Mediator modulates Gli3-dependent Sonic hedgehog signaling.
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.
Zhou
Haiying
H
Department of Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA.
Kim
Seokjoong
S
Ishii
Shunsuke
S
Boyer
Thomas G
TG
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D053823:Hedgehog Proteins / Q000378:metabolism*; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D056892:Mediator Complex; D008954:Models, Biological; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D011988:Receptors, Thyroid Hormone / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000074290:Zinc Finger Protein Gli3
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C065683:GLI3 protein, human; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; C413163:MDC1 protein, human; C460107:MED12 protein, human; D056892:Mediator Complex; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; C509239:SHH protein, human; D015534:Trans-Activators; D000074290:Zinc Finger Protein Gli3; D008156:Luciferases; D005982:Glutathione Transferase