Krüppel-like factor 7 (Klf7) has been extensively studied in the mammalian species, but its function in avian species is unclear. The objective of this study was to reveal the function of chicken Klf7 (Gallus gallus Klf7, gKlf7) in adipogenesis. The results of real-time reverse transcription polymerase chain reaction demonstrated that the relative mRNA level of chicken Klf7 (gKlf7/gβ-Actin) in the abdominal adipose tissue was significantly associated with the abdominal fat content and the age of broilers (P < 0.05), and gKlf7 was more highly expressed in preadipocytes than in mature adipocytes (P < 0.05). In addition, Oil red O staining showed that gKlf7 inhibited chicken preadipocyte differentiation, and MTT assay indicated that gKlf7 overexpression promoted preadipocyte proliferation. Additionally, luciferase assays showed that gKlf7 overexpression suppressed the chicken CCAAT/enhancer-binding protein α (C/ebpα), fatty acid synthase (Fasn), and lipoprotein lipase (Lpl) promoter activities (P < 0.05), and gKlf7 knockdown increased the chicken peroxisome proliferator-activated receptor γ (Pparγ), C/ebpα and fatty acid-binding protein 4 (Fabp4) promoter activities (P < 0.05). Together, our study demonstrated that chicken Klf7 inhibits preadipocyte differentiation and promotes preadipocyte proliferation.

Peroxisome proliferator-activated receptor γ is a master regulator of adipocyte differentiation and function. Expression of PPARγ in mammals is regulated by DNA methylation; however, it is currently unknown whether chicken PPARγ expression is regulated by DNA methylation. To enhance our understanding of molecular mechanisms underlying chicken adipose tissue development and adipogenesis, we investigated the promoter methylation status and gene expression of PPARγ gene in Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). Deoxyribonucleic acid methylation was analyzed by bisulfite sequencing method, and mRNA expression was detected by real-time quantitative real time reverse-transcription polymerase chain reaction (RT-PCR). The analyzed region located from -1,175 to -301 bp upstream of the translation start codon ATG contains 6 CpG dinucleotides, which are located at positions -1,014, -796, -625, -548, -435, and -383 bp, respectively. The results revealed that the 3 CpGs at positions -548, -435, and -383 bp showed differential methylation between the lean and fat chicken lines, but the other 3 CpG sites at positions -1,014, -796, and -625 bp did not. PPARγ gene promoter methylation in both chicken lines decreased with age, and PPARγ promoter methylation levels were significantly higher in lean than fat broilers at 2 wk of age (79.9 to 64.5%; P < 0.0001), at 3 wk of age (66.7 to 58.3%; P < 0.0001), and at 7 wk of age (50.0 to 42.7%; P = 0.0004). Real-time quantitative RT-PCR analysis showed that, negatively correlated with DNA methylation (Pearson's r = -0.653, P = 0.0057), PPARγ expression was increased with age and significantly lower in lean than fat chicken lines at 2, 3, and 7 wk of age (P < 0.0001). In conclusion, our findings suggest that chicken PPARγ is regulated by DNA methylation during adipose tissue development.

Peroxisome proliferator-activated receptor gamma regulates adipogenesis. The genomic structure of the chicken peroxisome proliferator-activated receptor gamma (cPPARγ) gene has not been fully characterized, and only one cPPARγ gene mRNA sequence has been reported in genetic databases. Using 5' rapid amplification of cDNA ends, we identified five different cPPARγ mRNAs that are transcribed from three transcription initiation sites. The open reading frame analysis showed that these five cPPARγ transcript variants (cPPARγ1 to 5) could encode two cPPARγ protein isoforms (cPPARγ1 and cPPARγ2), which differ only in their N-terminal region. Quantitative real-time RT-PCR analysis showed that, of these five cPPARγ transcript variants, cPPARγ1 was ubiquitously highly expressed in various chicken tissues, including adipose tissue, liver, kidney, spleen and duodenal; cPPARγ2 was exclusively highly expressed in adipose tissue; cPPARγ3 was highly expressed in adipose tissue, kidney, spleen and liver; cPPARγ4 and cPPARγ5 were ubiquitously weakly expressed in all the tested tissues, and comparatively, cPPARγ5 was highly expressed in adipose tissue, heart, liver and kidney. The comparison of the expression of the five cPPARγ transcript variants showed that adipose tissue cPPARγ1 expression was significantly higher in the fat line than in the lean line from 2 to 7 wk of age (P < 0.05 or P < 0.01). Adipose tissue cPPARγ3 expression was significantly higher in the fat line than in the lean line at 3, 5 and 6 wk of age (P < 0.01, P < 0.05), but lower at 4 wk of age (P < 0.05). Adipose tissue cPPARγ5 expression was significantly higher in the fat line than in the lean line at 3, 4, and 6 wk of age (P < 0.01) and at 2 and 7 wk of age (P < 0.05). This is the first report of transcript variants and protein isoforms of cPPARγ gene. Our findings provided a foundation for future investigations of the function and regulation of cPPARγ gene in adipose tissue development.