Dysregulation of Hedgehog (HH) signaling is linked to catastrophic birth defects in infants and is an underlying cause of the structural defects in a range of syndromes, including ciliopathies. The effects of HH signaling are primarily mediated through de-repression of target genes, but the mechanisms by which GLI proteins act as transcriptional repressors remain poorly understood. Recently, we found that GLI repressors prevent transcription of target genes by deactivating their own enhancers through chromatin-based histone deacetylation. We then found that GLI3 is present but apparently inert in early limb buds before the onset of HH signaling. We hypothesize that GLI repression is normally regulated by the expression of co-repressors rather than being the default state occurring in the absence of HH signaling. In Specific Aim 1 we will determine how GLI3 repressor activity is inactive prior to the onset of Hedgehog signaling. We will determine if GLI repression is inert because of sub-threshold protein kinase A activity or, alternatively, if it is triggered by the onset of HH expression. In parallel, we will determine if the lack of GLI3 repression is due to an unexpected early redundancy with GLI2. These findings will determine whether HH pathway members trigger GLI transcriptional repression. In Specific Aim 2 we will determine how GLI transcriptional repression regulates chromatin accessibility. GLI transcriptional repression causes both reductions in H3K27 acetylation as well as reductions in ATAC-seq accessibility at GLI enhancers. We will determine if the reduced accessibility occurs because of changes in nucleosomal density or because of reduced histone acetylation. Additionally, we hypothesize that GLI3 transcriptional repression occurs through the NuRD complex, which uniquely contains both the HDAC and chromatin remodeling activities that are observed with GLI3 repression. Our preliminary data supports this hypothesis, indicating that GLI3 binds to CHD4, a component of the NuRD repressor complex that mediates nucleosomal sliding. The results will provide a mechanistic understanding of how GLI transcriptional repression controls chromatin accessibility to regulate HH target gene expression. In Specific Aim 3, we will identify co-factors regulating GLI repressive activity. We hypothesize that GLI repression occurs through the recruitment of an unknown co-repressor complex containing HDAC activity. We will test several ranked candidate co-factors by determining if they bind to known GLI enhancers and if this binding is reduced in limb buds lacking GLI3. In a complementary, unbiased approach, we will identify factors that bind to GLI3 on chromatin using an endogenously biotinylated GLI3 allele. The identification and subsequent validation of a co-factor(s) mediating GLI repression will fill a major gap in our understanding of how HH signaling regulates gene expression. Overall, these results will be impactful in illuminating a novel layer ...