BILE ACID METABOLISM AND PLASMA CHOLESTEROL REGULATION

肝胆固醇7-α-羟化酶催化初始和速率- 在胆固醇转化为 胆盐因此，7-α-羟化酶在细胞内和细胞外都起着核心作用。 维持全身固醇平衡，其次， 调节血清脂蛋白浓度。 肝脏7-α- 羟化酶在转录水平上受胆汁 盐，脂肪酸，在某些物种中，胆固醇;然而， 这些主要的生理调节因子 改变7-α-羟化酶基因的转录， 未知 这在很大程度上是由于缺乏体外或细胞 复制所观察到的主要调节形式的文化系统 in vivo. 此外，含有启动子/报告基因的转基因大鼠， 构建高达4kb的7-α-羟化酶5 -侧翼DNA 表现出非常低的转基因表达水平， 由胆汁板条或胆固醇调节，表明其他序列 比存在于4kb的5 '侧翼DNA中的那些更需要 在体内正常表达和调节。 的总体目标 这项研究的目的是了解7-alpha- 羟化酶基因受胆汁盐和脂肪酸调节， 确定胆固醇上调的机制 7-α-羟化酶基因在某些物种（大鼠和 小鼠），但在其他动物（仓鼠）中没有。 利用细胞核和细胞核提取物 来自饲喂胆盐、胆盐螯合剂、脂肪酸或 胆固醇，我们建议对大鼠和仓鼠进行系统评价， 以DNA酶I超敏位点起始的7 -α-羟化酶基因座 定位以确定重要的调控序列，然后是DNA酶 I足迹和甲基化干扰研究，以确定核 因子结合位点。 将使用凝胶分析选定的足迹 迁移率变动分析和竞争性寡核苷酸来鉴定已知的 转录因子 以这种方式确定的潜在增强将 克隆到7-α-羟化酶近端启动子（或 异源启动子）和增强子活性的测试， 能够在体内赋予对生理调节剂的反应性， 腺病毒介导的基因转移。 在不太可能的情况下， 序列介导的主要生理反应 使用体内瞬时转染不能鉴定调节子 我们将使用转基因动物来绘制 序列负责调节。 转基因小鼠最初将 使用大鼠7-α-羟化酶基因的P1克隆产生。 然后将在以下指导下进行缺失诱变分析： DNase I超敏位点定位的结果。 这些研究将 提供关于转录调控的关键信息， 7-α-羟化酶基因在体内，并可能打开大门， 制定新的战略， 胆固醇转化为胆盐，降低心血管风险。

Hepatic cholesterol 7-alpha-hydroxylase catalyzes the initial and rate- limiting step in the major pathway where by cholesterol is converted to bile salts. As such, 7-alpha-hydroxylase plays a central role both in the maintenance of whole body sterol balance and, secondarily, in the regulation of serum lipoprotein concentrations. Hepatic 7-alpha- hydroxylase is regulated at the transcriptional level in response to bile salts, fatty acids and, in some species, cholesterol; however, the molecular mechanisms where by these major physiological regulators alter transcription of the 7-alpha-hydroxylase gene are completely unknown. This is due, in large part, to the lack of in vitro or cell culture systems that reproduce the major forms of regulation observed in vivo. Moreover, transgenic rats containing promoter/reporter constructs up to 4 kb of 7-alpha-hydroxylase 5 -flanking DNA manifested very low levels of transgene expression that were minimally regulated by bile slats or cholesterol indicating that sequences other than those present in the 4 kb of 5'-flanking DNA are required for normal expression and regulation in vivo. The overall goal of the proposed research is to understand how transcription of the 7-alpha- hydroxylase gene is regulated by bile salts and fatty acids and to determine the mechanism whereby cholesterol up regulates transcription of the 7-alpha-hydroxylase gene in some species (rats and mice) but not in others (hamsters). Using nuclei and nuclear extracts from animals fed bile salts, bile salt sequestrants, fatty acids or cholesterol, we propose a systematic evaluation of the rat and hamster 7 -alpha-hydroxylase gene loci starting with DNase I hypersensitive site mapping to identify important regulatory sequences followed by DNase I footprinting and methylation interference studies to identify nuclear factor binding sites. Selected footprints will be analyzed using gel mobility shift assays and competitor oligonucleotides to identify known transcription factors. Potential enhances identified in this manner will be cloned next to the 7-alpha-hydroxylase proximal promoter (or a heterologous promoter) and testing for enhancer activity and the ability to confer responsiveness to physiologic regulators in vivo using adenovirus-mediated gene transfer. In the unlikely event that the sequences mediating responsiveness to the major physiological regulators cannot be identified using the in vivo transient transfection studies outline above, we will use transgenic animals to map the sequences responsible for regulation. Transgenic mice will initially be generated using P1 clone of the rat 7-alpha-hydroxylase gene. Deletional mutagenesis analysis will then be carried out guided by the results of DNase I hypersensitive site mapping. These studies will provide critical information regarding the transcriptional regulation of the 7-alpha-hydroxylase gene in vivo and may open the door for the development of novel strategies aimed at enhancing the conversion of cholesterol to bile salts and reducing cardiovascular risk.