Open AccessThe activity of the carbamoyl phosphate synthase 1 promoter in human liver‐derived cells is dependent on hepatocyte nuclear factor 3‐beta
Author(s) -
Chen Zhanfei,
Tang Nanhong,
Wang Xiaoqian,
Chen Yanling
Publication year - 2017
Publication title -
journal of cellular and molecular medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.44
H-Index - 130
eISSN - 1582-4934
pISSN - 1582-1838
DOI - 10.1111/jcmm.13123
Subject(s) - microbiology and biotechnology , biology , transcription factor , electrophoretic mobility shift assay , hepatocyte nuclear factors , reporter gene , carbamoyl phosphate synthetase , biochemistry , gene expression , enzyme , gene
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, CPS 1 epigenetic regulation involves promoter analysis and the role of liver‐enriched transcription factors ( LETF s), which is not fully elucidated. In this work, the promoter region of hCPS 1 gene was cloned, and its activity was investigated. An LETF , hepatocyte nuclear factor 3‐beta ( HNF 3β), was found to promote the transcriptional expression of CPS 1 in liver‐derived cell lines. In addition, dual‐luciferase reporter assay shows that the essential binding sites of the HNF 3β may exist in the oligonucleotide −70 nt to +73 nt. Two putative binding sites are available for HNF 3β. Mutation analysis results show that the binding site 2 of HNF 3β was effective, and the transcriptional activity of CPS 1 promoter significantly decreased after mutation. Electrophoretic mobile shift assay ( EMSA ) and Ch IP assay confirmed that HNF 3β can interact with the binding site in the CPS 1 promoter region of −70 nt to +73 nt promoter region in vivo and in vitro to regulate the transcription of CPS 1. Moreover, HNF 3β overexpression enhanced the transcription of CPS 1 and consequently improved the mRNA and protein levels of CPS 1, whereas the knockdown of HNF 3β showed the opposite effects. Finally, urea production in cells was measured, and ammonia detoxification improved significantly in cells after transfection with HNF 3β. HNF 3β plays a vital role in regulation of CPS 1 gene and could promote the metabolism of ammonia by regulating CPS 1 expression.