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

单细胞和单碱基对分辨率下颅面增强子的功能表征

基本信息

批准号：
10383769
负责人：
Justin Lee Cotney
金额：
$ 38.56万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-03 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10383769
关键词：
ATAC-seq Address Affect Architecture Atlases Base Pairing Binding Sites Body part Branchial arch structure CRISPR interference Cell Culture Techniques Cells Cephalic Child Chromatin Code Complex Congenital Abnormality Coupled Craniofacial Abnormalities Craniosynostosis Defect Development Disease Embryo Embryonic Development Enhancers Exons Face Gene Expression Gene Expression Regulation Genes Genetic Genetic Enhancer Element Genetic Transcription Genome Genomics Heritability Human Incidence Individual Knowledge Language Link Live Birth Location Maps Measurement Measures Methods Modeling Mus Mutation Neural Crest Cell Nucleic Acid Regulatory Sequences Organ Other Genetics Pattern Phenotype Play Population Pregnancy Proteins Publishing Regulator Genes Regulatory Element Resolution Role Site Specific qualifier value Syndrome System TWIST1 gene Technology Testing Tissues Untranslated RNA Variant Work base cell type chromatin immunoprecipitation chromosome conformation capture craniofacial craniofacial development craniofacial tissue disorder risk exome sequencing experimental study falls gene interaction genetic association genome wide association study human tissue orofacial cleft prevent risk variant single-cell RNA sequencing tissue culture transcriptome

项目摘要

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.

抽象的颅面畸形是世界范围内最常见的人类出生缺陷之一，每年有多达 200,000 名出生的儿童患有某种类型的颅面缺陷。这些缺陷可以作为涉及多个组织和器官的复杂综合征的一部分而发生。综合症的形式这些疾病已成功与近 500 个基因相关，其中包括 TWIST1 颅缝早闭和 IRF6 用于口颌面裂。然而，更常见的是身体的其他部分没有直接受累（50% 的颅缝早闭，70% 的口颌裂）。全基因组关联研究表明此类缺陷具有遗传性，但绝大多数关联不属于基因范围这表明基因调控缺陷是此类缺陷发生的主要原因。基因调控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，使他们难以识别和预测他们控制的基因。本次活动的总体目标应用程序是识别发育中的人脸中存在的细胞类型和功能表征重要的调控元件，将它们指定为单碱基对分辨率。目标 1 我们建议使用基于单细胞的方法系统地识别细胞类型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 至 5 周的组织以及胚胎 10.5 和 11.5 天的小鼠。在目标 2 中，我们提议鉴定这些调控序列和靶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，我们将识别这些组织中的调控元件，这些元件可以在颅神经嵴细胞的细胞培养模型中进行测试。这些增强剂将被评估当抑制或从基因组中去除时对基因表达的影响。那些具有显着将测试每个变体对基因表达的影响，以确定其中的重要位置。我们提出的研究将对活跃在细胞中的细胞类型产生最全面的了解。开发人脸并揭示个体非编码变异对基因的贡献表达。