A novel intronic peroxisome proliferator-activated receptor gamma enhancer in the uncoupling protein (UCP) 3 gene as a regulator of both UCP2 and -3 expression in adipocytes

Abstract

Uncoupling Proteins (UCPs) are integral ion channels residing in the inner mitochondrial membrane. UCP2 is ubiquitously expressed, while UCP3 is found primarily in muscles and adipose tissue. Although the exact molecular mechanism of action is controversial, it is generally agreed that both homologues function to facilitate mitochondrial fatty acid oxidation. UCP2 and -3 expression is activated by the peroxisome proliferator-activated receptors (PPARs), but so far no PPAR response element has been reported in the vicinity of the Ucp2 and Ucp3 genes. Using genome-wide profiling of PPARgamma occupancy in 3T3-L1 adipocytes we demonstrate that PPARgamma associates with three chromosomal regions in the vicinity of the Ucp3 locus and weakly with a site in intron 1 of the Ucp2 gene. These sites are isolated from the nearest neighboring sites by >900 kb. The most prominent PPARgamma binding site in the Ucp2 and Ucp3 loci is located in intron 1 of the Ucp3 gene and is the only site that facilitates PPARgamma transactivation of a heterologous promoter. This site furthermore transactivates the endogenous Ucp3 promoter, and using chromatin conformation capture we show that it loops out to specifically interact with the Ucp2 promoter and intron 1. Our data indicate that PPARgamma transactivation of both UCP2 and -3 is mediated through this novel enhancer in Ucp3 intron 1.

FIGURE 1.

UCP2 and -3 are target genes of PPARγ in 3T3-L1 cells. A, mRNA expression of PPARγ, UCP3, and UCP2 during 3T3-L1 adipogenesis. Levels of mRNA was determined by real-time PCR and normalized to the corresponding TFIIB levels. The experiment was performed in duplicate, and the range is indicated. This experiment is representative of three individual experiments. B, ectopic expression of PPARγ2 in 3T3-L1 preadipocytes induces UCP2 and -3 mRNA expression. 3T3-L1- CAR preadipocytes were transduced with adenovirus expressing PPARγ2 (AdHA-PPARγ2) in the presence of 1 μm PPARγ agonist rosiglitazone, or with control adenovirus containing empty vector (AdEmpty) in the presence of vehicle (DMSO). Total RNA was harvested 8 h after transduction, and the mRNA expression of PPARγ, UCP3, and UCP2 was determined by real-time PCR and normalized to the corresponding TFIIB levels. The experiment was performed in triplicate, and the range is indicated.

FIGURE 2.

PPARγ/RXR ChIP-seq profile of the Ucp2 and Ucp3 loci. Screen shots from the genome-wide ChIP-seq profile of PPARγ/RXR binding sites in 3T3-L1 cells by Nielsen et al. (40) on days 0 and 6 of differentiation. A, close-up of the murine Ucp2 and -3 loci with a schematic representation indicating the positions of the PPARγ/RXR binding sites and DR1 elements relative to the transcription start sites shown below. B, overview of the PPARγ and RXR binding profiles on >2.3 Mb of chromosome 7 encompassing the Ucp2 and -3 genes. The genomic distances to the neighboring PPARγ/RXR binding sites relative to the sites in the Ucp3 and -2 locus are indicated.

FIGURE 3.

PPARγ and RXR binding sites are found in proximity and within the Ucp2 and -3 genes. PPARγ and RXR ChIP-PCR to confirm binding sites identified by ChIP-seq. Chromatin was harvested from cross-linked 3T3-L1 cells on days 0 and 6 of differentiation. ChIP was performed using antibodies against PPARγ or RXR. Occupancy (-fold enrichment above myoglobin promoter levels) at the binding sites found in Fig. 2 and at intermittent control regions was determined by real-time PCR. This experiment is representative of three independent experiments.

FIGURE 4.

Only the PPARγ/RXR binding site in intron 1 of the Ucp3 gene mediates PPARγ transactivation of a heterologous promoter. Approximately 500 bp surrounding the PPARγ/RXR binding sites in Ucp2 and -3 were cloned in front of a SV40 promoter in the pGL3-Promoter luciferase reporter vector and transfected into HEK293T cells together with increasing amounts of PPARγ2 expression vector in the presence of 1 μm rosiglitazone or the vehicle DMSO as indicated. Luciferase levels were normalized to the expression from a β-galactosidase control vector. Results are representative of three independent experiments, each performed in triplicate. Standard deviations are indicated.

FIGURE 5.

The +1950 PPARγ/RXR binding site in the Ucp3 gene enables PPARγ-transactivation of the endogenous Ucp3 promoter. A, the Ucp3 proximal promoter (∼450 bp) and the +1950 PPARγ/RXR binding site were cloned into the promoterless pGL3-Basic vector upstream and downstream of the luciferase gene, respectively. The DR1 element in the +1950 PPARγ/RXR binding site was subsequently mutated by the introduction of an ApaI site. B, transfections were performed as in Fig. 4. Results are representative of three independent experiments each performed in triplicates. Standard deviations are indicated.

FIGURE 6.

The PPARγ binding site in Ucp3 intron 1 loops out to specifically interact with the Ucp2 promoter and 5′-region in adipocytes. A, schematic representation of the mouse Ucp3 and -2 loci showing the position of the +1950 and +400 PPARγ/RXR bindings sites in the Ucp3 and Ucp2 intron 1, respectively (solid vertical lines). Sites recognized by the restriction enzyme Csp6I used in the 3C assay are indicated by punctuated lines, and arrows mark the positions of the primers used in the 3C analyses. B, agarose gel picture showing the 3C PCR amplicons generated with the BF-IF primer combination that detects looping between the bait (restriction fragment covering the +1950 PPARγ/RXR binding site in intron 1 of the Ucp3 gene) and the interactor (the restriction fragment covering the Ucp2 promoter and 5′-region, including the +400 PPARγ/RXR bindings site). C, agarose gel picture showing the 3C PCR amplicons generated with the indicated primer combinations (see A) designed to detect looping between the bait and the genomic regions immediately up- or downstream of the interactor fragment. Lanes are as follows: marker (M), no template control (NTC), Csp6I-digested 3T3-L1 genomic DNA (GD), standard curve of control template (2–16 fg), 3T3-L1 adipocytes day 6 (d6), and non-cross-linked ligated control (NCLC).

FIGURE 7.

Model for PPARγ transactivation of the UCP3 and -2 genes through intrachromosomal looping. Our data show that the +1950 PPRE in intron 1 of the Ucp3 genes is the main PPARγ (PPγ) binding site in the Ucp3 and -2 loci in murine adipocytes and indicate a model for transactivation of both loci by PPARγ/RXR binding to this PPRE. According to this model PPARγ/RXR bind to the PPRE in intron 1 of the Ucp3 gene and loop out to simultaneously interact indirectly with intron 1 of the Ucp2 gene through a transcription factor complex at +400 relative to the transcription start site.