. Delineating molecular mechanisms influenced by cis-regulatory variants is essential to understand gene regulation and ultimately disease pathophysiology. Combining bioinformatics and public domain chromatin information with quantitative proteomics supports prediction of cis-regulatory variants and enabled identification of allele-dependent binding of both, transcription factors and coregulators at the type 2 diabetes associated PPARG locus. We found rs7647481A nonrisk allele binding of Yin Yang 1 (YY1), confirmed by allele-specific chromatin immunoprecipitation in primary adipocytes. Quantitative proteomics also found the coregulator RING1 and YY1 binding protein (RYBP) whose mRNA levels correlate with improved insulin sensitivity in primary adipose cells carrying the rs7647481A nonrisk allele. Our findings support a concept with diverse cis-regulatory variants contributing to disease pathophysiology at one locus. Proteome-wide identification of both, transcription factors and coregulators, can profoundly improve understanding of mechanisms underlying genetic associations.
... The VDR also can interact with nuclear receptor, such as PPARs, thyroid hormone receptors, corepressors, coactivators, and many transcriptional factors [30,46]. On the contrary, UCP3 gene promoter harbors several cis-elements essential for expression [47][48][49]. The relative contribution of those in the regulatory regions to UCP3 gene expression remains to be clarified. ...
Background:
The impact of vitamin D3 (VD3) on obesity has been reported in the past. Our study was aimed at investigating the possible mechanisms by which VD3 affects obesity induced by a high fat diet.
Methods:
Eight-week-old C57BL/6 J male mice were fed a normal- or high-fat diet for 9 weeks and were treated with a gavage of vehicle (corn oil) or cholecalciferol (50 μg/kg, daily). Body weight, white adipose tissue weight, blood lipid and glucose levels were measured. In addition, we investigated the expression of 1,25(OH)2D3 (calcitriol)/VDR-regulated genes involved in energy and lipid metabolism, such as of uncoupling protein 3 (UCP3), by using qRT-PCR in the liver, adipose tissue, skeletal muscle and C2C12, L6, and H-EMC-SS cells. We also measured UCP3 promoter transcription in the same cell lines using a Dual Luciferase Assay. Furthermore, we analyzed the binding site consensus sequences of VDR on the UCP3 promoter.
Results:
Mice consuming a high-fat diet treated with cholecalciferol had lower body weight and adipose tissue weight and higher expression of UCP3 compared to the other treatment groups. Changes in the expression of genes correlated with calcitriol/VDR. Luciferase activity was dose-dependently associated with calcitriol/VDR levels. We confirmed the functional VDR binding site consensus sequences at -2200, -1561, -634, and +314 bp in the UCP3 promoter region.
Conclusion:
We suggest that VD3/VDR inhibits weight gain by activating UCP3 in the muscles.
... Sp3 regulates the expression of a number of genes; consistent with this, our data demonstrated that TFF2 binds to Sp3 in the cytoplasm, resulting in the increased expression of genes involved in the suppression of cell proliferation and the induction of apoptosis (23). The Sp3 protein exists in multiple isoforms: two long fragments and two short ones (24)(25)(26). Previous research on Sp3 has focused on its DNA-binding sites and gene regulation (27). ...
The trefoil factor family (TFF) is a group of short secretory peptides of gastric mucous neck cells. The loss of TFF2 protein expression enhances gastric inflammation and occurs in gastric cancer. In this study, we examined the effect of TFF2 on gastric cancer cell lines in vitro and characterized the interaction between TFF2 and Sp3, including the mechanisms that mediate this interaction, using genomics and proteomics approaches, as well as genetics techniques, such as RNA interference and gene knockdown. Assays were performed to examine the role of TFF2 and Sp3 in cancer cell proliferation, invasion and migration. We found that TFF2 expression inhibited the proliferation and invasion capacity of gastric cancer cells, and induced apoptosis. TFF2 interacted with the Sp3 protein, as shown by immunofluorescence staining and immunoprecipitation with western blot analysis. Sp3 knockdown in gastric cancer cells antagonized TFF2 anti-tumor activity. Additionally, TFF2 upregulated the expression of pro-apoptotic proteins, such as Bid, but downregulated the expression of NF-κB and the anti-apoptotic proteins, Bcl-xL and Mcl-1. By contrast, Sp3 knockdown significantly blocked TFF2 activity, affecting the expression of these proteins. The data from our study demonstrate that the antitumor activity of TFF2 is mediated by an interaction with the Sp3 protein in gastric cancer cells. Additional in vivo and ex vivo warrned in order to fully characterize this interaction.
... In summary, the results indicated that this difference was most likely conferred by different ways in which UCP3 promoter regions respond to the Myf5, Myf6 and MyoD transcription factors, and the promoter-binding TF profiling assay shows that these transcription factors bind directly to the UCP3 promoter. We only focus the UCP3 promoter in this study, so we can not exclude the possibility that the intoronic region (e.g., intron 1) is associated with the regulation of UCP3 gene [36,37]. ...
