Predicting Tissue Specific Gli3 Regulatory Activity Using Hand2

Project Summary The Hedgehog (Hh) signaling pathway is essential for normal embryonic development and when perturbed, frequently results in human disease, including those that impact development of the craniofacial complex. The Gli transcription factors are the downstream effectors of the pathway and have been the subject of much research as they are associated with a number of craniofacial syndromes (e.g., Grieg cephalopolysyndactyly) and can function as both transcriptional activators and repressors of the Hh pathway. Gli3 is most stable and abundant as a repressor. Despite this, our recent work identified a specific and necessary role for Gli3 activator (Gli3A) function during normal development of the mandible that requires additional regulatory inputs to convey a robust Gli3A response. Little is known regarding what is required for Gli3, as a bimodal transcription factor, to function as a potent activator during development. To address existing knowledge gaps in how full-length Gli3 is converted into an activator, we engineered a set of endogenously epitope tagged alleles for Gli3. With these novel tools we propose to: (Aim1) determine if/how chromatin accessibility modulates Gli3A function; (Aim2) investigate the role of co-factors and regulatory grammar in regulating enhancer output; and (Aim3) unbiasedly identify protein interactors of Gli3A within the nucleus. We will focus on craniofacial development, specifically development of the mandible, as a relevant model for testing these principles given the requirement for Gli activity during glossogenesis and mandibular skeletogenesis. This developmental system will allow us to determine the requirement for chromatin accessibility, exhaustively interrogate the regulatory grammar, and identify the constituents of the Gli3 activator complex. Recent technological advances will enable hypothesis testing through single-cell analysis of chromatin accessibility and transcription profiles from mutants predicted to have pioneering activity. To validate our findings, we will perform in vivo experiments to test enhancer activity and apply CRISPR/Cas9 mutagenesis to functionally assess native binding site requirements. Collectively, our studies will shed light on the regulatory principles governing Gli-directed cellular programs that when disrupted can result in range of human disorders ranging from structural birth defects to cancer. Understanding how these programs are deployed and interpreted during normal development has the potential to improve human health through the expansion of therapeutic interventions that can help mitigate pathway dysregulation.