PROJECT SUMMARY The overarching goal of my research program is to gain a comprehensive understanding of the variability in drug metabolism to make drug therapies more effective and less toxic. The cytochrome P450 drug- metabolizing enzymes (dmCYPs) are the main enzymes that metabolize nearly 70% of the drugs currently used, which are highly variable from person to person, necessitating personalized drug therapy. Genetic factors significantly affect the drug metabolism, and the variants of some dmCYPs are used as clinical biomarkers to predict the metabolizer status. However, the underlying principles of their variable expression/activity are far from clear. Variants that result in altered amino acid sequences have been studied extensively, but they fail to account for all the differences observed. On the other hand, technical challenges have thus far limited the studies on factors that affect mRNA and protein expression of dmCYPs. Most dmCYPs are multi-gene clusters with complex genomic architecture and diverse genetic variations. With the support of NIGMS funding, we have adopted innovative methodologies and dissected the complex genomic architecture of multiple gene clusters (e. g., CYP3A) and identified hidden cis-acting regulatory variants that affect gene expression (e. g., 3A4, 3A5 and 3A43). Our results demonstrate that a single variant can have opposing effects on two genes in the same locus (e. g, 3A4, and 3A43), while more than one variant can regulate a single gene in the same or opposite direction (e. g., CYP7A1 and CYP2D6) indicating complex domain-domain interactions within the gene cluster and epistasis between variants. Such complexity usually escapes genome wide association study based discovery and warrants a dedicated approach proposed here. With the success of dissecting the CYP3A cluster, we now plan to expand and analyze the other dmCYP gene clusters over the next five years. Furthermore, we have recently identified ligand-free estrogen receptor alpha (ESR1) as a master regulator of CYP3A4 and many other dmCYP genes. Genetic, epigenetic, and non-genetic factors that affect ESR1-centered regulatory network will likely affect dmCYP gene expression and enzyme activity via trans-acting mechanisms. We plan to use ESR1-knockout hepatocytes differentiated from iPSC to characterize ESR1-centered regulatory network and to identify factors regulating ESR1 and dmCYPs expression. The identified functional cis and trans-acting genetic variants and epigenetic factors, including non-coding RNAs, will be tested in human liver samples for their impact on mRNA and protein expression of dmCYPs. The clinical significance of the identified variants will be tested in clinical cohort via collaboration. With my track record of pharmacogenetics biomarker discovery, insights gained from studying complex multi-gene clusters, availability of novel technologies, and support from collaborators with expertise in bioinformatics, proteomics and pharmacogene...