Project Summary Caries is a common oral disease that will affect 90% of US adults in the course of their lifetime. Carious lesions of the tooth develop over time when microbes on the tooth surface respond to carbohydrates by producing acids that degrade enamel. In addition to lifestyle factors like diet and hygiene, studies have shown a heritable component of caries risk, which is hypothesized to lie in the enamel proteins made during tooth development. Genome wide association studies (GWAS) have identified 52 loci containing common variants associated with caries risk, the majority of which lie in enhancers. Enhancers are noncoding sequences containing binding motifs that recruit transcription factors and loop to the promoter of a target gene to enhance its transcription in a time, tissue, and cell type-specific manner to control spatiotemporal gene expression in development. Enhancer dysfunction can affect expression of target genes in a subset of expression domains in the body, resulting in tissue-isolated disease, similar to caries presentation. We have observed enrichment of caries SNPs in enhancers active in craniofacial tissue from Carnegie stages 13-17, which contain the developing tooth. We observed a similar enrichment of caries SNPs in conserved enhancers of mouse craniofacial regions from similar developmental stages, including in isolated mouse tooth buds. These results suggest a role for evolutionarily conserved active enhancers of the early tooth in caries predisposition. While we have associated specific tooth enhancers with caries risk, their target genes, which may be ultimately responsible for caries predisposition, remain unidentified. Additionally, these results do not differentiate between the diverse cell types of the tooth including the enamel knot, dental epithelial cells, and dental mesenchymal cells. Given the production of enamel matrix proteins by dental epithelial cells during development, we hypothesize that dysregulation of enamel matrix genes in epithelial cells of the developing tooth, driven by variants in the non-coding genome, predisposes one to caries development. In this proposal, we will identify cell type-specific enhancers at evolutionarily conserved loci associated with caries risk and predict their target genes in the early developing tooth. Aim 1 will identify cell type specific enhancers associated with caries, by performing scATAC-seq on bud stage mouse incisors and using functional chromatin annotations to identify cell type-specific enhancers, then applying linkage disequilibrium-based computational methods to find the subset of enhancers associated with caries risk. Aim 2 will determine caries-associated genes in the dental epithelium of the early developing tooth by integrating multiple “omics” data sets into an activity by contact computational model. With this investigation we will identify caries-associated enhancers and their target genes, which will facilitate quantitative and functional car...