Drug Metabolizing Enzymes In Humans

在一个新的项目中，酵母和哺乳动物双杂交筛选表明Med25是一种hnf4a结合蛋白，Med25是中介复合物的一个可变成员。我们已经证明Med25对于RNA聚合酶II的募集很重要，以选择hnf4a激活的启动子，如重要的药物代谢基因细胞色素P450 2C9 （CYP2C9）和CYP3A4。我们假设这涉及Med25和HNF4之间的直接相互作用，以改变CYP2C9基因的染色质构象，使其处于转录活性状态。构象的改变需要酶修饰组蛋白，这些酶被招募到靶基因。组蛋白修饰包括组蛋白n端末端赖氨酸和精氨酸氨基酸的甲基化或乙酰化。例如，组蛋白3赖氨酸4二甲基化（H3K4me2）与基因激活相关，而组蛋白3赖氨酸27三甲基化（H3K27me3）是基因沉默的标志。在本研究中，我们使用HepG2细胞来确定Med25在hnf4a依赖性CYP2C9表达的表观遗传调控中的作用。我们进行了染色质免疫沉淀，以鉴定与Med25蛋白水平相关的HNF4a结合位点组蛋白修饰。我们的研究结果表明，Med25表达的改变改变了组蛋白3上某些赖氨酸27的乙酰化和甲基化。当Med 25表达时，赖氨酸被乙酰化。然而，当用小发夹环RNAi沉默Med25时，H3K27被三甲基化，这是基因沉默的典型。这些结果表明Med25在CYP2C9近端HNF4a结合位点诱导了一种允许的染色质状态。同样，共聚焦显微镜显示Med25与关键的组蛋白修饰标记共定位。我们还使用甲醛辅助分离调控元件（FAIRE）测定了激活条件下开放CYP2C9染色质的水平。FAIRE数据表明，CYP2C9近端启动子HNF4a位点周围的染色质在激活核受体CAR、HNF4a和Med25存在时是开放的，但在Med25沉默时是关闭的。这些研究将在全球范围内扩展到更多的HNF4a诱导基因。一项新的研究表明，雌激素受体α (ERa)在配体和外源性Med25存在的情况下诱导CYP2C9启动子活性。免疫沉淀研究显示ERa和Med25在配体存在或不存在的情况下相互作用。染色质免疫沉淀研究表明，在ERa存在的情况下，Med25与ERE（雌激素反应元件）结合。在原代肝细胞中，Med25和ERa能提高CYP2C9的催化活性，但siMed25能抑制CYP2C9的活性。最近我们发现亲电试剂和氧化应激通过AP-1蛋白与两个AP-1位点相互作用诱导人原代肝细胞的CYP2C9和CYP2C19。当cJun和JunD分别占用两个站点时，两个站点之间存在循环。许多药物是亲电性的或已知对亲电性活化。这是一种新的CYP2C9和CYP2C19激活机制，不涉及异种感应受体CAR或PXR。在合作研究中，我们发现使用重组CYP2C19*10等位基因（我们实验室发现的）体外代谢抗血小板聚集药物Plavix（氯吡格雷）的清除率比正常CYP2C19低75%。因此，虽然它不是一个无活性的等位基因，但它的活性大大受损。不同CYP2C19底物的研究显示出不同程度的损伤。CYP2C19激活氯吡格雷从前药到活性药。CYP2C19*10等位基因也干扰了一些无效CYP2C19*2等位基因的基因分型测试。这提示临床研究应包括CYP2C19*10等位基因的基因分型。

In a new project, yeast and mammalian two-hybrid screens showed that Med25, a variable member of the mediator complex, is an HNF4a-binding protein. We have shown that Med25 is important for the recruitment of RNA Polymerase II to select sets of HNF4a-activated promoters such as the important drug-metabolizing gene cytochrome P450 2C9 (CYP2C9) and CYP3A4. We hypothesized that this involves direct interaction between Med25 and HNF4 to alter chromatin conformation of the CYP2C9 gene to a transcriptionally active state. Conformational change requires the modification of histones by enzymes that are recruited to target genes. Histone modifications include methylation or acetylation of lysine and arginine amino acids on histone N-terminal tails. For example, histone 3 lysine 4 dimethylation (H3K4me2) is associated with gene activation, while histone 3 lysine 27 trimethylation (H3K27me3) is a marker of gene silencing. In this study, we used HepG2 cells to determine the role of Med25 in the epigenetic regulation of HNF4a-dependent CYP2C9 expression. We performed chromatin immunoprecipitation to identify histone modifications at the HNF4a binding site in relation to Med25 protein levels. Our results indicate that altering Med25 expression modified acetylation and methylation of certain lysine 27 on histone 3. When Med 25 was expressed, this lysine was acetylated. However, when Med25 was silenced with small-hairpin looped RNAi, H3K27 was trimethylated which is prototypical in gene-silencing. These results indicate that Med25 induces a permissive chromatin state at the CYP2C9 proximal HNF4a binding site. Similarly, confocal microscopy revealed that Med25 colocalized with key histone modification markers. We have also determined levels of open CYP2C9 chromatin under activating conditions using formaldehyde-assisted isolation of regulatory elements (FAIRE). FAIRE data indicated that the chromatin around the HNF4a sites of the CYP2C9 proximal promoter was open in the presence of activating nuclear receptors CAR and HNF4a and Med25 but closed when Med25 was silenced. These studies will be extended globally to more HNF4a induced genes. A new study showed that the estrogen receptor alpha (ERa) induced CYP2C9 promoter activity in the presence of ligand and exogenous Med25. Immunoprecipitation studies showed interaction between ERa and Med25 in the presence or absence of ligand. Chromatin immunoprecipitation studies showed that Med25 bound to the ERE (estrogen responsive element in the presence of ERa. CYP2C9 catalytic activity was increased in primary hepatocytes by Med25 and ERa but silenced with siMed25. Recently we found that electrophiles and oxidative stress induce CYP2C9 and CYP2C19 in human primary hepatocytes through AP-1 proteins which interact with two AP-1 sites. There is looping between the two sites when occupied by cJun and JunD respectively. Many drugs are electrophilic or known to be activated to electrophiles. This is a new mechanism of activation of CYP2C9 and CYP2C19 not involving the xenosensing receptors CAR or PXR. In collaborative studies we showed that Plavix (clopidogrel) a drug which prevents platelet aggregation is metabolized with a 75% lower clearance in vitro using recombinant CYP2C19*10 allele (discovered in our laboratory) than normal CYP2C19. Thus, although it is not an inactive allele, its activity is greatly impaired. Studies with different CYP2C19 substrates showed different degrees of impairment. CYP2C19 activates clopidogrel from a prodrug to the active drug. The CYP2C19*10 allele also interferes with a number of genotyping tests for the null CYP2C19*2 allele. This suggests clinical studies should include genotyping of the CYP2C19*10 allele.