The 5′ End Cloning, Expression And Regulation Analysis In MRNA Of Chicken Peroxisome Proliferator-Activated Receptor Gamma

Peroxisome proliferator-activated receptor gamma(PPARγ) is a member of the superfamiliy of nuclear receptors. PPARγ has extensive founctions in mammals that it plays an important roal in the regulation of adipocyte differentiation and lipid metabolism, and closely related with obesity and obesity-associated diseases.The structure of the chicken PPARγ gene(c PPARγ) has not been fully characterized, and only one c PPARγ m RNA sequence has been reported in NCBI database. Using 5? rapid amplification of c DNA ends, we identified five different c PPARγ m RNAs that they differed only in the 5′-terminal sequence. The open reading frame analysis showed that these five c PPARγ transcript variants encoded the c PPARγ1 and c PPARγ2 protein isoforms, the sequences of which differed in the N-terminal region.We employed quantitative reverse transcription and polymerase chain reaction to detect the expression patterns of the c PPARγ transcript variants in various tissues of chicken. The results showed that the expressions of c PPARγ1 in adipose tissue, heart, liver, spleen, kidney and duodenum were very high. Meanwhile, c PPARγ1 had a small amount of expressions in the lung, brain and pancreas. In the breast and glandular stomach, however, c PPARγ1 expressed extremely low. Compared with c PPARγ1, the cPPARγ2, c PPARγ3, c PPARγ4, and c PPARγ5 transcript variants expressed at lower levels in all the tissues, including adipose tissue. The expression of cPPARγ2 was high and presented an adipose tissue specificity; c PPARγ3 was highly expressed in adipose tissue, kidney, spleen and liver; c PPARγ4 and c PPARγ5were ubiquitously weakly expressed in all the tested tissues, and comparatively, c PPARγ5 was highly expressed in adipose tissue, heart, liver and kidney. The level of c PPARγ1 expression in abdominal adipose tissue was significantly higher in the fat chicken breeding line than that in the lean chicken breeding line from 2 to 7 weeks of age(P < 0.01). The level of c PPARγ3 expression in abdominal adipose tissue was significantly higher in the fat chicken line than that in the lean chicken line at 3 and 5 weeks of age(P < 0.01) and at 4 and 6 weeks of age(P < 0.05). The level of cPPARγ5 expression in abdominal adipose tissue was significantly higher in the fat chicken line than that in the lean chicken line at 3, 4, and 6 weeks of age(P < 0.01) and at 2 and 7 weeks of age(P < 0.05).Bioinformation analysis results showed that both P1 and P2 had GC-box in the 2kb sequences of promoter region, in which P1 contained many transcription factor binding site which associated with adipocyte differentiation, such as C/EBP family, NFKPB and AP2, and P2 contained some transcription factors such as C/EBP family and AP2. Analysis of truncated mutation found that in the promotor region of P1(-577/+109)and P2(-671/+140)showed very strong luciferase activity, P < 0.01 and P < 0.05, respectively.We also found Cp G sites in the promoter region of chicken P1 and P2. We used Real-time PCR to detect the relative expressions of five c PPARγ in DF1 cell lines and preadipocytes which had treated with 5′-aza-2′-deoxycytidine at different times. The results showed that in the DF1 cell lines, five c PPARγs showed different degrees of rising. The relative expressions of c PPARγ1 and c PPARγ3 in DF1 cells were significantly higher than the negative(P<0.05) at 96 h. Besides, c PPARγ4 expressed extremely significantly higher than the negative(P<0.01) at 96 h. The expression level of c PPARγ1 in preadipocyte continued to rise at 24, 48,72 and 96 h, and showed a significant level at 96h(P<0.05), while others four c PPARγs just showed a tendency of rising.We employed the report gene to detect the relative activity of P1, P2 and P3 promoter which treat with M.Sss I. The results showed that the P1(M) and P2(M) were extremely significant repressed(P<0.01), and the P3(M) were significant repressed(P<0.05). The above results indicated that the DNA methylation regulated the activity of the promoters of chicken PPARγs. This is the first report of 5 m RNA variants and 2 protein isoforms of PPARγs in chicken. Our findings provide a foundation for future investigations of the function of c PPARγ in the regulation of adipose tissue development.