Do chromatin changes control polymorphic expression of drug metabolizing genes?

性状（由申请方提供）：CYP 3A 4细胞色素p450和UGT 1A 1和UGT 1A 6 UDP-葡萄糖醛酸转移酶是药物、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。这些酶的活性在个体之间变化高达40倍，这可能对药物疗效或毒性和癌症风险产生重大影响。这些基因的启动子中的遗传多态性可显著上调或下调其表达。然而，在许多情况下，这些转录效应没有明显的解释，因为多态性通常不会改变已知的转录因子结合位点。我们推测这些多态性可能通过改变启动子核小体的位置间接调节转录因子结合。DNA序列可以通过产生低能、默认的核小体结合位点来影响核小体位置，我们最初的计算预测表明，CYP 3A 4、UGT 1A 1和UGT 1A 6启动子多态性可以显著改变默认的核小体位置。我们的初步研究结果还表明，DNA序列决定了核小体的位置移动和/或结构改变的染色质重塑复合物人SWI/SNF，这些位置不同于默认的位置。尽管CYP 3A 4、UGT 1A 1和UGT 1A 6很重要，但目前还没有关于其野生型或变体启动子的染色质结构的详细信息。在这里提出的工作中，我们将检查这些启动子的默认和hSWI/SNF重塑的染色质结构，在体外系统和体内，在人肝细胞系（使用RNAi控制hSWI/SNF水平）。然后，我们将通过比较野生型和变体启动子在体外和体内的结果来检验调节多态性改变这种染色质结构的假设。这项工作是PA-06-149 R21资助机制的理想选择，因为它将探索一种新的假设，该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重要见解，并且因为它将开发研究染色质结构对转录影响的新技术。CYP 3A 4、UGT 1A 1和UGT 1A 6表达和活性的个体间变异性具有显著的临床相关性，因为这种变异性可以极大地影响许多药物的全身生物利用度和/或毒性，并且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相关。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，也可能揭示一种新的机制，即启动子多态性不直接改变转录因子结合位点，但可以极大地影响转录。这些见解可能会提出新的方法，药物代谢率可以在临床上控制，并可能会突出一个新的模式，基因控制转录调控。

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.