Dissecting Protein Interactions for Developmental Analysis of SO-Cofactor Complexes

剖析蛋白质相互作用以进行 SO-辅因子复合物的发育分析

• 批准号: 9896459
• 负责人: FRANCESCA PIGNONI
• 金额: $ 8.1万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-19 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9896459
• 关键词: Adult; Affect; Binding; Binding Proteins; Biological; Biological Assay; Brain; Cell Fate Control; Cells; Cephalic; Comparative Study; Complex; Confusion; Congenital Abnormality; Data; Development; Developmental Process; Disease; Dissection; Drosophila eye; Drosophila genus; Endocrine Glands; Eye; Family; Family member; FlyBase; Future; Gene Targeting; Genes; Genetic Transcription; Goals; Grant; Holoprosencephaly; Homeobox; Homologous Gene; Human; Hybrids; In Vitro; Internet; Investigation; Kidney; Knowledge; Lead; Link; Mammals; Maps; Mediating; Methods; Molecular; Morphology; Mus; Muscle; Mutagenesis; Mutation; Nature; Neuroglia; Online Mendelian Inheritance In Man; Optic Lobe; Organ; Organism; Outcome; Ovary; Peripheral Nervous System; Phenotype; Play; Preparation; Protein Binding Domain; Proteins; RNA Interference; Rana; Reagent; Reporter; Repression; Role; Schizencephaly; Sensory; Short Interspersed Nucleotide Elements; Somatic Cell; Testing; Testis; Tissues; Transcription Coactivator; Transcription Repressor; Transcriptional Regulation; Transgenic Organisms; Variant; Work; Yeasts; base; cofactor; fly; in vivo; insight; mutant; neuroblast; novel strategies; physical mapping; protein protein interaction; stem cells; symposium; tool; transcription factor; transcriptome; vision development

Project Summary/Abstract The sine oculis (SO) gene belongs to the evolutionarily conserved ‘SIX’ family of homeobox transcription factors. Family members control cell fate, morphology, proliferation and survival in multiple tissues and organs of metazoans, including humans. In Drosophila, SO functions during development of the visual system, specific neuroblasts and glia of the central and peripheral nervous systems, endocrine glands, and in specialized somatic cells of the testis and the ovary of the adult fly. Several vertebrate SIX genes, including the SO orthologue SIX1, are required for the normal development of the brain, cranial sensory organs and the kidney; thus mutations in human homologues of SO lead to birth defects, including BOS/BOR (OMIM 601205, 600963), Holoprosencephaly 2 and Schizencephaly (OMIM 603714). SO/SIX1 functions as a transcriptional regulator together with a number of protein cofactors. We and others have shown that these cofactors modify the transcriptional activity of SO in vitro; moreover, in vivo evidence suggests that SO-cofactor complexes contribute to development in specific ways. Thus, in order to understand SO function in specific developmental contexts, we need to define the function of the various complexes. Our preliminary findings provide fundamental evidence that targeted mutagenesis of SO can be used to separately and specifically disrupt two well-known SO-cofactor interactions, one with the transcriptional activator Eya/EYA and the other with the transcriptional repressor Gro/TLE/GRG. In addition, we show that it is possible to assess the function of these SO variants in the organism. We propose here to dissect the role of these complexes in vivo as well as initiate an analysis of all known transcriptional cofactors of SO. The goal of this grant is to demonstrate that we can generate ‘designer SO proteins’ that either lack or retain only one or a few protein-protein interactions, and that we can develop assays for their functional assessment both in vitro and in vivo - in preparation for an R01 submission. Since SO is highly conserved from fly to human and the mouse orthologues, SIX1 and SIX2, can substitute for SO in the fly; most changes that affect partner binding in SO will similarly impact the vertebrate proteins. Thus, the proposed work is directly relevant to the human proteins and their linked human disorders.

项目摘要/摘要 Sine Oculis(SO)基因属于进化上保守的同源盒‘6’家族 转录因子。家族成员控制着细胞的命运、形态、增殖和存活 后生动物的多种组织和器官，包括人类。在果蝇中，所以在 中枢和外周的视觉系统、特定的神经母细胞和胶质细胞的发育 神经系统、内分泌腺，以及睾丸和卵巢的特化体细胞 成虫苍蝇。一些脊椎动物的6个基因，包括SIX1的同源基因，是需要的 大脑、头颅感觉器官和肾脏的正常发育；因此， 人类的SO同源物会导致出生缺陷，包括BOS/BOR(OMIM 601205,600963)， 前脑完整畸形2和脑裂畸形(OMIM 603714)。 SO/SIX1与许多蛋白质辅因子一起发挥转录调节作用。 我们和其他人已经证明，这些辅因子在体外改变了SO的转录活性； 此外，体内证据表明，SO-辅因子复合体有助于 具体的方式。因此，为了理解so在特定发育环境中的作用，我们 需要定义各种复合体的功能。我们的初步发现提供了 SO的靶向突变可以单独和特异性地用于 干扰两个众所周知的辅因子相互作用，其中一个与转录激活因子Eya/EYA 另一个是转录抑制因子Gro/Tle/GRG。此外，我们还证明了它是 有可能评估这些SO变异体在生物体中的功能。我们建议在这里剖析 这些复合体在体内的作用以及启动对所有已知转录的分析 SO的辅因子。这项拨款的目的是证明我们可以产生这样的设计师 缺乏或保留一种或几种蛋白质-蛋白质相互作用的蛋白质，而我们可以 建立体外和体内功能评估方法--为R01做准备 呈件。 因为从苍蝇到人和小鼠的同源物SIX1和SIX2都高度保守， 可以在运行中取代SO；大多数影响合作伙伴绑定的更改将产生类似的影响 脊椎动物的蛋白质。因此，这项拟议的工作与人类蛋白质和 他们与人类相关的疾病。