Regulation of cholesterol catabolism by bile acids

描述(申请人提供)：这个项目的总体目标是描述胆汁酸抑制人类胆固醇7-a羟基酶(CYP7A1)基因转录的机制。细胞色素P7A1是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。胆汁酸在转录水平上通过反馈抑制来调节细胞色素P7A1的生物活性。众所周知，胆汁酸激活的FXR激活SHP的转录，进而抑制LRH-1介导的CYP7A1基因的反式激活。最近对SHP基因敲除小鼠的研究表明，SHP不依赖于胆汁酸抑制的途径也存在。虽然已经确定了主要的调节因子和胆汁酸反应元件，但调节蛋白复合体中的胆汁酸反应和天然人类CYP7A1启动子染色质结构的变化导致该基因在体内受到抑制的情况尚不清楚。 在初步的染色质免疫沉淀(ChIP)检测中，我们发现胆汁酸处理导致组蛋白乙酰化减少，并减少了辅活化组蛋白乙酰转移酶(HATS)p300和CBP与人CYP7A1启动子的结合。在这些令人兴奋的结果的基础上，我们假设胆汁酸通过调节HATS和组蛋白脱乙酰酶(HDACs)与启动子的结合而抑制CYP7A1的转录，从而导致染色质结构的变化，从而导致CYP7A1转录的抑制。我们建议用芯片试验来检验这一假说，在体内检查蛋白质与人CYP7A1启动子的关联，并在细胞培养和体内转录试验中确定与启动子相关的蛋白质的功能意义。具体地说，我们将研究胆汁酸处理在人类CYP7A1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，以及3)辅助激活因子和辅助抑制因子的结合。实验将开始将这些与染色质结构相关的变化与核受体(LRH-1/HNF-4)、染色质重塑复合体和RNA预起始复合体的关联变化进行比较。我们将使用尾静脉注射的方法，在培养细胞和活体小鼠肝细胞中研究已确定的辅活化子和辅抑制子对细胞色素P7A1转录的影响。为了研究SHP与启动子的相互作用，以及确定SHP在未经处理的细胞中过表达是否能介导胆汁酸处理的所有作用，将构建稳定表达SHP的HepG2细胞。这些研究将确定胆汁酸处理后体内发生在CYP7A1启动子上的分子变化，并为抑制该基因的机制提供新的见解，可能还有其他参与胆固醇稳态的基因。

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.