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.

果蝇肌发生中MEF2功能的遗传，分子和计算分析。该建议的目的是定义转录因子心肌细胞的分子机制增强子2（MEF2）激活靶基因表达。几项研究已经确定了关键作用 MEF2在形成肌肉和其他组织的分化中，包括免疫细胞和神经元。此外，人类MEF2直系同源物的变体与心脏病和自闭症有关。但是，尽管MEF2对肌肉形成和人类疾病的重要性很重要，但数量相对较少已经确定了与MEF2一起参与肌发生的功能的副因素，没有已经确定了系统或全基因组的方法，以了解MEF2如何控制基因表达。此外，尚不清楚MEF2如何与基础转录机械相互作用。在这个提案，我们将使用具有单个MEF2基因的果蝇系统的力量来执行三个识别和表征与MEF2相互作用的因素的抬头方法。在AIM 1中，我们将继续扩展遗传修饰符屏幕，以识别单倍不足易于增强MEF2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，并确定了许多潜在的共同因素要表征。在AIM 2中，我们将执行一个分子屏幕，以确定因素从胚胎裂解物中与MEF2共同纯化。在初步数据中，我们证明了MEF2相互作用使用CF2，这是我们早期对MEF2共同因素的研究中发现的。在AIM 3中，我们将继续生物信息 MEF2靶基因的分析，以识别富含MEF2目标增强子和的序列启动子，并确定和表征与这些序列相互作用的因素。在初步数据中我们通过证明我们的独立生物信息分析确定了这种方法的可行性两个已知的MEF2共同因素的站点，我们确定了可能也代表的其他约束活动 MEF2联合因素。在AIM 4中，我们将整理AIMS 1-3中确定的因素，然后选择进行机械分析那些有可能成为MEF2副因素的可能性。总的来说，我们提出的实验都是基于的有了强大的初步数据，将为MEF2在动物中发挥作用的机制提供新的见解。鉴于动物王国内MEF2的顺序和功能的强大保护，我们的发现将直接影响我们对哺乳动物发育和疾病中MEF2功能的理解。