Functional characterization of craniofacial enhancers at single cell and single base pair resolution

摘要 颅面畸形是世界上最常见的人类出生缺陷之一， 每年有多达20万名儿童出生时患有某种类型的头面部缺陷。这些缺陷可能 作为涉及多个组织和器官的复杂综合征的一部分而发生。血液病的证候形式 这些疾病已经成功地与包括Twist1在内的近500个基因联系在一起 颅缝融合和IRF6治疗口面部裂。然而，更常见的是，身体的其他部位都不是 直接受累(50%的颅缝畸形，70%的口面部裂隙)。全基因组关联研究 表明这种缺陷的遗传性，然而，绝大多数关联属于基因之外 这表明基因调控缺陷是此类缺陷发生的主要原因。基因 调节元件可以位于整个基因组中并且通常具有组织特异性活性， 这使得他们很难识别和预测他们控制的基因。这样做的总体目标是 应用是识别出现在发育中的人脸和功能上的细胞类型 描述将它们指定为单碱基对解析的重要调控元素。在目标1中 我们建议使用基于单细胞的方法系统地识别细胞类型的群体 测量人类颅面部基因组中的转录组和活性调控位点 妊娠4-5周的组织和胚胎10.5天和11.5天的小鼠。在《目标2》中，我们 建议确定这些调控序列和目标基因之间的物理相互作用 相同的组织类型。最后，在目标3中，我们将从这些组织中识别出能够 在脑神经脊细胞的细胞培养模型中进行测试。这些增强剂将被评估为 当被抑制或从基因组中移除时，对基因表达的影响。那些有显著意义的 将测试每个变种对基因表达的影响，以确定其中的重要位置。 我们建议的研究将生成最全面的关于活跃在 开发人脸并揭示个体非编码变异对基因的贡献 表情。

Abstract Craniofacial abnormalities are some of the most commonly occurring human birth defects worldwide, with up to 200,000 children born every year with some type of craniofacial defect. These defects can occur as part of complex syndromes that involve multiple tissues and organs. The syndromic forms of these disorders have been successfully linked to nearly 500 genes including TWIST1 for craniosynostosis and IRF6 for orofacial clefting. However more frequently no other part of the body is directly involved (50% of craniosynostoses, 70% of orofacial clefts). Genome wide association studies indicate heritability for such defects, however the vast majority of associations fall outside of genes suggesting defective gene regulation is a major contributor to incidence of such defects. Gene regulatory elements can be located throughout the genome and typically have tissue-specific activity, making them difficult to identify and predict what gene they control. The overall objective of this application is to identify the cell types present in the developing human face and functionally characterize important regulatory elements that specify them as single base pair resolution. In Aim 1 we propose to systematically identify populations of cell types using single-cell based methods for measuring the transcriptome and active regulatory sites across the genome in human craniofacial tissue from 4 to 5 weeks of gestation and mouse from embryonic days 10.5 and 11.5. In Aim 2 we propose to identify physical interactions between regulatory sequences and target genes in these same tissue types. Finally, in Aim 3 we will identify regulatory elements from these tissues that can be tested in a cell culture model of cranial neural crest cells. These enhancers will be assessed for effects on gene expression when repressed or removed from the genome. Those with significant effects on gene expression will be tested for every variant to identify important locations within them. Our proposed studies will generate the most comprehensive view of the cell types active in the developing human face and reveal the contributions individual noncoding variants make on gene expression.