Regulation of orofacial ectodermal polarity by the CLP protein, IRF6

CLP 蛋白 IRF6 对口面部外胚层极性的调节

• 批准号: 8525152
• 负责人: Emily Yin Chu
• 金额: $ 3.66万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8525152
• 关键词: Ablation; Adherens Junction; Adhesions; Affect; Ameloblasts; Apical; Area; Award; Basic Science; Behavior; Binding; Biological Assay; C-terminal; Cadherins; Cell Adhesion; Cell Adhesion Molecules; Cell physiology; Cleaved cell; Cleft Lip; Clinical; Co-Immunoprecipitations; Complex; Congenital Abnormality; Conjunctival Pterygium; Cytoplasm; Data; Defect; Dental Enamel; Dentists; Development; Electroporation; Embryo; Enamel Formation; Epithelial; Epithelial Cells; Epithelium; Exhibits; Face; Foundations; Gene Expression; General Population; Genes; Genetic; Human; Hypodontia; In Vitro; Incidence; Inherited; Interferon Regulatory Factor 2; Interferons; Laboratories; Lead; Lip structure; Live Birth; Maintenance; Mandible; Measures; Mentorship; Microarray Analysis; Modeling; Molecular Genetics; Molecular Profiling; Mus; Mutate; Mutation; NME1 gene; Neoplasm Metastasis; Nuclear; Nucleoside-Diphosphate Kinase; Oral; Palate; Patients; Phosphorylation; Play; Population; Post-Translational Protein Processing; Premaxillary palate; Primordium; Procedures; Protein Binding; Proteins; Public Health; Regulation; Reporting; Research; Research Project Grants; Role; Scientist; Sequence Homology; Shapes; Site-Directed Mutagenesis; Specificity; Staging; Surface; Syndrome; T-Cell Lymphoma; Tissues; Tooth Abnormalities; Tooth Germ; Tooth structure; Training; Transfection; Universities; Van der Woude syndrome; Variant; Washington; Yeasts; base; deciduous tooth; design; interest; keratinocyte; laser capture microdissection; mutant; nectin; novel; oral cavity epithelium; orofacial; palatal fusion; paralogous gene; protein protein interaction; public health relevance; research study; transcription factor; treatment strategy; yeast two hybrid system

DESCRIPTION (provided by applicant): Interferon regulatory factor 6 (IRF6) variants are associated with common isolated forms of cleft lip with or without clefting of the palate (CLP), as well as with dominantly inherited forms of CLP. Based on high sequence and structural homology with other IRFs, IRF6 has been predicted to act as a transcription factor, although studies in keratinocytes have found IRF6 primarily in the cytoplasm. Recent studies in the Cox laboratory have found that IRF6 binds, in a phosphorylation-enhanced manner, to NME2 (non-metastatic 2), which has been reported to interact with factors that activate the PAR (PARD3-PARD6) polarity complex. NME regulated PARD3 activation stimulates establishment of epithelial apical and basolateral domains (i.e. polarity), ultimately impacting epithelial cell adhesion, shape, and behavior. Thus, via protein-protein interactions, IRF6 is hypothesized to contribute to the regulation of epithelial polarity, which when disrupted, leads to CLP. Specific aims were developed to investigate the contribution of IRF6 to primary palate and tooth development. Using site-directed mutagenesis, reported VWS patient mutations will be introduced into yeast and mammalian expression constructs and subsequently compared with wild type IRF6 in their ability to interact with NME2 . Potential effects of the IRF6-NME2 interaction on epithelial polarity will be assessed by measuring activity of polarity effector proteins, assaying for nuclear:cytoplasmic ratios of polarity markers, and observing for epithelial morphological changes. To determine a relationship between IRF6-NME2 binding and primary palatal fusion, a novel in ovo procedure developed by the Cox laboratory will be used to specifically perturb IRF6/NME2 expression in embryonic chick orofacial epithelia, and effects on primary palatal development characterized. In human patients, CLP is often associated with tooth abnormalities, and in mice, IRF6 was detected in ameloblasts and has roles in tooth epithelial invagination. The tooth also presents another mechanism to investigate IRF6 contribution to epithelial polarity; ameloblasts are derived from the oral epithelium and exhibit observable polarity during the secretory stage. A murine model with IRF6 conditionally ablated in ameloblasts will be developed, and IRF6 epithelial polarity contribution will be evaluated based on changes in ameloblast polarity, enamel formation, and gene expression (laser capture microdissection followed by microarray analysis). Thus, these studies will also explore a possible relationship between CLP and tooth development. This proposed F30 project was developed to help provide a foundation for an aspiring dentist scientist and contribute to research centered on genetic and molecular causes of CLP. IRF6 has been targeted because of its prominent contribution to CLP incidence. Understanding regulatory mechanisms behind CLP will help identify susceptible populations, lower incidence, and develop treatment strategies. With strong institutional support from the University of Washington and excellent mentorship, we are confident that this F30 award will aid in the basic sciences and clinical training required to be a successful clinician scientist.

描述（由申请人提供）：干扰素调节因子6（IRF6）变体与常见的孤立形式的唇裂伴或不伴腭裂（CLP）相关，如 以及显性遗传形式的CLP。基于与其他IRFs的高度序列和结构同源性，IRF6被预测为转录因子，尽管在角质形成细胞中的研究发现IRF6主要存在于细胞质中。考克斯实验室的最新研究发现，IRF 6以磷酸化增强的方式与NME 2（非转移性2）结合，据报道，NME 2与激活PAR（PARD3-PARD6）极性复合物的因子相互作用。NME调节的PARD3活化刺激上皮顶端和基底外侧结构域（即极性）的建立，最终影响上皮细胞粘附、形状和行为。因此，通过蛋白质-蛋白质相互作用，假设IRF6有助于调节上皮极性，当被破坏时，导致CLP。 具体目标是研究IRF6对初级腭和牙齿发育的贡献。使用定点诱变，将报道的VWS患者突变引入酵母和哺乳动物表达构建体中，随后与野生型IRF 6在其与NME 2相互作用的能力方面进行比较。IRF6-NME 2相互作用对上皮极性的潜在影响将通过测量极性效应蛋白的活性、测定极性标志物的核：胞质比率以及观察上皮细胞的极性来评估。 形态变化为了确定IRF 6-NME 2结合与初级腭融合之间的关系，将使用由考克斯实验室开发的新的卵内程序来特异性地干扰胚胎鸡口面上皮中的IRF 6/NME 2表达，并表征对初级腭发育的影响。 在人类患者中，CLP通常与牙齿异常有关，在小鼠中，在成釉细胞中检测到IRF6，并在牙齿上皮内陷中发挥作用。牙齿还提出了另一种机制来研究IRF6对上皮极性的贡献;成釉细胞来源于口腔上皮，并在分泌阶段表现出可观察到的极性。将开发在成釉细胞中条件性消融IRF 6的小鼠模型，并将基于成釉细胞极性、釉质形成和基因表达的变化（激光捕获显微切割，然后进行微阵列分析）评价IRF 6上皮极性贡献。因此，这些研究也将探索CLP与牙齿发育之间的可能关系。 这个拟议的F30项目旨在为有抱负的牙医科学家提供基础，并为以CLP的遗传和分子原因为中心的研究做出贡献。IRF6由于其对CLP发病率的显著贡献而成为目标。了解CLP背后的调控机制将有助于识别易感人群，降低发病率，并制定治疗策略。凭借华盛顿大学的强大机构支持和优秀的导师，我们相信这个F30奖将有助于成为一名成功的临床科学家所需的基础科学和临床培训。