The Functional SNPs in the 5’ Regulatory Region of the Porcine <i>PPARD</i> Gene Have Significant Association with Fat Deposition Traits

<div><p>Peroxisome proliferator-activated receptor delta (<i>PPARD</i>) is a key regulator of lipid metabolism, insulin sensitivity, cell proliferation and differentiation. In this study, we identified two Single Nucleotide Polymorphisms (SNPs, g.1015 A>G and g.1018 T>C) constituting four haplotypes (GT, GC, AC and AT) in the 5’ regulatory region of porcine <i>PPARD</i> gene. Functional analysis of the four haplotypes showed that the transcriptional activity of the <i>PPARD</i> promoter fragment carrying haplotype AC was significantly lower than that of the other haplotypes in 3T3-L1, C2C12 and PK-15 cells, and haplotype AC had the lowest binding capacities to the nuclear extracts. Transcription factor 7-like 2 (TCF7L2) enhanced the transcription activities of promoter fragments of <i>PPARD</i> gene carrying haplotypes GT, GC and AT in C2C12 and 3T3-L1 cells, and increased the protein expression of <i>PPARD</i> gene in C2C12 myoblasts. TCF7L2 differentially bound to the four haplotypes, and the binding capacity of TCF7L2 to haplotype AC was the lowest. There were significant associations between -655A/G and fat deposition traits in three pig populations including the Large White × Meishan F₂ pigs, France and American Large White pigs. Pigs with genotype <i>GG</i> had significantly higher expression of <i>PPARD</i> at both mRNA and protein level than those with genotype <i>AG</i>. These results strongly suggested that the SNPs in 5’ regulatory region of <i>PPARD</i> genes had significant impact on pig fat deposition traits.</p></div

The SNPs significantly affect the expression of <i>PPARD</i> gene <i>in vivo</i>.

<p>(A) The relative mRNA expression levels of <i>PPARD</i> gene in muscle and adipose between genotype <i>AG</i> and <i>GG</i>. (B) The protein expression levels of <i>PPARD</i> gene in muscle and adipose between genotype <i>AG</i> and <i>GG</i>.</p

Allele distribution of <i>g</i>.<i>1015 A>G</i> polymorphism in thirteen pig populations.

<p>Allele distribution of <i>g</i>.<i>1015 A>G</i> polymorphism in thirteen pig populations.</p

Association results of <i>g</i>.<i>1015 A>G</i> polymorphism with carcass traits in Large White × Meishan F₂ pig population.

<p>BFT1: backfat thickness at shoulder; ABT: average backfat thickness; BFT2: backfat thickness at thorax-waist; BFT3: backfat thickness at buttock; 67RIBBF: backfat thickness between 6^th and 7^th ribs; RN: rib numbers; CL1: Carcass body length 1; CL2: Carcass body length 2; LFP: Leaf fat percentage; IFR: Internal fat rate. Data were shown as means ± S.D. (standard deviation). Different superscript small letters in one row indicate significance level at <i>P</i><0.05; Different superscript large letters in one row indicate significance level at P<0.01; Asterisk (*) represents significance level at <i>P</i><0.05.</p><p>Association results of <i>g</i>.<i>1015 A>G</i> polymorphism with carcass traits in Large White × Meishan F₂ pig population.</p

Transcriptional activities analysis of deletion constructs of <i>PPARD</i> promoter carrying the four haplotypes.

<p>(A) Transcriptional activities of a series of deletion fragments determined by luciferase assay in C2C12 myoblasts. Left panel, schematic representation of the deleted fragments linked with the luciferase gene in the pGL3 vector. The nucleotides were numbered relative to the transcription start site that was assigned as + 1. Right panel, the relative activities of a series of deletion fragments of pGL3-1880GT vector determined by luciferase assay. (B)(C)(D) The relative activities of a series of deletion fragments of <i>PPARD</i> promoter carrying the different haplotypes in C2C12, 3T3-L1, and PK cells. Error bar represents mean ± S.D. (three independent replicates per group). Asterisk (*) and (**) represent the significance level at <i>P</i> < 0.05 and <i>P</i> < 0.01, respectively (The same below).</p

The SNPs in the 5’ regulatory region of <i>PPARD</i> gene affected transcriptional activation of TCF7L2.

<p>(A) The relative mRNA expression profiles of the porcine <i>PPARD</i> and <i>TCF7L2</i> gene in nine different tissues. (B) Transcriptional activities of four haplotypes determined by luciferase assay before and after <i>TCF7L2</i> overexpression in C2C12 myoblasts and 3T3-L1 cells. (C) The protein expression levels of <i>TCF7L2</i> and <i>PPARD</i> before and after <i>TCF7L2</i> overexpression in C2C12 myoblasts. C2C12 myoblasts were transfected with pcDNA3.1-TCF7L2 vector and empty vector, and then the total protein extracted after 48 h transfection. The protein levels of <i>TCF7L2</i> and <i>PPARD</i> genes were analyzed by western blotting. (D) Relative <i>PPARD</i> protein expression levels represented by ratio of detected protein to <i>β-actin</i> protein expression level after <i>TCF7L2</i> overexpression.</p

Nuclear extracts differentially bind to the promoter fragments carrying the four haplotypes.

<p>(A) (B) (C) EMSA results showing the binding capacities of four haplotypes to the nuclear extracts in C2C12 myoblasts, 3T3-L1 cells, and porcine LD muscle. (D) The EMSA results showing the binding capacities of nuclear extracts to the promoter fragments with haplotypes AC and GC, Lane 1 and Lane 8 were negative control; Lane 2–4 and 5–7 in turn were sample reactions, mutation competitive reactions, and cold competitive reactions for AC and GC reaction groups, respectively. The probes were incubated with nuclear extracts in the absence or presence of 1-fold excess of various competitor probes (mutant or non-labeled probe). The specific DNA-protein complex bands were indicated by arrows. The sequences of various probes were shown under the panel.</p

DNA pull-down results showing the interaction of TCF7L2 with the four haplotypes in 3T3-L1 cell <i>in vivo</i>.

<p>3T3-L1 cells were seeded in 10-cm dishes for 48 h, harvested and pull-downed by antibodies. Lane GT, AT, GC, and AC: immunoprecipitation results with anti-TCF7L2 monoclonal antibody. Lane total protein: the analysis of total cell lysates before immunoprecipitation to verify expression of <i>TCF7L2</i>. The sequences of four probes were shown under the panel.</p