Modeling p63-associated human birth defects with systems developmental biology approaches

利用系统发育生物学方法对 p63 相关人类出生缺陷进行建模

基本信息

批准号：
10705852
负责人：
Rui Yi
金额：
$ 46.36万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-16 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10705852
关键词：
Address Adult Affect Alleles Basement membrane Binding Biological Process CRISPR/Cas technology Cell Adhesion Cell Lineage Cells Child Chromatin Cleft Palate Clustered Regularly Interspaced Short Palindromic Repeats Competence Congenital Abnormality DNA Binding Domain Data Defect Development Developmental Biology Dryness Ectodermal Dysplasia Embryonic Development Enhancers Epithelial Cells Epithelium Etiology Event Extracellular Matrix Failure Gene Expression Genes Genetic Genetic Models Genetic Transcription Genome Genomics Goals Growth Hair Hair follicle structure Hand Heterozygote Homo Human Human Development Hypopigmentation Impairment Individual Knock-in Knowledge Limb Development Maintenance Mediating Mesenchymal Messenger RNA Mission Modeling Molecular Morphogenesis Mus Mutant Strains Mice Mutation National Institute of Child Health and Human Development Natural regeneration Neonatal Patients Phenotype Point Mutation Reporting Research Role Scalp structure Signal Pathway Signal Transduction Skin Specific qualifier value Structural Congenital Anomalies Syndrome System Systems Biology Testing Tissues WNT Signaling Pathway appendage cleft lip and palate computerized tools ectrodactyly-ectodermal dysplasia-clefting syndrome epithelial stem cell foot gene regulatory network genetic analysis in vivo insight loss of function malformation mouse model mutant novel novel therapeutics orofacial cleft precise genome editing promoter single cell analysis single cell technology single-cell RNA sequencing skin organogenesis stemness success tool transcription factor transcriptome

项目摘要

Project Summary: The overarching goal of the proposed research is to understand how point mutations in the DNA binding domain (DBD) of p63 cause different phenotypes in Ectrodactyly, Ectodermal Dysplasia, and Cleft lip/palate (EEC) syndrome. The transcription factor p63 is the most critical regulator governing epithelial fate specification and the maintenance of stemness in adult epithelial tissues. Loss of p63 expression during mouse embryonic development leads to widespread failure of epithelial and limb development. In humans, heterozygous p63 mutations causes birth defects that include ectodermal dysplasia, orofacial clefting and hand/foot malformation. Interestingly, p63 mutations in the DBD often cause different phenotypes in the skin of human patients. This suggest the existence of p63 DBD mutation-sensitive enhancers and their regulated genes. Our preliminary studies have revealed that: 1) p63 profoundly changes transcriptome, chromatin accessibility and signaling competence of epithelial cells during skin development; 2) signaling pathways that are directly relevant to hair morphogenesis, including Wnt and EDAR signaling, are direct targets of p63. This establishes a molecular basis to examine the mechanism of hair defects observed in EEC; 3) our novel R279C and R280C het and homo mutant mice show different defects than p63 KO, revealing distinct biological functions of each mutation; 4) R279C and R280C het mutations differentially affect critical genes involved in epidermal fate specification and induction of hair follicles; 5) R279C het mutation affects the H3K27Ac levels of a subset of p63 bound enhancers. Our hypothesis is that a subset of p63 enhancers is sensitive to p63 DBD mutations, and their dysregulated genes underlie the defects in the skin of p63 mutants. To investigate this hypothesis, we propose to use systems developmental biology tools including scRNA-seq, scATAC-seq and their associated computational tools, combining with a high-fidelity CRISPR-mediated knockin approach, to examine p63-controlled enhancers and gene regulatory networks (GRNs) in murine skin. We propose to 1) investigate the role of EEC-associated p63 mutations in epidermal fate specification; 2) Investigate the role of EEC-associated p63 mutations in hair morphogenesis; 3) elucidate the impact of p63 DBD mutations on open chromatin accessibility, enhancer activity and genome organization. Success of these aims will provide genetic, genomic and molecular insights into the etiology of birth defects caused by p63 mutations.

项目概要：拟议研究的总体目标是了解点突变如何 p63 的 DNA 结合域 (DBD) 导致外指畸形、外胚层畸形的不同表型发育不良和唇裂/腭裂 (EEC) 综合征。转录因子p63是最关键的控制上皮命运规范和成人干性维持的调节因子上皮组织。小鼠胚胎发育过程中 p63 表达缺失会导致上皮和肢体发育普遍失败。在人类中，杂合 p63 突变导致出生缺陷，包括外胚层发育不良、口面部裂和手/足畸形。有趣的是，DBD 中的 p63 突变通常会导致不同的表型。人类患者的皮肤。这表明 p63 DBD 突变敏感增强子的存在及其调控基因。我们的初步研究表明：1）p63 深刻改变转录组，皮肤发育过程中上皮细胞的染色质可及性和信号传导能力； 2）与毛发形态发生直接相关的信号通路，包括 Wnt 和 EDAR 信号传导，是 p63 的直接目标。这为检查该机制奠定了分子基础 EEC 中观察到的毛发缺陷； 3) 我们的新型 R279C 和 R280C het 和同源突变小鼠显示与 p63 KO 不同的缺陷，揭示了每个突变的不同生物学功能； 4）R279C 和 R280C het 突变对参与表皮命运规范的关键基因有不同影响和毛囊诱导； 5) R279C het 突变影响部分子集的 H3K27Ac 水平 p63 结合增强子。我们的假设是 p63 增强子的一个子集对 p63 DBD 敏感突变及其失调基因是 p63 突变体皮肤缺陷的基础。到研究这一假设，我们建议使用系统发育生物学工具，包括 scRNA-seq、scATAC-seq 及其相关计算工具，结合高保真度 CRISPR 介导的敲入方法，用于检查 p63 控制的增强子和基因调控小鼠皮肤中的网络（GRN）。我们建议 1) 研究 EEC 相关 p63 的作用表皮命运规范的突变； 2) 研究 EEC 相关 p63 突变的作用在头发形态发生中； 3）阐明p63 DBD突变对开放染色质的影响可及性、增强子活性和基因组组织。这些目标的成功将提供对 p63 引起的出生缺陷病因学的遗传学、基因组学和分子学见解突变。