title: Characterization of the porcine peptidylarginine deiminase type VI gene (PADI6) promoter: Sp1 regulates basal transcription of the porcine PADI6.
abstract: It is a general consensus that oocyte quality is the key to embryo survival in pig reproduction. Thus, study on regulation of the ovary-associated gene is of great significance in pig breeding. Peptidylarginine deiminases (PADs) are a family of enzymes which catalyze the conversion of arginine to citrulline in proteins. The peptidylarginine deiminases type VI gene (PADI6) is mainly expressed in the ovary, and plays an important role in oocyte growth, fertilization and early embryo development. However, until now, little is known about its transcriptional regulation mechanism. Here, we firstly isolated and characterized the 5'-flanking region of porcine PADI6 gene. We determined the transcription start site using 5'-rapid amplification of cDNA ends (RACE) analysis, and identified the minimal promoter (-85/+68) that drove the basal expression of PADI6 by constructing various progressive deletions. Mutational analysis and electrophoretic mobility shift assays demonstrated Sp1 bound to the -56/-47 region of the PADI6 promoter. Furthermore, overexpression of Sp1 significantly increased the promoter activity and promoted PADI6 gene expression, and accordingly, inhibition of Sp1 expression with specific siRNA significantly reduced the promoter activity and suppressed the PADI6 expression. In addition, inhibition of Sp1 binding by Mithramycin A treatment reduced the transcriptional activity of PADI6 in a dose-dependent manner. Taken together, these data indicate that Sp1 is essential for the transcriptional regulation of PADI6.
authors.affiliation: Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: xxl8365@163.com.
authors.forename: Xiaoliang
authors.initials: X
authors.lastname: Xia
authors.affiliation: Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: yanchi_2005@126.com.
authors.forename: Chi
authors.initials: C
authors.lastname: Yan
authors.affiliation: Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: wuwangjun2012@njau.edu.cn.
authors.forename: Wangjun
authors.initials: W
authors.lastname: Wu
authors.affiliation: Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: zhouyingamy@126.com.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: mnhouliming@126.com.
authors.forename: Liming
authors.initials: L
authors.lastname: Hou
authors.affiliation: Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: zuobo@mail.hzau.edu.cn.
authors.forename: Bo
authors.initials: B
authors.lastname: Zuo
authors.affiliation: Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: dequanxu@mail.hzau.edu.cn.
authors.forename: Dequan
authors.initials: D
authors.lastname: Xu
authors.affiliation: Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: renzq@mail.hzau.edu.cn.
authors.forename: Zhuqing
authors.initials: Z
authors.lastname: Ren
authors.affiliation: Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: xiongyuanzhu@hotmail.com.
authors.forename: Yuanzhu
authors.initials: Y
authors.lastname: Xiong
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D004252:DNA Mutational Analysis; D005260:Female; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D006867:Hydrolases / Q000235:genetics*; D008926:Plicamycin / Q000031:analogs & derivatives / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013552:Swine / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D016329:Sp1 Transcription Factor; C066851:mithramycin A; D006867:Hydrolases; D008926:Plicamycin
keywords: Cis-acting elements; EMSA; Trans-acting factors; Transcriptional regulation