| Mediator is an evolutionarily conserved multiprotein adaptor complex that represents a common interface between gene-specific activator and repressor proteins and the eukaryotic RNA polymerase II general transcription machinery. In this capacity, Mediator has thus been proposed to function as a general conduit and integrator of regulatory signals that converge on the promoters of protein-coding genes. In agreement with this idea, recent studies have revealed that, in metazoans, diverse signaling pathways require specific Mediator subunits to properly regulate transcription when activated. These include MED1 for nuclear receptor, MED14 for interferon-gamma, MED23 for MAP kinase, and MED15 for TGF-beta signaling pathways.; MED12 is a 230KD Mediator subunit for which identifiable orthologs exist throughout the animal kingdom. In Saccharomyces cerevisiae, the MED12 ortholog SRB8 was initially identified through genetic screens for negative regulators of transcription and subsequently shown to be a component of Mediator. In Schizosaccharomyces pombe, global gene expression profiling analysis suggested that SRB8/MED12 is required for both positive and negative transcriptional regulation of a small, defined set of genes. In metazoans, no such systematic analysis of MED12-dependent gene expression profiles has heretofore been described. Nonetheless, the phenotypes of MED12 mutations in model metazoan organisms are complex and often partially resemble mutations in multiple developmental signaling pathways, including the WNT, Shh and Notch pathways. However, whether these observations reflect a direct or indirect requirement for MED12 in these diverse developmental signaling pathways remains to be established.; To gain further insight into the contribution of MED12 to global transcriptional regulation in human cells, I employed a gene expression profiling approach to identify MED12-regulated genes. To this end, I examined the influence of RNAi-mediated MED12 suppression on the global gene expression profile of BG-1 human ovarian cancer cells. This analysis identified ∼200 genes whose expression levels were consistently up- or down-regulated in three independent coupled siRNA/microarray experiments. Gene Ontology analysis indicated that MED12-regulated genes participate in diverse functional pathways including signal transduction, cell proliferation, cell adhesion, and neurodevelopmental pathways. Notably, a set of MED12-regulated genes identified in this analysis were previously reported to be targets of the WNT signal transduction pathway, to which MED12 has recently been linked through genetic analyses in lower model metazoan organisms.; Signal transduction within the canonical WNT signaling pathway drives development and carcinogenesis through programmed or un-programmed changes in gene transcription. Central to the ability of WNT signaling to regulate the transcriptional profile of target cells is a transcriptional activator protein, beta-catenin, whose stability is regulated by WNT signaling. While the upstream events linked to signal-induced activation of beta-catenin in the cytoplasm have been deciphered in considerable detail, much less is known regarding the mechanism by which beta-catenin stimulates target gene transcription in the nucleus. The observation that MED12 depletion affects the expression profile of specific WNT-target genes coupled with the role of Mediator as an established signal transducer led me to investigate whether and how MED12 is directly involved in WNT/beta-catenin signaling. Here I show that beta-catenin physically and functionally targets the MED12 subunit in Mediator to activate transcription. The beta-catenin transactivation domain binds directly to isolated MED12 and intact Mediator both in vitro and in vivo, and Mediator is recruited to Wnt-responsive genes in a beta-catenin-dependent manner. Disruption of the beta-catenin-MED12 interaction through dominant negative interference or RNAi-mediat... |
