The activity of the carbamoyl phosphate synthase 1 promoter in human liver-derived cells is dependent on hepatocyte nuclear factor 3-beta.
The activity of the carbamoyl phosphate synthase 1 promoter in human liver-derived cells is dependent on hepatocyte nuclear factor 3-beta.
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人肝源性细胞中氨基甲酰磷酸合酶 1 启动子的活性依赖于肝细胞核因子 3-β
DOI：
10.1111/jcmm.13123
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发表时间：
2017-09
影响因子：
5.3
通讯作者：
Chen Y
中科院分区：
文献类型：
--
作者：
Chen Z;Tang N;Wang X;Chen Y
氨甲酰磷酸合成酶1 （CPS1）是尿素循环第一步的限速酶，也是人体肝脏代谢中不可缺少的酶。然而，CPS1的表观遗传调控涉及启动子分析和肝脏富集转录因子（LETFs）的作用，目前尚未完全阐明。本研究克隆了hCPS1基因的启动子区域，并对其活性进行了研究。肝细胞核因子3β (HNF3β)可促进CPS1在肝源性细胞系中的转录表达。此外，双荧光素酶报告分析表明，HNF3β的基本结合位点可能存在于- 70 nt至+73 nt的寡核苷酸中。HNF3β有两个可能的结合位点。突变分析结果表明，HNF3β结合位点2有效，突变后CPS1启动子转录活性显著降低。电泳移动位移实验（EMSA）和ChIP实验证实，HNF3β在体内和体外均可与CPS1启动子区−70 nt ~ +73 nt的结合位点相互作用，调控CPS1的转录。此外，HNF3β过表达增强了CPS1的转录，从而提高了CPS1的mRNA和蛋白水平，而HNF3β过表达则表现出相反的效果。最后，检测细胞的尿素产量，转染HNF3β后，细胞的氨解毒能力显著提高。HNF3β在CPS1基因调控中发挥重要作用，通过调控CPS1的表达促进氨代谢。
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.
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