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

项目总结 颅面发育障碍，如腭裂和下颌后垂(较小的下颌骨)，通常是由于 神经脊细胞发育缺陷分别影响每700名和1,500名活产儿中的1名。裂隙 在诸如Treacher Collins的情况下，腭部和颌后畸形通常与其他颅面畸形一起出现 综合征和迪乔治综合征。尽管遗传分析已经确定了导致一些 颅面畸形，绝大多数有未确诊的分子病因和细胞发病机制。在……里面 经ENU突变筛选，Med23基因被鉴定为对颅面发育至关重要。一分 Med23基因突变会导致头面部和血管缺陷，导致妊娠中期胚胎死亡。 Med23属于介体复合体的尾部模块，介体复合体是基因的全球转录辅助调节因子 Med23在小鼠胚胎中普遍表达，从而提高了一个 普遍表达的蛋白质如何调节组织特异性发育的重要问题 胚胎发生，尤其是在头面部发育过程中。了解Med23在颅面外科中的作用 和神经脊细胞发育，我产生了神经脊细胞特异性的Med23条件基因敲除 表现为腭裂、颌下垂、舌下垂和锁骨颅骨发育不良。有趣的是，内皮细胞特异性 Med23的条件性基因敲除也会导致头面部缺陷和血管缺陷。在这份提案中， 我将测试最重要的假设，即Med23和Mediator的其他尾部模块亚基具有重要的 神经脊细胞和神经营养因子在颅面发育中的转录调控功能 内皮细胞转录本。具体地说，我将解决以下目标。(目标1)确定分子 Med23神经脊细胞特异性突变体颅面缺陷的机制分析 通过鉴定Med23 DNA和蛋白质结合伙伴，这些突变体中的转录水平发生了变化。(目标 2)研究Med23及其内皮细胞特异性突变体中颅面缺损的发病机制。 通过对颅面转录组的分析来揭示其分子机制。此外，我将测试 Med23介导调控颅面部发育的关键基因及其机制 与头面部疾病有关。具体地说，我将通过它的分子基础来研究Med23功能 下颌间充质中Runx2和β-catenin的调控(目标3)产生损失并描述损失的特征 介体尾部亚模块蛋白MED24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械理解，这些网络是全球 转录辅助因子介体复合体蛋白在基本细胞过程和发育中的作用

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.