title: The activity of the carbamoyl phosphate synthase 1 promoter in human liver-derived cells is dependent on hepatocyte nuclear factor 3-beta.
abstract: Carbamoyl phosphate synthase 1 (CPS1) is the rate-limiting enzyme in the first step of the urea cycle and an indispensable enzyme in the metabolism of human liver. However, CPS1 epigenetic regulation involves promoter analysis and the role of liver-enriched transcription factors (LETFs), which is not fully elucidated. In this work, the promoter region of hCPS1 gene was cloned, and its activity was investigated. An LETF, hepatocyte nuclear factor 3-beta (HNF3β), was found to promote the transcriptional expression of CPS1 in liver-derived cell lines. In addition, dual-luciferase reporter assay shows that the essential binding sites of the HNF3β may exist in the oligonucleotide -70 nt to +73 nt. Two putative binding sites are available for HNF3β. Mutation analysis results show that the binding site 2 of HNF3β was effective, and the transcriptional activity of CPS1 promoter significantly decreased after mutation. Electrophoretic mobile shift assay (EMSA) and ChIP assay confirmed that HNF3β can interact with the binding site in the CPS1 promoter region of -70 nt to +73 nt promoter region in vivo and in vitro to regulate the transcription of CPS1. Moreover, HNF3β overexpression enhanced the transcription of CPS1 and consequently improved the mRNA and protein levels of CPS1, whereas the knockdown of HNF3β showed the opposite effects. Finally, urea production in cells was measured, and ammonia detoxification improved significantly in cells after transfection with HNF3β. HNF3β plays a vital role in regulation of CPS1 gene and could promote the metabolism of ammonia by regulating CPS1 expression.
authors.affiliation: Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, Fuzhou, China.
authors.forename: Zhanfei
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, Fuzhou, China.|Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Research Center for Molecular Medicine, Fujian Medical University, Fuzhou, China.
authors.forename: Nanhong
authors.initials: N
authors.lastname: Tang
authors.affiliation: Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, Fuzhou, China.
authors.forename: Xiaoqian
authors.initials: X
authors.lastname: Wang
authors.affiliation: Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, Fuzhou, China.|Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Research Center for Molecular Medicine, Fujian Medical University, Fuzhou, China.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000641:Ammonia / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D051542:Hepatocyte Nuclear Factor 3-beta / Q000378:metabolism*; D006801:Humans; D008658:Inactivation, Metabolic; D008099:Liver / Q000166:cytology*; D009711:Nucleotides / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D014158:Transcription, Genetic; D014508:Urea / Q000378:metabolism
chemical_list: D009711:Nucleotides; D034741:RNA, Small Interfering; D051542:Hepatocyte Nuclear Factor 3-beta; D000641:Ammonia; D014508:Urea; D002222:Carbamoyl-Phosphate Synthase (Ammonia)
keywords: ammonia detoxification; carbamoyl phosphate synthetase 1; hepatocyte nuclear factor 3-beta; liver-derived cell; promoter