Functional Characterization of Mediator Complex Proteins in Neural Crest and Craniofacial Development

神经嵴和颅面发育中介导复合蛋白的功能表征

• 批准号: 10448751
• 负责人: Soma Dash
• 金额: $ 10万
• 依托单位: STOWERS INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10448751
• 关键词: Address; Affect; Binding; Binding Proteins; Bioinformatics; Birth; Blood Vessels; Bone Development; Branchial arch structure; Candidate Disease Gene; Cartilage; Cell Communication; Cell Line; Cell physiology; Cells; Cellular Assay; ChIP-seq; Chondrogenesis; Cleft Palate; Cleidocranial Dysplasia; Complex; Congenital Abnormality; Congenital Disorders; Craniofacial Abnormalities; DNA; DNA Binding; Data; Defect; Development; Developmental Process; DiGeorge Syndrome; Ectoderm; Embryo; Embryonic Development; Endothelial Cells; Enhancers; Ethylnitrosourea; Etiology; Exhibits; Frontonasal Prominence; Gene Proteins; Genes; Genetic Enhancer Element; Genetic Transcription; Germ Layers; Head; Histology; Human; Immunoprecipitation; Jaw; Knowledge; Link; Mandible; Mandibulofacial Dysostosis; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Mesenchyme; Micrognathism; Molecular; Morphogenesis; Multiprotein Complexes; Mus; Mutagenesis; Neural Crest; Neural Crest Cell; Osteogenesis; Pathogenesis; Phenotype; Point Mutation; Pregnancy; Proteins; RNA; RNA Polymerase II; Regulation; Regulator Genes; Role; Signal Pathway; Signal Transduction; Specificity; Tail; Testing; Tissues; Training; Transcription Coregulator Gene; Triad Acrylic Resin; Work; base; beta catenin; bone; cartilage development; cellular development; chromatin immunoprecipitation; conditional knockout; craniofacial; craniofacial bone; craniofacial development; craniofacial disorder; craniofacial tissue; developmental disease; gene function; genetic analysis; improved; in vivo; innovation; insight; loss of function; mouse model; mutant; neural patterning; neuroregulation; novel; novel therapeutics; protein complex; single-cell RNA sequencing; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

PROJECT SUMMARY Craniofacial developmental disorders such as cleft palate and retrognathia (smaller lower jaw) often arise due to defects in neural crest cell development and affect 1 in 700 and 1 in 1,500 live human births, respectively. Cleft palate and retrognathia often present with other craniofacial anomalies in conditions such as Treacher Collins syndrome and DiGeorge syndrome. Although genetic analyses have identified the genes responsible for some craniofacial anomalies, the vast majority have an undiagnosed molecular etiology and cellular pathogenesis. In an ENU mutagenesis screen, the Med23 gene was identified to be critical for craniofacial development. A point mutation in Med23 results in craniofacial and vascular defects, leading to embryonic lethality at mid-gestation. Med23 belongs to the tail module of the Mediator complex, which is a global transcription coregulator for genes transcribed by RNA Polymerase II. Med23 is ubiquitously expressed in mouse embryos, thereby, raising an important question of how a ubiquitously expressed protein regulates tissue specific development during embryogenesis, particularly in craniofacial development. To understand the function of Med23 in craniofacial and neural crest cell development, I generated neural crest cell specific conditional knockouts of Med23 that exhibit cleft palate, retrognathia, glossoptosis and cleidocranial dysplasia. Interestingly, endothelial cell specific conditional knockouts of Med23 also result in craniofacial defects together with vascular defects. In this proposal, I will test the overarching hypothesis that Med23 and other tail module subunits of Mediator have important and distinct transcriptional regulatory functions in craniofacial development via the control of neural crest cell and endothelial cell transcriptomes. Specifically, I will address the following aims. (Aim 1) Identify the molecular mechanism underlying the craniofacial defect in neural crest cell specific mutants of Med23 by analyzing transcriptomic changes in these mutants as well as by identifying Med23 DNA and protein binding partners. (Aim 2) Characterize the pathogenesis of craniofacial defects in endothelial cell specific mutants of Med23 and their underlying molecular mechanisms by analysis of the craniofacial transcriptome. Furthermore, I will test the mechanism of Med23-mediated control of key regulatory genes that function in craniofacial development and are linked to craniofacial disorders. Specifically, I will investigate the molecular basis of Med23 function via its control of Runx2 and β-Catenin in the mandibular mesenchyme. (Aim 3) Generate and characterize loss of function mutants of the Mediator tail submodule protein, Med24. The broad impact of this innovative proposal will advance our mechanistic understanding of the gene and protein networks underlying the function of global transcription co-factor Mediator complex proteins in fundamental cellular processes and development.

项目摘要 颅面发育障碍，如腭裂和下颌后缩（下颌较小），往往是由于 神经嵴细胞发育缺陷，分别影响1/700和1/1，500活产婴儿。裂 在Treacher柯林斯等情况下，腭和下颌后缩通常与其他颅面异常一起出现 DiGeorge综合征。虽然基因分析已经确定了一些基因负责 颅面异常，绝大多数有一个未确诊的分子病因和细胞发病机制。在 通过ENU诱变筛选，Med 23基因被鉴定为颅面发育的关键基因。的点 Med 23突变导致颅面和血管缺陷，导致妊娠中期胚胎死亡。 Med 23属于Mediator复合体的尾模块，Mediator复合体是基因的全局转录辅助调节因子 由RNA聚合酶II转录。Med 23在小鼠胚胎中普遍表达，因此， 一个普遍表达的蛋白质如何调节组织特异性发育的重要问题， 胚胎发育，特别是颅面发育。目的：了解Med 23在颅面神经中的作用 和神经嵴细胞发育，我产生了Med 23的神经嵴细胞特异性条件性敲除， 出现腭裂、缩颌、舌下垂和锁骨颅发育不良。有趣的是，内皮细胞特异性 Med 23的条件性敲除也导致颅面缺陷以及血管缺陷。在这一提议中， 我将测试总体假设，即Med 23和其他尾模块亚基的调解人有重要的， 通过控制神经嵴细胞在颅面发育中具有独特的转录调节功能， 内皮细胞转录组。具体而言，我将讨论以下目标。(Aim（1）分子识别 通过分析Med 23的神经嵴细胞特异性突变体中颅面缺陷的潜在机制， 这些突变体中的转录组学变化以及通过鉴定Med 23 DNA和蛋白质结合配偶体。（目标 2)描述Med 23的内皮细胞特异性突变体中颅面缺损的发病机制及其与骨缺损的关系。 通过分析颅面转录组的潜在分子机制。此外，我将测试 Med 23介导的控制颅面发育关键调控基因的机制， 与颅面疾病有关具体来说，我将通过其研究Med 23功能的分子基础 下颌间充质中Runx 2和β-连环蛋白的控制。(Aim 3）产生和表征 介体尾亚模块蛋白Med 24的功能突变体。这一创新提案的广泛影响 将促进我们对基因和蛋白质网络的机制性理解，这些网络是全球生物多样性功能的基础。 转录辅因子介导复合体蛋白在基本细胞过程和发展。