Regulation of cholesterol catabolism by bile acids

胆汁酸调节胆固醇分解代谢

• 批准号: 7084471
• 负责人: Jongsook Kim Kemper
• 金额: $ 22.99万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7084471
• 关键词: acyltransferase; amidohydrolases; bile; biotransformation; chenodeoxycholate; cholanate compound; cholesterol; chromatin; endonuclease; gene induction /repression; genetic promoter element; genetic transcription; histones; immunoprecipitation; laboratory mouse; lipid metabolism; liver cells; nuclear receptors; nucleosomes; tissue /cell culture

DESCRIPTION (provided by applicant): The overall aim of this project is to delineate the mechanism by which bile acids repress transcription of the human cholesterol 7-a hydroxylase (CYP7A1) gene. CYP7A1 catalyzes the initial, rate-limiting step of the classical pathway for bile acid biosynthesis and is a key enzyme in maintaining cholesterol homeostasis. Bioactivity of CYP7A1 is regulated by feedback inhibition at the level of transcription by bile acids. It is known that bile acid-activated FXR activates transcription of SHP which in turn suppresses LRH-1 mediated transactivation of the CYP7A1 gene. Recent studies of SHP knock-out mice suggest that SHP independent pathways of bile acid repression exist as well. Although the master regulators and the bile acid response elements have been identified, the changes in response to bile acids in the regulatory protein complex and in the chromatin structure at the native human CYP7A1 promoter that lead to suppression of the gene in vivo are not known. In preliminary chromatin immunoprecipitation (CHIP) assays, we found that bile acid treatment resulted in decreased histone acetylation, and decreased association of coactivator histone acetyltransferases (HATs) p300 and CBP with the human CYP7A1 promoter. On the basis of these exciting results, we hypothesize that bile acids repress CYP7A1 transcription by modulating the association of HATs and histone deacetylases (HDACs) with the promoter resulting in chromatin structural changes which lead to repression of the CYP7A1 transcription. We propose to test this hypothesis using the ChIP assay to examine the association of proteins with the human CYP7A1 promoter in vivo and transcriptional assays in cell culture and in vivo to determine the functional significance of proteins shown to associate with the promoter. Specifically, we will examine the temporal effects of bile acid treatment at the human CYP7A1 promoter on: 1) endonuclease sensitivity of the nucleosomal structures, 2) histone acetylation and 3) the association of coactivators and corepressors. Experiments will be initiated to compare these changes related to chromatin structure with changes in the association of the nuclear receptors (LRH-1/HNF-4), chromatin remodeling complexes, and the RNA preinitiation complex. The effects of identified coactivators and corepressors on CYP7A1 transcription will be studied in cultured cells and in mouse hepatocytes in vivo using a tail vein injection method. Stably transfected HepG2 cells will be constructed for the study of the interaction of SHP with the promoter and for determination of whether overexpression of SHP in untreated cells can mediate all the effects of bile acid treatment. These studies should define the molecular changes that occur at the CYP7A1 promoter in vivo after bile acid treatment and provide new insight into the mechanism of repression of this gene, and possibly other genes, involved in cholesterol homeostasis.

描述（由申请人提供）：本项目的总体目的是描述胆汁酸抑制人胆固醇7-a羟化酶（CYP 7A 1）基因转录的机制。CYP 7A 1催化胆汁酸生物合成经典途径的起始限速步骤，是维持胆固醇稳态的关键酶。CYP 7A 1的生物活性通过胆汁酸在转录水平上的反馈抑制来调节。已知胆汁酸激活的FXR激活SHP的转录，SHP反过来抑制LRH-1介导的CYP 7A 1基因的反式激活。最近对SHP基因敲除小鼠的研究表明，也存在不依赖于SHP的胆汁酸抑制途径。虽然已经鉴定了主调节因子和胆汁酸反应元件，但尚不清楚调节蛋白复合物和天然人CYP 7A 1启动子处的染色质结构中对胆汁酸的反应变化导致体内基因抑制。 在初步的染色质免疫沉淀（CHIP）试验中，我们发现胆汁酸处理导致组蛋白乙酰化降低，并减少了辅激活剂组蛋白乙酰转移酶（HATs）p300和CBP与人CYP 7A 1启动子的关联。基于这些令人兴奋的结果，我们假设胆汁酸通过调节HAT和组蛋白脱乙酰酶（HDAC）与启动子的结合，导致染色质结构变化，从而抑制CYP 7A 1转录，从而抑制CYP 7A 1转录。我们建议使用ChIP检测来检验这一假设，以检查蛋白质与人CYP 7A 1启动子在体内的相关性，并在细胞培养和体内的转录检测来确定与启动子相关的蛋白质的功能意义。具体而言，我们将研究胆汁酸处理在人CYP 7A 1启动子上的时间效应：1）核小体结构的核酸内切酶敏感性，2）组蛋白乙酰化和3）辅激活子和辅阻遏子的关联。将启动实验，以比较这些与染色质结构相关的变化与核受体（LRH-1/HNF-4）、染色质重塑复合物和RNA前起始复合物相关的变化。将采用尾静脉注射方法，在培养细胞和小鼠肝细胞中研究已鉴别的共激活因子和共阻遏因子对CYP 7A 1转录的影响。将构建稳定转染的HepG 2细胞，用于研究SHP与启动子的相互作用，并用于确定未处理细胞中SHP的过表达是否可以介导胆汁酸处理的所有效应。这些研究应确定胆汁酸处理后体内CYP 7A 1启动子发生的分子变化，并对该基因以及可能参与胆固醇稳态的其他基因的抑制机制提供新的见解。