Zhang Ruirui, Feng Xiaoting, Zhan Mengsi, Huang Cong, Chen Kun, Tang Xiaoyin, Kang Tingting, Xiong Yuanzhu, Lei Minggang
Key Laboratory of Swine Genetics and Breeding of Agricultural Ministry & Key Laboratory 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 Life Science and Technology, Wuhan Bioengineering Institute, Wuhan 430070, China.
Int J Mol Sci. 2016 Jan 15;17(1):112. doi: 10.3390/ijms17010112.
Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) gene plays a crucial role in maintaining genomic stability, tumorigenesis and myogenesis. However, little is known about the regulatory elements governing the transcription of porcine ROCK1 gene. In the current study, the transcription start site (TSS) was identified by 5'-RACE, and was found to differ from the predicted one. The region in ROCK1 promoter which is critical for promoter activity was investigated via progressive deletions. Site-directed mutagenesis indicated that the region from -604 to -554 bp contains responsive elements for Sp1. Subsequent experiments showed that ROCK1 promoter activity is enhanced by Sp1 in a dose-dependent manner, whereas treatment with specific siRNA repressed ROCK1 promoter activity. Electrophoretic mobility shift assay (EMSA), DNA pull down and chromatin immunoprecipitation (ChIP) assays revealed Sp1 can bind to this region. qRT-PCR and Western blotting research followed by overexpression or inhibition of Sp1 indicate that Sp1 can affect endogenous ROCK1 expression at both mRNA and protein levels. Overexpression of Sp1 can promote the expression of myogenic differentiation 1(MyoD), myogenin (MyoG), myosin heavy chain (MyHC). Taken together, we conclude that Sp1 positively regulates ROCK1 transcription by directly binding to the ROCK1 promoter region (from -604 to -532 bp) and may affect the process of myogenesis.
Rho相关的卷曲螺旋蛋白激酶1（ROCK1）基因在维持基因组稳定性、肿瘤发生和肌生成中起关键作用。然而，关于猪ROCK1基因转录调控元件的了解甚少。在本研究中，通过5'-RACE鉴定了转录起始位点（TSS），发现其与预测的位点不同。通过逐步缺失研究了ROCK1启动子中对启动子活性至关重要的区域。定点诱变表明，-604至-554 bp区域含有Sp1的反应元件。随后的实验表明，Sp1以剂量依赖的方式增强ROCK1启动子活性，而用特异性siRNA处理则抑制ROCK1启动子活性。电泳迁移率变动分析（EMSA）、DNA下拉和染色质免疫沉淀（ChIP）分析表明Sp1可以结合该区域。在过表达或抑制Sp1后进行的qRT-PCR和蛋白质印迹研究表明，Sp1可以在mRNA和蛋白质水平上影响内源性ROCK1的表达。Sp1的过表达可以促进肌分化1（MyoD）、肌细胞生成素（MyoG）、肌球蛋白重链（MyHC）的表达。综上所述，我们得出结论，Sp1通过直接结合ROCK1启动子区域（-604至-532 bp）正向调节ROCK1转录，并可能影响肌生成过程。
