Abstract
Peroxisome proliferator-activated receptor-γ (PPARγ) is a master regulator of adipogenesis, and alterations in its function are associated with various pathological processes related to metabolic syndrome. Recently, we found that the chicken PPARγ gene is regulated by three alternative promoters (P1, P2 and P3), producing five different transcript isoforms and two protein isoforms. In this study, the P1 promoter structure was characterized. Bioinformatics identified six putative nuclear respiratory factor 1 (NRF1) binding sites in the P1 promoter, and a reporter assay showed that NRF1 inhibited the activity of the P1 promoter. Of the six putative NRF1 binding sites, individual mutations of three of them abolished the inhibitory effect of NRF1 on P1 promoter activity. Furthermore, a ChIP assay indicated that NRF1 directly bound to the P1 promoter, and real-time quantitative RT-PCR analysis showed that NRF1 mRNA expression was negatively correlated with PPARγ1 expression (Pearson’s r = -0.148, p = 0.033). Further study showed that NRF1 overexpression inhibited the differentiation of the immortalized chicken preadipocyte cell line (ICP1), which was accompanied by reduced PPARγ1 mRNA expression. Taken together, our findings indicated that NRF1 directly negatively regulates the P1 promoter of the chicken PPARγ gene and inhibits adipogenesis.
Highlights
Adipogenesis plays central roles in energy homeostasis and is significantly associated with obesity
To understand the transcriptional regulation of the chicken Peroxisome proliferator-activated receptor-γ (PPARγ) P1 promoter, an ∼2-kb genomic DNA fragment spanning 1,891 bp upstream and 108 bp downstream of the transcription start site of PPARγ1 was amplified by PCR and cloned into luciferase reporter vector pGL3-Basic
The P1 promoter was active in these three different cell lines, consistent with our previous finding that PPARγ1 was widely expressed in various chicken tissues (Duan et al, 2015)
Summary
Adipogenesis plays central roles in energy homeostasis and is significantly associated with obesity. Ectopic expression of PPARγ is sufficient to induce adipocyte differentiation in fibroblasts, and no factor has been reported to promote adipogenesis in the absence of PPARγ (Lee and Ge, 2014). A number of transcription factors and coregulators have been identified that regulate the alternative promoters of the PPARγ gene in humans and mice. E2F transcription factor 1 (E2F1), early B-cell factor 1 (EBF1), and sterol regulatory element-binding protein-1 (SREBP1) directly bind to mouse PPARγ1 promoter and enhance the expression of the PPARγ1 transcript (Lee and Ge, 2014). Forkhead box class O1 (FOXO1) can directly bind to and reduce human PPARγ2 promoter activity, and in addition, it can indirectly inhibit human PPARγ1 promoter activity (Armoni et al, 2006)
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