Uncovering Caries Risk in the Noncoding Genome of the Developing Tooth

项目摘要 龋齿是一种常见的口腔疾病，将影响90%的美国成年人在他们的一生。龋损 当牙齿表面的微生物对碳水化合物产生反应时， 会降解牙釉质除了饮食和卫生等生活方式因素外，研究表明， 龋齿风险的组成部分，这是假设在于牙釉质蛋白质在牙齿 发展全基因组关联研究（GWAS）已经确定了52个含有常见变异的基因座 与龋齿风险有关，其中大部分是增强剂。增强子是非编码序列 含有结合基序，该基序募集转录因子并环到靶基因的启动子上， 其转录以时间、组织和细胞类型特异性的方式控制时空基因表达， 发展增强子功能障碍可以影响靶基因在表达结构域的子集中的表达， 身体，导致组织孤立的疾病，类似于龋齿的介绍。我们观察到， Carnegie阶段13-17的颅面组织中活性增强剂中的龋齿SNP，其包含发育中的 牙.我们在小鼠颅面区域的保守增强子中观察到类似的龋齿SNP富集 从相似的发育阶段，包括在孤立的小鼠牙芽。这些结果表明， 龋齿易感性中早期牙齿进化上保守的活性增强剂。虽然我们已经联系到 具有龋齿风险的特定牙齿增强剂，其靶基因，其可能最终导致龋齿 易感性，仍无法确定。此外，这些结果不能区分不同的细胞类型， 包括牙釉质结、牙上皮细胞和牙间充质细胞。鉴于生产 牙釉质基质蛋白质的调节异常，我们推测， 釉基质基因在上皮细胞的发育牙齿，驱动的变异，在非编码 基因组，易患龋齿。在本提案中，我们将确定细胞类型特异性 增强子在进化上保守的基因座与龋齿的风险，并预测其靶基因， 早期发育的牙齿目的1将通过以下方法鉴定与龋齿相关的细胞类型特异性增强子： 在芽期小鼠切牙上进行scATAC-seq并使用功能性染色质注释来鉴定细胞 类型特异性增强子，然后应用基于连锁不平衡的计算方法来找到子集 与龋齿风险相关的增强剂。目的2将确定龋齿相关基因在牙齿 通过将多个“组学”数据集整合到接触活动中， 计算模型通过这项研究，我们将确定龋齿相关的增强子及其靶基因， 这将有助于本提案范围之外的定量和功能性龋齿风险测定。的 在这个奖学金提出的实验将提供机会，以扩大我在基因组学和单 细胞技术的职业生涯作为一个临床科学家在牙科发展和疾病领域。

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 caries risk assays outside the scope of this proposal. The experiments proposed in this fellowship will provide the opportunity to expand my skills in genomics and single cell techniques for a career as a clinician-scientist in the field of dental development and disease.