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Genetic, molecular and computational analysis of MEF2 function in Drosophila myogenesis

MEF2 在果蝇肌发生中的功能的遗传、分子和计算分析

基本信息

批准号：
9369457
负责人：
Richard Matthew Cripps
金额：
$ 30.13万
依托单位：
UNIVERSITY OF NEW MEXICO
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9369457
关键词：
Adult Affect Animals Architecture Autistic Disorder Binding Binding Proteins Binding Sites Biochemical Biochemical Genetics Bioinformatics Brain Cell Differentiation process Cells Characteristics Chromosomes Complement Computer Analysis DNA DNA Binding Data Development Developmental Process Disease Drosophila Proteins Drosophila genus Elements Embryo Enhancers Gene Expression Gene Targeting Genes Genetic Genetic Screening Genetic Transcription Genome Genomic Segment Goals Heart Diseases Human Immune Individual Learning Mammals Molecular Molecular Analysis Muscle Muscle Development Mutation Neuronal Differentiation Neurons Nuclear Extract Orthologous Gene Phenotype Probability Process Protein Isoforms Proteins Public Health Rest Role Series Site System Therapeutic Intervention Tissues Variant Work base cofactor experimental study gene complementation gene discovery genetic analysis genome-wide human disease insight knock-down mutant myocyte-specific enhancer-binding factor 2 myogenesis novel novel strategies programs promoter transcription factor

项目摘要

Genetic, molecular and computational analysis of MEF2 function in Drosophila myogenesis. The goal of this proposal is to define the molecular mechanisms by which the transcription factor Myocyte enhancer factor-2 (MEF2) activates target gene expression. Several studies have identified critical roles for MEF2 in formation of the musculature and in differentiation of other tissues including immune cells and neurons. Moreover, variants in MEF2 orthologs in humans are associated with cardiac disease and autism. However despite the importance of MEF2 to muscle formation and human disease, a relatively small number of co-factors have been identified that function alongside MEF2 to participate in myogenesis, and no systematic or genome-wide approaches have been identified to understand how MEF2 controls gene expression. Moreover, it is not clear how MEF2 interacts with the basal transcription machinery. In this proposal, we will use the power of the Drosophila system, that has a single Mef2 gene, to execute a three- pronged approach to identify and characterize factors that interact with MEF2. In Aim 1, we will continue and expand a genetic modifier screen to identify genes for which haploinsufficiency enhances a Mef2 mutant phenotype. In preliminary data we demonstrate the feasibility of this approach, and identify a number of potential co-factors to be characterized. In Aim 2, we will carry out a molecular screen to identify factors that co-immune purify with MEF2 from embryonic lysates. In preliminary data we demonstrate that MEF2 interacts with CF2, that was identified in our earlier studies of MEF2 co-factors. In Aim 3, we will continue a bioinformatic analysis of MEF2 target genes, to identify sequences that are enriched in MEF2 target enhancers and promoters, and to identify and characterize the factors that interact with these sequences. In preliminary data we demonstrate the feasibility of this approach by showing that our independent bioinformatic analyses identify sites for two known MEF2 co-factors, and we identify additional binding activities that might also represent MEF2 co-factors. In Aim 4, we will collate the factors identified in Aims 1-3 and select for mechanistic analysis those that have high probability to be MEF2 co-factors. Overall our proposed experiments, which are all based upon strong preliminary data, will provide new insight into mechanisms by which MEF2 functions in animals. Given the strong conservation in the sequence and function of MEF2 within the animal kingdom, our findings will have direct impact upon our understanding of MEF2 function in mammalian development and disease.

MEF 2在果蝇肌肉发生中功能的遗传、分子和计算分析。本研究的目的是确定转录因子Myocyte 增强子因子-2（MEF 2）激活靶基因表达。一些研究已经确定了 MEF 2在肌肉组织的形成和包括免疫细胞在内的其他组织的分化中的作用，神经元此外，人类MEF 2直系同源物的变异与心脏病和自闭症有关。然而，尽管MEF 2对肌肉形成和人类疾病的重要性，已经鉴定出的辅助因子与MEF 2一起参与肌生成，已经确定了系统的或全基因组的方法来理解MEF 2如何控制基因表达，表情此外，MEF 2如何与基础转录机制相互作用尚不清楚。在这我们将利用果蝇系统的力量，它有一个单一的Mef 2基因，执行一个三- 一种方法来识别和表征与MEF 2相互作用的因素。在目标1中，我们将继续，扩大遗传修饰筛选以鉴定单倍不足增强Mef 2突变体的基因表型在初步数据中，我们证明了这种方法的可行性，并确定了一些待表征的潜在辅助因子。在目标2中，我们将进行分子筛选，以确定与来自胚胎裂解物的MEF 2共免疫纯化。在初步的数据中，我们证明MEF 2相互作用 CF2，这是在我们早期的MEF 2辅因子研究中确定的。在目标3中，我们将继续进行生物信息学分析MEF 2靶基因，以鉴定富含MEF 2靶增强子的序列，启动子，并鉴定和表征与这些序列相互作用的因子。初步数据显示，我们证明了这种方法的可行性，表明我们的独立生物信息学分析确定，两个已知的MEF 2辅因子的位点，我们确定了额外的结合活性，也可能代表 MEF 2辅助因子。在目标4中，我们将整理目标1-3中确定的因素，并选择进行机理分析那些具有高概率是MEF 2辅因子的那些。总的来说，我们提出的实验，这都是基于在强大的初步数据，将提供新的洞察机制，MEF 2在动物中的功能。考虑到MEF 2在动物王国中的序列和功能的高度保守性，我们的发现将对我们理解MEF 2在哺乳动物发育和疾病中的功能产生直接影响。