Mediator Modulates Gli3-Dependent Sonic Hedgehog Signaling

Abstract

The physiological and pathological manifestations of Sonic hedgehog (Shh) signaling arise from the specification of unique transcriptional programs dependent upon key nuclear effectors of the Ci/Gli family of transcription factors. However, the underlying mechanism by which Gli proteins regulate target gene transcription in the nucleus remains poorly understood. Here, we identify and characterize a physical and functional interaction between Gli3 and the MED12 subunit within the RNA polymerase II transcriptional Mediator. We show that Gli3 binds to MED12 and intact Mediator both in vitro and in vivo through a Gli3 transactivation domain (MBD; MED12/Mediator-binding domain) whose activity derives from concerted functional interactions with both Mediator and the histone acetyltransferase CBP. Analysis of MBD truncation mutants revealed an excellent correlation between the in vivo activation strength of an MBD derivative and its ability to bind MED12 and intact Mediator in vitro, indicative of a critical functional interaction between the Gli3 MBD and the MED12 interface in Mediator. Disruption of the Gli3-MED12 interaction through dominant-negative interference inhibited, while RNA interference-mediated MED12 depletion enhanced, both MBD transactivation function and Gli3 target gene induction in response to Shh signaling. We propose that activated Gli3 physically targets the MED12 interface within Mediator in order to functionally reverse Mediator-dependent suppression of Shh target gene transcription. These findings thus link MED12 to the modulation of Gli3-dependent Shh signaling and further implicate Mediator in a broad range of developmental and pathological processes driven by Shh signal transduction.

We thank the following individuals for their generous gifts of reagents used in this study: Susan Mackem for Gli3 antibody, Michael Garabedian for MED14 antibody, Paul Lieberman for mammalian and baculovirus expression vectors for human CBP, Hiroshi Sasaki for the 8X3′Gli-BS Luc reporter plasmid, Andreas Hecht for GAL4- and GST-β-catenin expression plasmids, and Arnold Berk for the GAL4-E1A and GAL4-VP16 expression plasmids. We are grateful to Pao-Tien Chuang and Alan Brooks for advice regarding production of Shh-conditioned medium. We also thank Amy Trauernicht, Ning Ding, Xuan Xu, Sejin Kim, Paula Garza, Wei Tan, and P. Renee Yew for advice, discussion, and comments.

This work was supported by Public Health Service grant CA-0908301 from the National Cancer Institute (T.G.B.) and by U.S. Army Department of Defense BCRP grants DAMD17-03-1-0272 and DAMD17-02-1-0584 (T.G.B.).