Zhao Zhichao, Liu Guangbin, Li Xinyun, Huang Ji, Xiao Yujing, Du Xiaoyong, Yu Mei
Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
College of Animal Science, South China Agricultural University, Guangzhou, 510642, China.
PLoS One. 2016 Apr 21;11(4):e0153936. doi: 10.1371/journal.pone.0153936. eCollection 2016.
The keratin-associated proteins (KAPs) are the structural proteins of hair fibers and are thought to play an important role in determining the physical properties of hair fibers. These proteins are activated in a striking sequential and spatial pattern in the keratinocytes of hair fibers. Thus, it is important to elucidate the mechanism that underlies the specific transcriptional activity of these genes. In this study, sheep KRTAP 3-3 and KRTAP11-1 genes were found to be highly expressed in wool follicles in a tissue-specific manner. Subsequently, the promoter regions of the two genes that contained the 5' flanking/5' untranslated regions and the coding regions were cloned. Using an in vivo transgenic approach, we found that the promoter regions from the two genes exhibited transcriptional activity in hair fibers. A much stronger and more uniformly expressed green fluorescent signal was observed in the KRTAP11-1-ZsGreen1 transgenic mice. In situ hybridization revealed the symmetrical expression of sheep KRTAP11-1 in the entire wool cortex. Consistently, immunohistochemical analysis demonstrated that the pattern of ZsGreen1 expression in the hair cortex of transgenic mice matches that of the endogenous KRTAP11-1 gene, indicating that the cloned promoter region contains elements that are sufficient to govern the wool cortex-specific transcription of KRTAP11-1. Furthermore, regulatory regions in the 5' upstream sequence of the sheep KRTAP11-1 gene that may regulate the observed hair keratinocyte specificity were identified using in vivo reporter assays.
角蛋白相关蛋白（KAPs）是毛发纤维的结构蛋白，被认为在决定毛发纤维的物理特性方面发挥重要作用。这些蛋白在毛发纤维的角质形成细胞中以显著的顺序和空间模式被激活。因此，阐明这些基因特定转录活性的潜在机制很重要。在本研究中，发现绵羊KRTAP 3-3和KRTAP11-1基因在毛囊中以组织特异性方式高度表达。随后，克隆了包含5'侧翼/5'非翻译区和编码区的这两个基因的启动子区域。使用体内转基因方法，我们发现这两个基因的启动子区域在毛发纤维中表现出转录活性。在KRTAP11-1-ZsGreen1转基因小鼠中观察到更强且表达更均匀的绿色荧光信号。原位杂交显示绵羊KRTAP11-1在整个羊毛皮质中呈对称表达。一致地，免疫组织化学分析表明转基因小鼠毛发皮质中ZsGreen1的表达模式与内源性KRTAP11-1基因的表达模式匹配，表明克隆的启动子区域包含足以控制KRTAP11-1羊毛皮质特异性转录的元件。此外，使用体内报告基因测定法鉴定了绵羊KRTAP11-1基因5'上游序列中可能调节观察到的毛发角质形成细胞特异性的调控区域。