Uncoupling protein 3 (UCP3) is mainly expressed in muscle. It plays an important role in muscle, but less research on the regulation of cattle UCP3 has been performed. In order to elucidate whether cattle UCP3 can be regulated by muscle-related factors, deletion of cattle UCP3 promoter was amplified and cloned into pGL3-basic, pGL3-promoter and PEGFP-N3 vector, respectively, then transfected into C2C12 myoblasts cells and UCP3 promoter activity was measured using the dual-Luciferase reporter assay system. The results showed that there is some negative-regulatory element from -620 to -433 bp, and there is some positive-regulatory element between -433 and -385 bp. The fragment (1.08 kb) of UCP3 promoter was cotransfected with muscle-related transcription factor myogenic regulatory factors (MRFs) and myocyte-specific enhancer factor 2A (MEF2A). We found that UCP3 promoter could be upregulated by Myf5, Myf6 and MyoD and downregulated by MyoG and MEF2A.
... However, L-165,041 successfully stimulated firefly luciferase expression from EP1. These observations are in line with previous findings showing that a UCP3 enhancer contained in the first intron regulates the activity of the core promoter (44). The positive effect of the PPAR/␦ agonist was significantly attenuated when co-incubated with insulin (Fig. 6C). ...
... On the contrary, PPAR␥ binds to a remote downstream element located in the first intron of the murine Ucp3 locus (56). This remote region was recently found to also contain MyoG/MyoD and SP1/SP3 binding elements (44). The investigators have proposed that this region forms an enhancer complex where SP1/SP3 acts as a gatekeeper by recruiting other components of the complex and/or by mediating interaction with the proximal promoter and the transcriptional start site. ...
The risk for heart failure and death after myocardial infarction is abnormally high in diabetic subjects. We and others have previously shown that the mitochondrial uncoupling protein (UCP)3 improves functional recovery of the rodent heart during reperfusion. Here, we demonstrate that pharmacological induction of hyperinsulinemia in mice downregulates myocardial UCP3. Decreased UCP3 expression was linked to the development of selective insulin resistance in the heart, characterized by decreased basal activity of Akt but preserved activity of the p44/42 mitogen-activated protein kinase, and over-activation of the sterol regulatory element-binding protein (SREBP)-1-mediated lipogenic program. In cultured myocytes, insulin treatment and SREBP-1 overexpression decreased, while SREBP-1 interference increased, peroxisome proliferator-activated receptor-stimulated expression of UCP3. Promoter deletion and site-directed mutagenesis identified three functional sterol regulatory elements in the vicinity of a known complex intronic enhancer. Increased binding of SREBP-1 to this DNA region was confirmed in the heart of hyperinsulinemic mice. In conclusion, we describe a hitherto unknown regulatory mechanism by which insulin inhibits cardiac UCP3 expression through activation of the lipogenic factor SREBP-1. Sustained downregulation of cardiac UCP3 by hyperinsulinemia may partly explain the poor prognosis of type 2 diabetic patients post myocardial infarction.
... The various expression of UCP3 in BAT and muscles may be explained by the recently described differential gene regulation in both tissues (40). In BAT, the UCP3 transcription is stimulated by an additional intronic enhancer with SP1/3 and PPARγ as core factors for ucp3 gene expression. ...
UCP1 and UCP3 are members of the uncoupling protein (UCP) subfamily and are localized in the inner mitochondrial membrane. Whereas UCP1's central role in non-shivering thermogenesis is acknowledged, the function and even tissue expression pattern of UCP3 are still under dispute. Because UCP3 properties regarding transport of protons are qualitatively identical to those of UCP1, its expression in brown adipose tissue (BAT) alongside UCP1 requires justification. In this work, we tested whether any correlation exists between the expression of UCP1 and UCP3 in BAT by quantification of protein amounts in mouse tissues at physiological conditions, in cold-acclimated and UCP1 knockout mice. Quantification using recombinant UCP3 revealed that the UCP3 amount in BAT (0.51ng/(μg total tissue protein)) was nearly one order of magnitude higher than that in muscles and heart. Cold-acclimated mice showed an approximate three-fold increase in UCP3 abundance in BAT in comparison to mice in thermoneutral conditions. Surprisingly, we found a significant decrease of UCP3 in BAT of UCP1 knockout mice, whereas the protein amount in skeletal and heart muscles remained constant. UCP3 abundance decreased even more in cold-acclimated UCP1 knockout mice. Protein quantification in UCP3 knockout mice revealed no compensatory increase in UCP1 or UCP2 expression. Our results do not support the participation of UCP3 in thermogenesis in the absence of UCP1 in BAT, but clearly demonstrate the correlation in abundance between both proteins. The latter is important for understanding UCP3's function in BAT.
