Hepatocyte nuclear factor 4␣ (HNF4␣) controls the expression of many critical metabolic pathways, and the Mediator complex occupies a central role in recruiting RNA polymerase II (Pol II) to these gene promoters. An impaired transcriptional HNF4␣ network in human liver is responsible for many pathological conditions, such as altered drug metabolism, fatty liver, and diabetes. Here, we report that Med25, an associated member of the Mediator complex, is required for the association of HNF4␣ with Mediator, its several cofactors, and RNA Pol II. Further, increases and decreases in endogenous Med25 levels are reflected in the composition of the transcriptional complex, Pol II recruitment, and the expression of HNF4␣-bound target genes. A novel feature of Med25 is that it imparts "selectivity." Med25 affects only a significant subset of HNF4␣ target genes that selectively regulate drug and lipid metabolism. These results define a role for Med25 and the Mediator complex in the regulation of xenobiotic metabolism and lipid homeostasis.The liver is the central organ that controls/regulates such metabolic processes as lipid metabolism (lipogenesis and fatty acid ␤-oxidation [FAO]), energy homeostasis (glycolysis and gluconeogenesis), bile acid synthesis, and drug metabolism (4,48,49,57). Metabolism in the liver is governed by a highly dynamic transcriptional regulatory network whose key receptors may be classical (glucocorticoid), adopted (peroxisome proliferator activated receptor, farnesoid X receptor, liver specific X receptor, and constitutively active receptor/pregnane X receptor), or orphan (hepatocyte nuclear factor 4␣ [HNF4␣]) (6). HNF4␣, also known as NR2A1, is an important member of the orphan nuclear receptor superfamily (51, 53). When HNF4␣ binds to DNA, it regulates the expression of both constitutive genes, such as apolipoprotein genes (37), CYP7A1 and CYP8B1 (25, 27), the FXII and XIIIB genes (26), the proline oxidase gene (30), CYP2D6 (5), CYP2A6 (42), UGT1A9 (1), SULTA1 (31), and the glucose-6-phosphatase (G6PC) and Pck1 genes (47), and also xenobiotic inducible genes, such as CYP3A4 (56), CYP2C9 (8), and CYP2B6 (24). Malfunctions in the regulation of genes by HNF4␣ are responsible for alterations in drug metabolism and metabolic disorders, such as atherosclerosis, diabetes, hemophilia, hypoxia, medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) deficiency, and ornithine transcarbamylase (OTC) deficiency (16,22).Like most nuclear receptors, HNF4␣ displays a typical DNA binding domain (DBD), including a double zinc finger motif, a ligand binding domain (LBD), and two activation domains, AF1 and AF2, located in the N-and C-terminal regions, respectively (21, 53). The DNA binding domain binds to a cisacting element which optimally consists of a direct repeat of AGGTCA with a 1-or 2-nucleotide spacer (DR1/DR2) to which HNF4␣ binds as a homodimer (52). The ligand binding domain is structurally homologous to other receptors of the retinoid receptor family, with a well-defined hydrophobic pocket capable of ...