Do chromatin changes control polymorphic expression of drug metabolizing genes?

描述(申请人提供)：CYP3A4细胞色素P450和UGT1A1和UGT1A6 UDP-葡萄糖醛酸基转移酶是药物、外源性致癌物和内源性化合物(如类固醇激素)代谢的一些最重要的酶。这些酶的活性在不同的人之间差异高达40倍，这可能对药物疗效或毒性和癌症风险产生重大影响。这些基因启动子的遗传多态可以显著上调或下调它们的表达。然而，在许多情况下，对这些转录效应没有明显的解释，因为这些多态通常不会改变已知的转录因子结合位点。我们推测这些多态可能通过改变启动子核小体的位置来间接调节转录因子的结合。DNA序列可以通过创建低能量的默认核小体结合位点来影响核小体位置，我们的初步计算预测表明，CYP3A4、UGT1A1和UGT1A6启动子的多态可以显著改变默认核小体位置。我们的初步结果还揭示了DNA序列决定了由染色质重塑复合体人SWI/SNF移动和/或结构改变的核小体的位置，并且这些位置与默认位置不同。尽管CYP3A4、UGT1A1和UGT1A6很重要，但目前还没有关于它们的野生型或变异型启动子染色质结构的详细信息。在这里提出的工作中，我们将在体外系统和活体人类肝细胞系(使用RNAi控制hSWI/SNF水平)中检查这些启动子的默认和hSWI/SNF重塑的染色质结构。然后，我们将通过比较野生型和变异型启动子在体外和体内的结果来检验调控多态改变染色质结构的假设。这项工作是PA-06-149 R21授予机制的理想选择，因为它将探索一种新的假设，可能为药物代谢基因表达中肝细胞变异性的起源提供必要的见解，并且因为它将开发新的技术来研究染色质结构对转录的影响。CYP3A4、UGT1A1和UGT1A6表达和活性的个体间变异具有重要的临床意义，因为这种变异可以极大地影响许多药物的系统生物利用度和/或毒性，并与许多形式的癌症以及内源性化合物代谢不当引起的疾病有关。这些研究的成功完成将提供关于这些基因的启动子染色质结构的第一个详细信息，并可能揭示一种新的机制，即不直接改变转录因子结合位点的启动子多态仍然可以极大地影响转录。这些见解可能会为临床上控制药物代谢率提供新的方法，并可能突出转录调控的基因控制的新范例。

DESCRIPTION (provided by applicant): The CYP3A4 cytochrome p450 and UGT1A1 and UGT1A6 UDP-glucuronosyl transferases are some of the most important enzymes for the metabolism of drugs, exogenous carcinogens and endogenous compounds such as steroid hormones. The activities of these enzymes vary up to 40-fold between individuals, and this can have a major effect on drug efficacy or toxicity and cancer risk. Genetic polymorphisms in the promoters of these genes can significantly upregulate or downregulate their expression. In many cases, however, there is no obvious explanation for these transcriptional effects, since the polymorphisms often do not alter known transcription factor binding sites. We hypothesize that these polymorphisms may regulate transcription factor binding indirectly, by altering the positions of promoter nucleosomes. DNA sequence can affect nucleosome positions by creating low energy, default nucleosome binding sites, and our initial computational predictions indicate that CYP3A4, UGT1A1 and UGT1A6 promoter polymorphisms can significantly alter default nucleosome positions. Our preliminary results have also revealed that DNA sequence determines the locations of nucleosomes moved and/or structurally-altered by the chromatin remodeling complex human SWI/SNF, and that these positions differ from the default positions. Despite the importance of CYP3A4, UGT1A1 and UGT1A6, there is currently no detailed information about the chromatin structures of their wild type or variant promoters. In the work proposed here, we will examine the default and hSWI/SNF-remodeled chromatin structures of these promoters in both a defined in vitro system and in vivo, in a human liver cell line (using RNAi to control hSWI/SNF levels). We will then test the hypothesis that regulatory polymorphisms alter this chromatin structure by comparing the results of wild-type and variant promoters in vitro and in vivo. This work is ideal for the PA-06-149 R21 grant mechanism because it will explore a novel hypothesis which may provide essential insights into the origins of hepatocellular variability in drug metabolizing gene expression, and because it will develop novel techniques for studying the effects of chromatin structure on transcription. Interindividual variability in CYP3A4, UGT1A1 and UGT1A6 expression and activity is of significant clinical relevance, since this variability can greatly influence the systemic bioavailability and/or toxicity of many drugs, and is linked to many forms of cancer as well as diseases arising from improper metabolism of endogenous compounds. The successful completion of these proposed studies will provide the first detailed information about the promoter chromatin structure of these genes, and may also reveal a novel mechanism by which promoter polymorphisms that do not directly alter transcription factor binding sites can nonetheless greatly affect transcription. These insights may suggest new ways by which drug metabolism rates can be controlled clinically, and may highlight a novel paradigm for the genetic control of transcriptional regulation.