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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.

MEF2 在果蝇肌发生中的功能的遗传、分子和计算分析。该提案的目标是定义转录因子 Mycell 的分子机制增强子因子 2 (MEF2) 激活靶基因表达。多项研究已经确定了关键作用 MEF2 参与肌肉组织的形成和其他组织的分化，包括免疫细胞和神经元。此外，人类 MEF2 直向同源物的变异与心脏病和自闭症有关。然而，尽管 MEF2 对肌肉形成和人类疾病很重要，但相对较少的数量已鉴定出与 MEF2 一起参与肌生成的辅助因子，并且没有已确定系统或全基因组方法来了解 MEF2 如何控制基因表达。此外，尚不清楚 MEF2 如何与基础转录机制相互作用。在这个提案中，我们将利用具有单个 Mef2 基因的果蝇系统的力量来执行三项任务：多管齐下的方法来识别和表征与 MEF2 相互作用的因素。在目标 1 中，我们将继续并扩展遗传修饰筛选以确定单倍体不足增强 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，以确定影响因素使用 MEF2 从胚胎裂解物中进行联合免疫纯化。在初步数据中，我们证明 MEF2 相互作用与 CF2 相关，这是我们在早期对 MEF2 辅助因子的研究中发现的。在目标 3 中，我们将继续进行生物信息学研究分析 MEF2 靶基因，以确定富含 MEF2 靶增强子的序列和启动子，并鉴定和表征与这些序列相互作用的因子。初步数据显示我们通过独立的生物信息学分析确定了这种方法的可行性两个已知 MEF2 辅助因子的位点，并且我们确定了可能也代表的其他结合活性 MEF2 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制分析那些很可能是 MEF2 辅因子的。总的来说，我们提出的实验都是基于基于强有力的初步数据，将为 MEF2 在动物中发挥作用的机制提供新的见解。鉴于动物界中 MEF2 的序列和功能具有很强的保守性，我们的发现将直接影响我们对 MEF2 在哺乳动物发育和疾病中功能的理解。