Pbx Homeodomain Proteins in Skeletal Muscle Differentiation

描述（由申请人提供）： 肌肉前体细胞分化为收缩性骨骼肌纤维是正常肌肉发育和再生所必需的，当有缺陷时，会导致肌肉骨骼疾病，如肌营养不良症和肌肉肿瘤。骨骼肌分化程序由碱性螺旋-环-螺旋家族的转录因子控制。这些因子之一，Myod，通过顺序激活基因组，包括编码肌肉收缩蛋白的基因，启动骨骼肌分化。该提案的长期目标是阐明肌肉调节因子如Myod如何以时间和空间控制的方式激活其转录靶点以驱动分化。该提议的具体假设是Pbx同源结构域蛋白将Myod引导至其转录靶点的子集，从而调节肌肉前体细胞分化的能力。两个发现支持这一假设：1。Pbx蛋白与肌细胞生成素启动子上的Myod结合成复合物。2. Pbx蛋白是Myod激活斑马鱼胚胎中肌细胞生成素表达和快速收缩肌肉特异性分化程序所必需的。这些发现提供了一个独特的机会，利用骨骼肌发育，以确定同源结构域蛋白如何指导特定的细胞分化程序。了解Pbx和Myod因子如何相互作用，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产生影响。该提案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，以允许对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。该提案的具体目标是：目标1。测试Pbx蛋白是否是哺乳动物骨骼肌分化过程中基因表达子集所必需的。将在Pbx缺陷小鼠胚胎中分析骨骼肌发育，以确定哪些小鼠Pbx基因是肌细胞生成素表达和快肌或慢肌分化所需的。为了全面确定哺乳动物Pbx依赖性肌肉基因表达的子集，将使用对来自Pbx敲除小鼠胚胎的RNA的表达阵列分析。目标二。区分Myod靶点Pbx调节的指导性或许可性机制。为了测试Myod结合和染色质修饰是否需要肌细胞生成素启动子中的Pbx结合位点，将使用小鼠细胞系中的报告基因测定。斑马鱼胚胎中的遗传相互作用实验将确定Pbx蛋白在克服快肌阻遏物Prdm 1的活性方面是否具有指导性或许可性。为了测试Pbx蛋白是否广泛结合或仅特异性结合肌肉基因启动子的子集，将使用全局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： 项目叙述肌肉前体细胞分化为收缩性骨骼肌纤维对于正常的肌肉发育和再生是必要的，如果有缺陷，会导致肌肉骨骼疾病，例如肌肉营养不良和肌肉肿瘤。该提案将确定驱动肌肉前体细胞分化为肌纤维的分子机制。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有关。

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.