Molecular mechanisms of Gli protein activation in vertebrate Hedgehog signaling and Hedgehog-driven cancers.

• Applicant: Dr. Hermann Broder Schmidt
• Subject Area: Cell Biology ; Biochemistry ; Developmental Biology
• Term: from 2015 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 282919647

The transcription factors of the Gli family are the final executors of the Hedgehog (Hh) signaling pathway, which orchestrates the patterning of the digits during limb development and establishes cell identify along the dorso-ventral axis of the developing neural tube. Unrestrained Hh signaling however results in constitutively active Gli proteins and drives the formation of various malignant cancers in children and adults. Normally, Gli proteins are subject to three levels of regulation. In the absence of Hh signals, full-length Gli (GliFL) is in a complex with its negative regulator Suppressor of Fused (SuFu) and thereby sequestered in the cytoplasm. This allows the efficient phosphorylation of GliFL by Protein Kinase A (PKA) and causes its subsequent partial degradation by the proteasome system. The remaining intact fragment travels to the nucleus and acts as a transcriptional repressor (GliR). Upon activation of the Hh pathway, the GliFL-SuFu complex escapes to the primary cilium, where the two proteins are presumably dissociated. GliFL then leaves the cilium and enters the nucleus, now functioning as a transcriptional activator (GliA). Notably, nuclear GliFL is hyper-phosphorylated and rapidly degraded by the proteasome. Despite of its fundamental importance for development and cancer, our understanding of Gli protein activation is far from complete. This is in particular the case for the process of GliFL release from SuFu and the control of GliA activity in the nucleus. This proposal strives to decipher the mechanisms underlying Gli transcription factor regulation in the cytoplasm, cilium and nucleus. First, I will test how the phosphorylation of GliFL by PKA in the cytoplasm regulates complex formation with SuFu and ciliary transport. This will be done by creating phospho-defective and -mimetic mutant version of GliFL and testing their (i) subcellular localization, (ii) association with SuFu and (iii) ability to execute Hh signaling. Second, I will use a biochemical approach to clarify the role of hyper-phosphorylation and proteasome-mediated metastability of GliFL in the nucleus and how these features are required to convert GliFL into a transcriptional activator (GliA). Finally, I will perform unbiased genetic loss- and gain-of-function screens to identify novel factors involved in the regulation of Gli proteins (using state-of-the-art CRISPR technologies). Taken together, my work will not only advance our knowledge of Gli protein regulation, but also our insight into the processes of ciliary trafficking and proteasome-mediated fine-tuning of transcriptional responses. In addition, it will focus on introducing novel therapeutic and diagnostic strategies to combat Hh-driven tumors.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. Rajat Rohatgi