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Cytochrome P450 3A4 isoform ( CYP3A4 ) transcription is controlled by hepatic transcription factors (TFs), but how TFs dynamically interact remains uncertain. We hypothesize that several TFs form a regulatory network with nonlinear, dynamic, and hierarchical interactions. To resolve complex interactions, we have applied a computational approach for estimating Sobol's sensitivity indices (SSI) under generalized linear models to existing liver RNA expression microarray data (GSE9588) and RNA-seq data from genotype-tissue expression (GTEx), generating robust importance ranking of TF effects and interactions. The SSI-based analysis identified TFs and interacting TF pairs, triplets, and quadruplets involved in CYP3A4 expression. In addition to known CYP3A4 TFs, estrogen receptor α (ESR1) emerges as key TF with the strongest main effect and as the most frequently included TF interacting partner. Model predictions were validated using small interfering RNA (siRNA)/short hairpin RNA (shRNA) gene knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-mediated transcriptional activation of ESR1 in biliary epithelial Huh7 cells and human hepatocytes in the absence of estrogen. Moreover, ESR1 and known CYP3A4 TFs mutually regulate each other. Detectable in both male and female hepatocytes without added estrogen, the results demonstrate a role for unliganded ESR1 in CYP3A4 expression consistent with unliganded ESR1 signaling reported in other cell types. Added estrogen further enhances ESR1 effects. We propose a hierarchical regulatory network for CYP3A4 expression directed by ESR1 through self-regulation, cross regulation, and TF-TF interactions. We also demonstrate that ESR1 regulates the expression of other P450 enzymes, suggesting broad influence of ESR1 on xenobiotics metabolism in human liver. Further studies are required to understand the mechanisms underlying role of ESR1 in P450 regulation. SIGNIFICANCE STATEMENT: This study focuses on identifying key transcription factors and regulatory networks for CYP3A4, the main drug metabolizing enzymes in liver. We applied a new computational approach (Sobol's sensitivity analysis) to existing hepatic gene expression data to determine the role of transcription factors in regulating CYP3A4 expression, and used molecular genetics methods (siRNA/shRNA gene knockdown and CRISPR-mediated transcriptional activation) to test these interactions in life cells. This approach reveals a robust network of TFs, including their putative interactions and the relative impact of each interaction. We find that ESR1 serves as a key transcription factor function in regulating CYP3A4, and it appears to be acting at least in part in a ligand-free fashion.

Ligand-Free Estrogen Receptor α (ESR1) as Master Regulator for the Expression of CYP3A4 and Other Cytochrome P450 Enzymes in the Human Liver