The liver is the primary organ that responds to a variety of stimuli to regulate drug and lipid metabolism, including detoxification, storage, transport, and elimination (46
). Hepatocyte nuclear factor 4α is implicated in regulating the genes involved in metabolic processes (22
). Dysfunctional signaling in the HNF4α network is linked to several metabolic disorders, including maturity-onset diabetes of the young (MODY) (20
), diabetes, and atherosclerosis, and to altered drug metabolism and clearance (6
). Several coactivators belonging to different subcomplexes have been discovered and implicated in gene regulation controlling the various metabolic processes, either directly interacting with HNF4α (47
) or interacting in tandem with other nuclear receptors (CAR, GR, and C/EBP) (40
). While many of these cofactors were found to bind directly or indirectly to nuclear receptors to form the transcriptional complex, how they are targeted to specific loci on chromatin to give the regio- and genospecificity remains poorly understood. In the present study, we have identified Med25 as an interacting partner of HNF4α that transforms the inactive transcriptional complex to an active transcriptional complex and uncovered a role for Med25 in drug and lipid metabolism in the human liver. Med25 is variably associated with the Mediator complex (36
). Ectopic expression of HNF4α and Med25 stimulates many drug metabolism pathways, while the reduction of Med25 decreases them, clearly linking Med25 to the HNF4α-signaling network.
Mediators are a complex of coactivators anchored by Med1 (28
) that are able to synchronize gene expression to a variety of environmental signals regulating diverse biological processes (55
). However, evidence suggests that not all NR-mediated transcriptional activity is dependent on Med1 (35
), and transcriptional complexes could be formed in the absence of Med1 (18
). In our quest to identify other cofactors that regulate HNF4α signaling, we have identified Med25. Interestingly, although many cofactors bind with all nuclear receptors, Med25 is an exception since it fails to bind with two xenobiotic sensing receptors, CAR and PXR, or the vitamin D receptor (32
Several known coactivators have been identified in the immunoprecipitates of HNF4α belonging to the CBP-HAT complex (CBP and SRC-1), the Mediator complex (Med8), and the ASCOM complex (NCOA6), in addition to PGC-1α and RNA Pol II. We have now identified Flag-Med25 and Med8 as additional coactivators. Interestingly, most of these coactivators were found to bind directly with Med25 in GST pulldown in vitro
interaction assays, but the sites of interaction on Med25 are quite different. HNF4α binds to Med25 through the LXXLL motif, whereas chromatin modifiers such as CBP interact with the middle of the Med25-spanning PTOV domain. PGC-1α interacts with the C-terminal region but not through the LXXLL motif. Med1, SRC-1, and GRIP interact in the N-terminal region (see Fig. S2C, diagram at bottom, at http://www.niehs.nih.gov/research/atniehs/labs/ltp/human/docs/med25supplemental-data.pdf
). Although NCOA6 was identified in the HNF4α complex, it does not bind with Med25 directly. HNF4α and the majority of these coactivators colocalize with Med25 in the nucleus, whereas NCOA6 does not colocalize, suggesting that although these two proteins belong to the HNF4α-bound complex, they may exist as separate subcomplexes or be recruited in a sequential order.
Med25 enhances the transactivation potential of HNF4α. However, the synergistic activation of two target genes, CYP2C9 and CYP3A4, by CAR and HNF4α is further boosted by expression of Med25. The CAR ligand CITCO enhanced the synergistic transcriptional effect, suggesting that Med25 might be the final piece of the puzzle explaining the transcriptional activation of these target genes by xenobiotics.
The effect of Med25 appears to be solely mediated through a functional HNF4α protein, and reduction of Med25 levels inhibited the HNF4α-dependent activation of CYP2C9 but not CAR-mediated transcriptional activity. The synergistic activity of CAR and HNF4α was also blunted when endogenous Med25 levels were reduced, as seen from CYP2C9 and CYP3A4 promoter activation assays and mRNA induction assays. Our studies show that Med25 not only is essential for mediating HNF4α-dependent upregulation of target genes but also dramatically influences CAR-dependent upregulation through HNF4α (Fig. ). These transcriptional effects were seen in HepG2 cells and in primary hepatocytes, suggesting that the Med25 effects are not cell line specific and are thus applicable to human liver drug metabolism.
After establishing Med25 as a coactivator of HNF4α, we explored the purpose of Med25 in the transcriptional complex. On immunoprecipitation with HNF4α, nuclear extracts depleted of Med25 bound a different transcriptional complex than when Med25 was ectopically expressed. Mass spectral identification of all the proteins indicated that Med25 depletion resulted in a transcriptional complex, as indicated by the presence of NCOR and MLL4, whereas in the presence of Med25, we observed a transcriptional complex with coactivators such as Med1, Med8, Med14, MLL3, and RPA-1. We suggest that Med25 is essential for converting the NCOR-bound HNF4α complex (inactive) to the Mediator-bound HNF4α transcriptional (active) complex. At the molecular level, although it has been speculated that the Med1-anchored Mediator complex was required for the recruitment of RNA polymerase II, EMSA and ChIP data from HepG2 cells and primary human hepatocytes clearly showed that for a subset of genes typified by CYP2C9 and CYP3A4, it is Med25 and not a Med1-anchored Mediator complex that is essential for Pol II recruitment to the target promoter. Given the fact that Med25 is not part of the Mediator complex but an associated protein, these data clearly show that Med25 is essential for the recruitment. Moreover, from qPCR analysis we found that when Med25 was depleted, PPARα levels were decreased, whereas when HNF4α levels were increased, PPARα levels increased. In addition, PPARα would also require Med25 for transcription regulation of its target genes, namely, those encoding Acox, Ehhadh, etc. These results suggest that PPARα-mediated fatty acid oxidation may be regulated by Med25, leading to alterations in lipid metabolism, one of the two pathways identified in the IPA analysis. The effects of a reduction in Med25 only partially parallel those of a reduction in HNF4α. Clearly, as the Venn diagram indicates, there is only a partial overlap between the genes that are upregulated by overexpressing HNF4α and those that are downregulated by silencing Med25.
Interestingly, the expression of HNF4α target genes belonging to pathways such as Pck1 and G6PC involved in glucose homeostasis was not affected by reduced availability of Med25. These results suggest that Med25 acts in concert with HNF4α in regulating only genes involved in specific pathways. In the future, it will be of interest to investigate why Med25 regulates only some genes containing HNF4α binding sites in the promoter region. For the genes that were selected for further analysis by qPCR, we note that an HNF4α response element was quite close to the transcription start site, whereas in the nonresponsive genes the binding site was far from the start site. In the future, an unbiased genome-wide bioinformatics approach could help identify the mechanism that determines the selectivity of Med25 in regulating only certain HNF4α-dependent pathways.
In summary, we have shown that Med25 plays an important role in the regulation of drug and lipid metabolism in primary human hepatocytes as well as in human liver cell lines. While there are many known coactivators described for modulating a plethora of signaling pathways, we show that Med25 confers selectivity and plays a vital role in the recruitment of various other cofactors through HNF4α binding sites to mediate the effects of HNF4α on metabolism of drugs and lipids.