Pbx Homeodomain Proteins in Skeletal Muscle Differentiation

骨骼肌分化中的 Pbx 同源结构域蛋白

• 批准号: 7713922
• 负责人: Lisa Maves
• 金额: $ 8.8万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7713922
• 关键词: BHLH Protein; Binding; Binding Sites; Biochemical; Biological Assay; Cell Culture Techniques; Competence; Complex; Contractile Proteins; Embryo; Family; Fibrinogen; Gene Expression; Gene Targeting; Genes; Genetic; Genomics; Goals; Homeodomain Proteins; Knockout Mice; Mammalian Cell; Modeling; Molecular; Mouse Cell Line; Mus; Muscle; Muscle Development; Muscle Fibers; Muscular Dystrophies; Musculoskeletal Diseases; MyoD Protein; Myoblasts; Myogenin; Myomatous neoplasm; Natural regeneration; Protein Binding; Proteins; RNA; Regulation; Reporter; Research; Skeletal Muscle; Testing; Zebrafish; chromatin immunoprecipitation; chromatin modification; developmental genetics; genetic analysis; genome-wide; improved; programs; promoter; public health relevance; research study; skeletal muscle differentiation; therapy development

DESCRIPTION (provided by applicant): PROJECT SUMMARY The differentiation of muscle precursor cells into contractile skeletal muscle fibers is necessary for normal muscle development and regeneration and, when defective, leads to musculoskeletal diseases such as muscular dystrophies and muscle tumors. The skeletal muscle differentiation program is controlled by transcription factors of the basic helix-loop-helix family. One of these factors, Myod, initiates skeletal muscle differentiation by sequentially activating groups of genes, including those encoding muscle contractile proteins. The long term goal of this proposal is to elucidate how muscle regulatory factors like Myod activate their transcriptional targets in a temporally and spatially controlled manner to drive differentiation. The specific hypothesis of this proposal is that Pbx homeodomain proteins direct Myod to a subset of its transcriptional targets, thereby regulating the competence of muscle precursor cells to differentiate. Two findings support this hypothesis: 1. Pbx proteins bind in a complex with Myod on the Myogenin promoter. 2. Pbx proteins are required for Myod to activate Myogenin expression and the fast- twitch muscle-specific differentiation program in zebrafish embryos. These findings present a unique opportunity to use skeletal muscle development to determine how homeodomain proteins direct specific cellular differentiation programs. Understanding how Pbx and Myod factors interact may ultimately be useful in improving the control over muscle precursor differentiation, with implications for treating musculoskeletal diseases. This proposal will utilize a combination of approaches in mouse and zebrafish embryos as well as in mammalian cell culture to allow a comprehensive biochemical, genomic and developmental genetic analysis of how Pbx regulates skeletal muscle differentiation and Myod activity. The Specific Aims of this proposal are: Aim 1. Test whether Pbx proteins are required for a subset of gene expression during mammalian skeletal muscle differentiation. Skeletal muscle development will be analyzed in Pbx-deficient mouse embryos to determine which mouse Pbx genes are required for Myogenin expression and fast- or slow-twitch muscle differentiation. To comprehensively determine the subset of mammalian Pbx-dependent muscle gene expression, expression array analyses on RNA from Pbx knockout mouse embryos will be used. Aim 2. Distinguish between an instructive or permissive mechanism for Pbx regulation of Myod targets. To test whether the Pbx binding site in the Myogenin promoter is required for Myod binding and chromatin modifications, reporter assays in mouse cell lines will be used. Genetic interaction experiments in zebrafish embryos will determine whether Pbx proteins are instructive or permissive in overcoming the activity of the fast-muscle repressor Prdm1. To test whether Pbx proteins bind broadly to, or specifically to only a subset of, muscle gene promoters, genome-wide Pbx binding sites will be determined using a global chromatin immunoprecipitation approach. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE The differentiation of muscle precursor cells into contractile skeletal muscle fibers is necessary for normal muscle development and regeneration and, when defective, leads to musculoskeletal diseases such as muscular dystrophies and muscle tumors. This proposal will identify molecular mechanisms that drive muscle precursor cells into differentiated muscle fibers. Understanding these mechanisms ultimately may be useful in improving the ability to control muscle precursor differentiation and, therefore, relevant to developing therapies for treating musculoskeletal diseases.

描述(由申请人提供)： 肌肉前体细胞分化为可收缩的骨骼肌纤维是正常肌肉发育和再生所必需的，当缺陷时，会导致肌肉营养不良和肌肉肿瘤等肌肉骨骼疾病。骨骼肌分化程序由基本螺旋-环-螺旋家族的转录因子控制。其中一种因子，Myod，通过顺序激活一组基因来启动骨骼肌分化，包括那些编码肌肉收缩蛋白的基因。这一提议的长期目标是阐明肌肉调节因子如Myod如何以时间和空间受控的方式激活它们的转录靶标以驱动分化。这一建议的具体假设是，PBX同源结构域蛋白将Myod导向其转录靶点的一个子集，从而调节肌肉前体细胞分化的能力。支持这一假说的有两个发现：1.PBX蛋白与Myod基因启动子上的Myod结合。2.在斑马鱼胚胎中，Myod需要PBX蛋白来激活肌肉生成素的表达和快速抽动肌肉特异性分化程序。这些发现为利用骨骼肌发育来确定同源结构域蛋白如何指导特定的细胞分化程序提供了一个独特的机会。了解PBX和Myod因子是如何相互作用的，最终可能有助于改善对肌肉前体分化的控制，并对治疗肌肉骨骼疾病有所启示。这项建议将利用小鼠和斑马鱼胚胎以及哺乳动物细胞培养中的方法组合，对PBX如何调节骨骼肌分化和Myod活性进行全面的生化、基因组和发育遗传分析。这一建议的具体目的是：目的1.测试在哺乳动物骨骼肌分化过程中，是否需要PBX蛋白来表达基因的一个子集。将对PBX缺陷小鼠胚胎的骨骼肌发育进行分析，以确定哪些小鼠PBX基因是肌肉生成素表达和快慢抽动肌肉分化所必需的。为了全面确定哺乳动物PBX依赖的肌肉基因表达亚集，将使用PBX基因敲除小鼠胚胎的RNA表达阵列分析。目的2.区分PBX调节Myod靶点的指导性或允许性机制。为了测试Mygenin启动子中的PBX结合位点是否需要Myod结合和染色质修饰，将使用小鼠细胞系中的报告分析。斑马鱼胚胎中的遗传相互作用实验将确定PBX蛋白在克服快速肌肉抑制因子Prdm1的活性方面是有指导意义的还是允许的。为了测试PBX蛋白是否广泛地与肌肉基因启动子结合，或仅与肌肉基因启动子的子集特异结合，将使用全球染色质免疫沉淀方法确定全基因组PBX结合位点。 公共卫生相关性： 项目简介肌肉前体细胞分化为可收缩的骨骼肌纤维对于正常的肌肉发育和再生是必要的，当缺陷时，会导致肌肉营养不良和肌肉肿瘤等肌肉骨骼疾病。这项提议将确定驱动肌肉前体细胞分化为肌肉纤维的分子机制。了解这些机制最终可能有助于提高控制肌肉前体分化的能力，因此，与开发治疗肌肉骨骼疾病的疗法相关。