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 Mediatordependent 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.

MED12 mutations link intellectual disability syndromes with dysregulated GLI3-dependent Sonic Hedgehog signaling

“…Our prior work revealed that an essential function of the GLI3 MBD in gene activation induced by Shh is to physically bind the MED12 interface in Mediator to functionally reverse a MED12/Mediator-imposed constraint on its transactivation activity (12). Notably, the isolated GLI3 MBD, when fused to a heterologous (i.e., Gal4) DNAbinding domain, will drive high levels of activated transcription in the absence of Shh through an identical mechanism (12), because the principal function of Shh is to promote the nuclear accumulation of a full-length GLI3 activator carrying its C-terminal MBD. This Gal4-MBD-dependent transactivation assay thus offers a facile means to assess the functional consequences of the FG and Lujan mutations in MED12 on GLI3 transactivator function.…”

“…Nonetheless, relatively few studies have focused on the mechanism by which GLI3 stimulates target gene transcription in the nucleus. In this regard, we previously showed that SHHactivated GLI3, through a unique C-terminal transactivation domain (MED12/Mediator-binding domain, MBD) physically targets both Mediator and the histone acetyltransferase CBP to activate transcription (12). Furthermore, we found that the GLI3 MBD targets Mediator through both an unidentified subunit(s), likely required for MBD-directed Mediator recruitment onto GLI3-target genes, and MED12 within the kinase module.…”

“…Surprisingly, however, RNAi-mediated MED12 depletion enhanced GLI3 transactivation induced by SHH, implicating MED12/Mediator in suppression of GLI3 transactivation activity. Based on these findings, we proposed a model in which SHH-activated GLI3, through its MBD, targets the MED12 interface in Mediator to reverse a Mediator-imposed constraint on GLI3 MBD transactivation activity (12). Given the physical and functional interaction between GLI3 and MED12 coupled with the fact that mutations in each of these interacting proteins elicit congenital anomaly syndromes with overlapping phenotypes, we hypothesized that pathogenic mutations in MED12 leading to FG and Lujan syndromes elicit dysregulated GLI3-dependent SHH signaling.…”

“…Within the Mediator kinase module, MED12 is a critical transducer of regulatory information conveyed by signal-activated transcription factors linked to diverse developmental pathways, including the EGF, Notch, Wnt, and Hedgehog pathways (10)(11)(12). Furthermore, MED12 has been implicated in vertebrate neural development, and genetic variation in MED12 is associated with neuoropsychiatric illness, including schizophrenia and psychoses, in humans (5,13).…”

“…However, the underlying basis by which genetic disruption of MED12 elicits the broad spectrum of clinical phenotypes observed in FG and Lujan syndromes remains unknown. Notably, several phenotypes associated with FG and/or Lujan syndromes including macrocephaly, corpus callosal defects, hypertolerism, syndactyly, and cognitive impairment, overlap with a subset of those variously appearing in Greig cephalopolysyndactyly syndrome (GCPS) and/or Pallister-Hall syndrome (PHS) arising from mutations in GLI3, a Sonic Hedgehog (SHH) signaling effector and direct interaction target of MED12 (12,14).…”