Differential expression of KRT83 regulated by the transcript factor CAP1 in Chinese Tan sheep.

Keratin 83 (KRT83) is an important keratin protein in hair development. In this study, expression of KRT83 was compared among different tissues and between 1-month-old lambs and 48-month adult of Chinese Tan sheep, which showed different fleece phenotypes. The results showed that KRT83 was only expressed in skin, and KRT83 mRNA level in skin was significantly higher in Tan lambs than in adult sheep. To further understand the expression regulation of KRT83 by transcription factors in Tan sheep, amplified sequences coving different ranges of KRT83 promoter region were inserted into a pGL3-basic vector and then transfected into sheep primary fibroblast cells. Luciferase assay indicated that the sequence from -218bp to -10bp in the KRT83 promoter induced the highest transcription activity of the vector in the fibroblast cells. Transcription factor adenylate cyclase-associated protein 1 (CAP1) was predicted by online tools within this region. Electrophoretic mobility shift assay (EMSA) confirmed binding of the purified CAP1 protein to the target core region from -88bp to -10bp, because mutation in the target core sequence resulted in failure of CAP1 binding to the target region. Moreover, overexpression of CAP1 protein led to repression of the KRT83 promoter activity in sheep primary fibroblast cells, and expression of CAP1 was lower in lambs than in adult sheep. Therefore, we concluded that CAP1 is a key transcription factor involved in negative regulation of KRT83 expression in Tan sheep skin. Our study provides new insights into the transcriptional regulation of KRT83 and further hints of its critical role in curly hair phenotype in sheep.