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

• 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
• 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

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.

果蝇肌肉发生过程中MEF2功能的遗传、分子和计算分析。 这项提议的目标是确定转录因子肌细胞 增强因子-2(MEF2)激活靶基因表达。几项研究已经确定了 MEF2在肌肉系统的形成和其他组织的分化中，包括免疫细胞和 神经元。此外，人类MEF2同源基因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 已被确定与MEF2一起参与肌肉发生的辅助因子，而NO 已经确定了系统的或全基因组的方法来了解MEF2是如何控制基因的 表情。此外，MEF2如何与基础转录机制相互作用尚不清楚。在这 提议，我们将利用只有一个Mef2基因的果蝇系统的力量，执行三个- 确定和表征与MEF2相互作用的因素的综合方法。在目标1中，我们将继续和 扩展遗传修饰符屏幕以识别单倍体不足可增强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 潜在的共性因素有待描述。在目标2中，我们将进行分子筛选，以确定 胚胎裂解液中MEF2的联合免疫纯化。在初步数据中，我们证明了MEF2相互作用 对于CF2，这在我们早期对MEF2辅助因素的研究中得到了确认。在目标3中，我们将继续生物信息学 分析MEF2靶基因，以确定富含MEF2靶向增强剂和 启动子，并鉴定和表征与这些序列相互作用的因素。在初步数据中 我们通过我们的独立生物信息学分析证明了这种方法的可行性。 两个已知的MEF2辅助因子的结合位点，我们发现其他结合活性也可能代表 MEF2的共同因素。在目标4中，我们将整理目标1-3中确定的因素，并选择进行机械分析 那些有很大可能是MEF2共同因素的人。总体而言，我们建议的实验都基于 基于强大的初步数据，将为MEF2在动物中发挥作用的机制提供新的见解。 鉴于MEF2在动物界的序列和功能上具有很强的保守性，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的功能的理解。