One of the most common craniofacial birth defects is craniosynostosis (CS), where connective tissue of the sutures that normally separate skull bones is prematurely replaced by bone. While most CS cases are of unknown origin, about 20% can be attributed to single gene mutations. Among these, heterozygous inactivating mutations in ERF cause CS of variable severity, affecting multiple sutures and sometimes manifesting later in childhood. Erf is an ETS2-related transcription factor (TF), but unlike most family members is a transcriptional repressor. It is thought to compete with activating ETS2 factors for binding to the consensus site GGAA and prevent induction of target genes. Despite widespread expression of ERF and the critical role that ETS2 signaling plays in normal development, haploinsufficiency for ERF leads to a very limited set of defects. Our proposal is aimed at discovering the underlying mechanisms leading to CS in a setting of reduced Erf. We have created a zebrafish erf mutant which displays severe multi–sutural CS, demonstrating deep conservation of the role of Erf in normal skull development. We previously identified three enhancers from the human genome which direct expression to skeletal precursor cells in transgenic zebrafish, two associated with RUNX2 and one with BMPER, that bind ERF in a yeast 1–hybrid assay. Both genes are independently implicated in CS risk, suggesting that their increased expression in ERF mutants contributes to the pathogenesis of CS. We propose to test specific hypotheses about the pathogenesis of CS caused by Erf reduction. In Aim 1, we will use live imaging to follow the progression of CS in erf mutant fish. We will determine the growth rate of the skull bones before and after suture formation, the timing of suture fusion, and how variable the process is among mutants. These data will allow us to distinguish among models of CS pathogenesis and focus our epigenomic profiling at the appropriate stages. In Aim 2 we will assess genome–wide changes in accessible chromatin and gene transcription in erf mutants during craniofacial development. We hypothesize that CS in erf mutants results from increased expression of a small number of critical genes. By correlating open chromatin regions containing Erf binding sites with upregulated genes in the erf mutants, we aim to identify critical genes that are direct targets of Erf regulation. Finally, in Aim 3 we will test the hypothesis that loss of Erf results in increased expression, of RUNX2 and BMPER and of additional candidate genes implicated in Aim 2, by determining the activity of identified enhancers in erf mutants. We will characterize the timing and tissue specificity of enhancer activities and quantify changes in expression in erf mutants. Successful completion of our Aims will provide detailed insight into the genetic and cellular mechanisms underlying craniosynostosis in the setting of reduced Erf and the conserved role of Erf in normal craniofacial dev...