Despite their architectural diversity, epithelial organs such as the vertebrate hair use the Sonic hedgehog (Shh) signaling system to expand multipotent progenitor cells during organ self-renewal. Shh signaling, mediated through the Gli family of transcription factors, plays a crucial role in human development with inappropriate pathway activity associated with a variety of human birth defects and up to 25% of human cancers. How progenitor cells receive precise Shh signal strength, without becoming transformed, remains poorly understood. Work from our lab support the hypothesis that cell-intrinsic Shh target gene induction in hair follicle progenitors is regulated by specific signals from the surrounding stromal cells. Our results point to two cell-intrinsic mechanisms that regulate Gli activity and Shh target gene induction in the epithelium: regulation of Gli protein destruction, and regulation of Gli activity through the presence of the accessory transcription factor Missing in Metastasis (MIM). We have demonstrated that progenitor proliferation depends on epithelial Gli protein accumulation. Gli is destroyed in a proteasome-dependent manner through two distinct, conserved destruction signals (degrons); removal of these degrons stabilizes GN1 and rapidly accelerates progenitor proliferation and BCCs in mice. We have also shown that MIM is required for Shh signaling and mediates Shh- dependent growth and invasion by forming complexes with Gli and the tumor-suppressor, Suppressor of Fused. The goal of this proposal is to gain a deeper understanding of the epithelial signaling inputs that control Gli protein stability and activity. We will: 1) Elucidate the mechanisms of Gli destruction in follicle progenitors by defining the contribution of each degron to Gli stability and activity in vivo and identifying the molecular components of signaling pathways that regulate Gli protein stability; 2) Determine how MIM antagonizes Suppressor of Fused by examining MIM activity in different states of the Shh pathway, determining which Sufu activity MIM antagonizes, and characterizing the Sufu protein surface with which MIM interacts; 3) Determine the impact of Gli regulators on murine hair follicle progenitors by ascertaining whether Gli3 is the major MIM regulator in vivo, determining the requirements for MIM in progenitor cells, determining how MIM levels impact Sufu-dependent hair progenitor proliferation, and determine how MIM and Sufu affect stabilized Gli1 activity. This effort is based on the premise that understanding the regulation of Gli function will lead to new insights into hedgehog signaling, with a goal toward new therapeutics for skin regeneration and epithelial tumors. [unreadable] [unreadable] [unreadable]