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

项目总结： 这项拟议的研究的首要目标是了解点突变是如何在 P63的DNA结合域(DBD)导致外指、外胚层不同的表型 发育不良和唇腭裂(EEC)综合征。转录因子p63是最关键的 调控上皮细胞命运的调节因子与成人干细胞的维持 上皮组织。小鼠胚胎发育过程中p63表达缺失导致 广泛的上皮和肢体发育障碍。在人类中，杂合子p63突变 导致出生缺陷，包括外胚层发育不良、口裂和手/脚 畸形。有趣的是，DBD中的p63突变通常会导致不同的表型 人类病人的皮肤。这表明存在对p63 dbd突变敏感的增强子。 以及它们的调控基因。 我们的初步研究表明：1)p63深刻改变了转录组， 皮肤发育过程中上皮细胞染色质可及性和信号传递能力；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的直接靶点。这为研究这一机制奠定了分子基础。 在EEC中观察到的毛发缺陷；3)我们的新型R279C和R280C het和ho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突变不同地影响与表皮命运相关的关键基因 和毛囊的诱导；5)R279C het突变影响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。 突变及其失调的基因是p63突变体皮肤缺陷的根源。至 为了研究这一假设，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，与高保真 CRISPR介导的敲门法检测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相关的p63的作用 表皮命运规范中的突变；2)研究EEC相关p63突变的作用 3)阐明p63DBD突变对开放染色质的影响 可获得性、增强子活性和基因组组织。这些目标的成功将提供 对p63所致出生缺陷病因的遗传学、基因组和分子方面的见解 突变。

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.