Recent collaborative studies showed that Plavix (clopidogrel) a drug which prevents platelet aggregation is metabolized with a 75% lower clearance in vitro using recombinant CYP2C19*10 allele (a defect discovered in our laboratory) than by wild-type CYP2C19 enzyme (1). 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 future clinical studies should include genotyping of the CYP2C19*10 allele. 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 (2). This year we reported that Med25 is important for the recruitment of RNA Polymerase II to select sets of HNF4a-activated promoters such as the important drug-metabolizing genes cytochrome P450 2C9 (CYP2C9) and CYP3A4 (2). We showed 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. 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. 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 (3). 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. Another collaborative study (4) 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. Recent studies in press show the effects of hypolipidemic drugs through the peroxisome proliferator-activated receptor alpha.

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Support Year
34
Fiscal Year
2015
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Indirect Cost
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U.S. National Inst of Environ Hlth Scis
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Englert, Neal A; Luo, George; Goldstein, Joyce A et al. (2015) Epigenetic modification of histone 3 lysine 27: mediator subunit MED25 is required for the dissociation of polycomb repressive complex 2 from the promoter of cytochrome P450 2C9. J Biol Chem 290:2264-78
Langaee, Taimour Y; Zhu, Hao-Jie; Wang, Xinwen et al. (2014) The influence of the CYP2C19*10 allele on clopidogrel activation and CYP2C19*2 genotyping. Pharmacogenet Genomics 24:381-6
Shi, Zhe; Yang, Wenjun; Goldstein, Joyce A et al. (2014) Med25 is required for estrogen receptor alpha (ER?)-mediated regulation of human CYP2C9 expression. Biochem Pharmacol 90:425-31
Makia, Ngome L; Surapureddi, Sailesh; Monostory, Katalin et al. (2014) Regulation of human CYP2C9 expression by electrophilic stress involves activator protein 1 activation and DNA looping. Mol Pharmacol 86:125-37
Rana, Ritu; Coulter, Sherry; Kinyamu, Harriet et al. (2013) RBCK1, an E3 ubiquitin ligase, interacts with and ubiquinates the human pregnane X receptor. Drug Metab Dispos 41:398-405
Joy, Melanie S; La, Mary; Wang, Jinzhao et al. (2012) Cyclophosphamide and 4-hydroxycyclophosphamide pharmacokinetics in patients with glomerulonephritis secondary to lupus and small vessel vasculitis. Br J Clin Pharmacol 74:445-55
Sam, Wai-Johnn; Chamberlain, Christine E; Lee, Su-Jun et al. (2012) Associations of ABCB1 and IL-10 genetic polymorphisms with sirolimus-induced dyslipidemia in renal transplant recipients. Transplantation 94:971-7
Lee, Su-Jun; Goldstein, Joyce A (2012) Comparison of CYP3A4 and CYP3A5: the effects of cytochrome b5 and NADPH-cytochrome P450 reductase on testosterone hydroxylation activities. Drug Metab Pharmacokinet 27:663-7
Makia, N L; Amunom, I; Falkner, K C et al. (2012) Activator protein-1 regulation of murine aldehyde dehydrogenase 1a1. Mol Pharmacol 82:601-13
Zhang, Shu-Yun; Surapureddi, Sailesh; Coulter, Sherry et al. (2012) Human CYP2C8 is post-transcriptionally regulated by microRNAs 103 and 107 in human liver. Mol Pharmacol 82:529-40

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