Functional Analysis of Sheep POU2F3 Isoforms.

POU domain class 2 transcription factor 3 (POU2F3) plays an important role in keratinocyte proliferation and differentiation. Our previous study identified four sheep POU2F3 transcript variants (POU2F3-1, POU2F3-2, POU2F3-3, and POU2F3-4), encoding three POU2F3 protein isoforms (POU2F3-1, POU2F3-2, and POU2F3-3). However, the functional differences among the three POU2F3 isoforms remain unknown. The objective of this study was to determine the tissue expression pattern of the four POU2F3 transcript variants in sheep and to investigate the functional differences in cell proliferation among the three POU2F3 isoforms. Quantitative RT-PCR analysis showed that the four POU2F3 transcripts were ubiquitously expressed in all tested adult sheep tissues, and POU2F3-1 exhibited higher expression level than the other three POU2F3 transcript variants in skin (P < 0.05). Cell proliferation assay showed that overexpression of any one of the three POU2F3 isoforms significantly inhibited the proliferation of sheep fetal fibroblasts and HaCaT cells at 48 and 72 h after transfection (P < 0.05). POU2F3-3 had less inhibitory effect on cell proliferation than POU2F3-1 and POU2F3-2 (P < 0.05), and POU2F3-1 and POU2F3-2 had similar inhibitory effects (P > 0.05). Dual luciferase reporter assays demonstrated that overexpression of any one of the three POU2F3 isoforms significantly inhibited the promoter activities of keratin 14 (KRT14) and matrix metalloproteinase 19 (MMP19) genes (P < 0.05). POU2F3-3 had less inhibitory effect on the promoter activities of KRT14 and MMP19 genes than POU2F3-1 and POU2F3-2 (P < 0.05), and POU2F3-1 and POU2F3-2 had similar inhibitory effects (P > 0.05). These results suggest three sheep POU2F3 isoforms have similar functional effects, but to a different extent.

An Upstream Open Reading Frame Represses Translation of Chicken PPARγ Transcript Variant 1.

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipogenesis. The PPARγ gene produces various transcripts with different 5'-untranslated regions (5' UTRs) because of alternative promoter usage and splicing. The 5' UTR plays important roles in posttranscriptional gene regulation. However, to date, the regulatory role and underlying mechanism of 5' UTRs in the posttranscriptional regulation of PPARγ expression remain largely unclear. In this study, we investigated the effects of 5' UTRs on posttranscriptional regulation using reporter assays. Our results showed that the five PPARγ 5' UTRs exerted different effects on reporter gene activity. Bioinformatics analysis showed that chicken PPARγ transcript 1 (PPARγ1) possessed an upstream open reading frame (uORF) in its 5' UTR. Mutation analysis showed that a mutation in the uORF led to increased Renilla luciferase activity and PPARγ protein expression, but decreased Renilla luciferase and PPARγ1 mRNA expression. mRNA stability analysis using real-time RT-PCR showed that the uORF mutation did not interfere with mRNA stability, but promoter activity analysis of the cloned 5' UTR showed that the uORF mutation reduced promoter activity. Furthermore, in vitro transcription/translation assays demonstrated that the uORF mutation markedly increased the translation of PPARγ1 mRNA. Collectively, our results indicate that the uORF represses the translation of chicken PPARγ1 mRNA.

Nuclear Respiratory Factor 1 Negatively Regulates the P1 Promoter of the Peroxisome Proliferator-Activated Receptor-γ Gene and Inhibits Chicken Adipogenesis.

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.

Polymorphisms of FST gene and their association with wool quality traits in Chinese Merino sheep.

Follistatin (FST) is involved in hair follicle morphogenesis. However, its effects on hair traits are not clear. This study was designed to investigate the effects of FST gene single nucleotide polymorphisms (SNP) on wool quality traits in Chinese Merino sheep (Junken Type). We performed gene expression analysis, SNP detection, and association analysis of FST gene with sheep wool quality traits. The real-time RT-PCR analysis showed that FST gene was differentially expressed in adult skin between Chinese Merino sheep (Junken Type) and Suffolk sheep. Immunostaining showed that FST was localized in inner root sheath (IRS) and matrix of hair follicle (HF) in both SF and Suffolk sheep. Sequencing analysis identified a total of seven SNPs (termed SNPs 1-7) in the FST gene in Chinese Merino sheep (Junken Type). Association analysis showed that SNP2 (Chr 16. 25,633,662 G>A) was significantly associated with average wool fiber diameter, wool fineness SD, and wool crimp (P < 0.05). SNP4 (Chr 16. 25,633,569 C>T) was significantly associated with wool fineness SD and CV of fiber diameter (P < 0.05). Similarly, the haplotypes derived from these seven identified SNPs were also significantly associated with average wool fiber diameter, wool fineness SD, CV of fiber diameter, and wool crimp (P < 0.05). Our results suggest that FST influences wool quality traits and its SNPs 2 and 4 might be useful markers for marker-assisted selection and sheep breeding.