Genetic Regulation of Skeletal Muscle Repair

骨骼肌修复的基因调控

基本信息

批准号：
7633335
负责人：
Michael A Rudnicki
金额：
$ 20.44万
依托单位：
OTTAWA HOSPITAL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-07-15 至 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7633335
关键词：
Adult Affinity Chromatography Alternative Splicing Binding Binding Sites Biochemical Biological Assay Boxing Chromatin Co-Immunoprecipitations Complex DNA Data Degenerative Disorder Development Dissection Dominant-Negative Mutation Elements Fibroblasts Gene Targeting Genes Genetic Genomics Histones Human Immune Sera Knowledge Laboratories Lead Length Maps Mass Spectrum Analysis Mediator of activation protein Modality Molecular Molecular Genetics Mus Muscle Myoblasts Nucleic Acid Regulatory Sequences PTPRC gene Paired box protein Pax-7 Pan Genus Pattern Plasmids Principal Investigator Procedures Process Protein Isoforms Proteins RNA Interference Recovery Recruitment Activity Regulation Reporter Research Role Signal Transduction Skeletal Muscle Skeletal Muscle Satellite Cells Surveys Therapeutic Intervention Time Transfection Transferase adult stem cell base cell type chromatin immunoprecipitation chromatin modification comparative density endonuclease gene function genome-wide histone modification insight knock-down muscle regeneration myogenesis novel progenitor programs promoter protein complex regenerative repaired representational difference analysis research study response tissue trauma transcription factor

项目摘要

DESCRIPTION (provided by applicant): Regenerative myogenesis is the formation of muscle progenitors during the repair of adult skeletal muscle following tissue trauma. In this application, we outline a research program to molecularly define the genetic mechanisms through which Pax7 enforces lineage commitment during regenerative myogenesis. To fully define the Pax7 regulome in primary myoblasts, Pax7 bound to fragmented chromatin will be immunoprecipitated and the DNA isolated and subjected to a subtractive PCR-based procedure to selectively amplify Pax7 binding sites. Direct targets will be identified by a comparative analysis between our existing microarray data, and regulatory regions that recruit Pax7. The function of a select set of prioritized candidate target genes will be investigated using molecular and genetic approaches. Real-time PCR will be used to characterize cell-type and differentiation patterns of expression. Knock down experiments will be performed with RNAi, and full-length cDNAs over-expressed in myoblasts. Lastly, the binding of Pax7 alternative splice forms to candidate promoters will be evaluated. To investigate the hypothesis that Pax7 recruits co-factors to facilitate appropriate regulation of target genes, the ability of Pax7 to direct acetyl-transferase activity will be investigated, and chromatin immunoprecipitation assays will be used to characterize the histone modifications over Pax7 target promoters. A genome wide assessment of histone modifications using microarrays will be conducted. To elucidate Pax7 function, we will isolate and identify protein components of the Pax7 transcriptional complex by mass spectroscopy. These experimental approaches will facilitate a molecular understanding of Pax7 function and the genomic definition of the chromatin domains established by Pax7. Understanding the molecular mechanism regulating gene function during muscle regeneration will provide insights that will potentially lead to new modalities of therapeutic intervention for the treatment of muscle degenerative disease.

描述（由申请人提供）：再生肌发生是在组织创伤后修复成年骨骼肌期间肌肉祖细胞的形成。在此应用中，我们概述了一项研究计划，以分子定义PAX7在再生肌发生过程中执行谱系承诺的遗传机制。为了完全定义原代成肌细胞中的PAX7调节体，将对与碎片染色质结合的PAX7进行免疫沉淀，DNA分离并进行基于减去PCR的程序，以选择性地扩增PAX7结合位点。直接目标将通过我们现有的微阵列数据与募集PAX7的监管区域之间的比较分析来确定。将使用分子和遗传方法研究一组优先候选目标基因的一组优先级候选靶基因的功能。实时PCR将用于表征细胞类型和表达的分化模式。敲除实验将使用RNAi进行，并且在成肌细胞中过表达的全长cDNA。最后，将评估PAX7替代剪接形式与候选启动子的结合。为了研究PAX7募集辅助因子以促进靶基因的假设，将研究PAX7直接乙酰基转移酶活性的能力，并将使用染色质免疫沉淀测定法来表征组蛋白在PAX7靶启动子上的修饰。使用微阵列对组蛋白修饰的基因组进行了广泛的评估。为了阐明PAX7功能，我们将通过质谱分离和鉴定PAX7转录复合物的蛋白质成分。这些实验方法将促进对PAX7功能的分子理解以及PAX7建立的染色质结构域的基因组定义。了解调节肌肉再生过程中调节基因功能的分子机制将提供洞察力，从而有可能导致治疗肌肉退行性疾病的新型治疗干预措施。