ABSTRACT: Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis. Our previous study revealed that chicken PPARγ has 3 alternative promoters named as P1, P2, and P3, and the DNA methylation of promoter P3 was negatively associated with PPARγ mRNA expression in abdominal adipose tissue (AAT). However, the methylation status of promoters P1 and P2 is unclear. Here we assessed promoter P1 methylation status in AAT of Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). The results showed that promoter P1 methylation differed in AAT between the lean and fat lines of NEAUHLF at 7 wk of age (p < 0.05), and AAT expression of PPARγ transcript 1 (PPARγ1), which was derived from the promoter P1, was greatly higher in fat line than in lean line at 2 and 7 wk of age. The results of the correlation analysis showed that P1 methylation was positively correlated with PPARγ1 expression at 7 wk of age (Pearson's r = 0.356, p = 0.0242), suggesting P1 methylation promotes PPARγ1 expression. To explore the underlying molecular mechanism of P1 methylation on PPARγ1 expression, bioinformatics analysis, dual-luciferase reporter assay, pyrosequencing, and electrophoresis mobility shift assay (EMSA) were performed. The results showed that transcription factor NRF1 repressed the promoter activity of the unmethylated P1, but not the methylated P1. Of all the 4 CpGs (CpG48, CpG49, CpG50, and CpG51), which reside within or nearby the NRF1 binding sites of the P1, only CpG49 methylation in AAT was remarkably higher in the fat line than in lean line at 7 wk of age (3.18 to 0.57, p < 0.05), and CpG49 methylation was positively correlated with PPARγ1 expression (Pearson's r = 0.3716, p = 0.0432). Furthermore, EMSA showed that CpG49 methylation reduced the binding of NRF1 to the P1. Taken together, our findings illustrate that P1 methylation promotes PPARγ1 expression at least in part by preventing NRF1 from binding to the promoter P1.
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