Genetic Regulation of Skeletal Muscle Repair

骨骼肌修复的基因调控

基本信息

批准号：
8068855
负责人：
Michael A Rudnicki
金额：
$ 24.74万
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 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8068855
关键词：
Activator Appliances Adult Affect Arginine Binding Binding Proteins Binding Sites Biological Biological Process Biology Cell Cycle Progression Cell Polarity Cell Survival Cell physiology Cells Cellular biology Chromatin Complex DNA Binding Development Dominant-Negative Mutation Epigenetic Process Gene Expression Gene Targeting Genetic Genetic Transcription Genome Genomics Growth Histones In Vitro LacZ Genes Lead MLL2 gene Maps Mass Spectrum Analysis Mediating Methylation Modality Molecular Mus Muscle Muscle satellite cell Muscular Dystrophies Mutation Myoblasts N-terminal Nature Pathway interactions Patients Phosphorylation Phosphotransferases Play Positioning Attribute Process Proliferating Proteins Recombinants Recruitment Activity Regulation Regulator Genes Research Role Seminal Serine Signal Pathway Signal Transduction Site Skeletal Muscle Skeletal Muscle Satellite Cells Small Interfering RNA Stem cells Therapeutic Intervention Transcriptional Activation Up-Regulation Wasting Syndrome age related base comparative genome-wide histone methyltransferase in vivo insight knock-down loss of function muscle regeneration mutant myogenesis novel novel therapeutic intervention postnatal programs public health relevance regenerative repaired research study sarcopenia satellite cell stem stem cell division transcription factor

项目摘要

DESCRIPTION (provided by applicant): Muscle satellite cells are responsible for the postnatal growth and repair capacity of adult skeletal muscle. The transcription factor Pax7 is expressed in satellite cells and is required for satellite cell survival and function through satellite cell development. We have shown that Pax7 activates target genes such as Myf5 through recruitment of the Ash2l/Wdr5/MLL2 histone methyltransferase complex. In addition, we found that 10% of satellite cells have never expressed Myf5 (based on Myf5-Cre lineage marking), and this subset of cells (termed satellite stem cells) are capable of repopulating the satellite cell niche. Using mass spectrometry, we identified Carm1 as a Pax7 binding protein. Carm1 directly methylates Pax7 on N-terminal arginines. Mutation of these sites results in a markedly reduced ability of Pax7 to upregulate Myf5 transcription, but does not affect DNA binding. Importantly, our experiments indicate that Carm1 methylation of Pax7 is necessary for Pax7 to bind MLL2. We therefore hypothesize that Carm1 positively controls transcriptional-activation by Pax7 through regulating the ability of Pax7 to bind MLL2 (and recruitment of the Wdr5/Ash2L/MLL2 histone methyltransferase), to activate Pax7-target genes. In this application, we propose a research program to molecularly define the role of Carm1 in regulating Pax7 function. To investigate the hypothesis that Pax7 mediates entry into the myogenic program through epigenetic mechanisms, we will undertake a comparative analysis of Pax7, Carm1 and Ash2L binding sites between satellite stem and myogenic cells, as well as map the distribution of modified histones as a function of gene expression. The enzymatic activity of Carm1 is regulated by phosphorylation at serine 229. Therefore, we will express an activated mutant version of Carm1 in satellite stem cells to ask whether this is sufficient to induce Myf5 transcription. To investigate at which levels of the satellite cell developmental program Carm1 is regulating Pax7 function, we will investigate the modulation of the Pax7-MLL2 interaction in quiescent satellite stem cells and myogenic cells, in activated satellite cells, during cell-cycle progression of proliferating myoblasts, and during terminal differentiation. We will undertake a multiplex in vitro kinase screen using recombinant Carm1 protein as substrate to identify the kinase(s) that are capable of phosphorylating Carm1. We will perform mass spectrometric analysis to identify additional regulators of Carm1. Lastly, we will employ siRNA knock-downs and conditional loss-of-function analysis in mice to characterize the biological significance of the Carm1-mediated regulation of Pax7 function. These studies will provide important new information about the molecular regulation of satellite cell function. Potentially, such insights will lead to novel therapeutic interventions for muscle- wasting diseases such as muscular dystrophy and sarcopenia. PUBLIC HEALTH RELEVANCE: Understanding how regulatory genes control the growth and repair of skeletal muscle, in particular the formation, activation, proliferation, and terminal differentiation of myogenic stem cells, is highly relevant to understanding the regenerative processes that occur in patients with muscle wasting diseases such as muscular dystrophy or age-related sarcopenia. We believe that our proposed studies will provide novel insights into the biology of muscle regeneration. Potentially, such insights will lead to new modalities of therapeutic intervention.

描述（由申请人提供）：肌肉卫星细胞负责成人骨骼肌的产后生长和修复能力。转录因子PAX7在卫星细胞中表达，是卫星细胞存活和通过卫星细胞发育功能所必需的。我们已经表明，PAX7通过募集ASH2L/WDR5/MLL2组蛋白甲基转移酶络合物复合物来激活靶基因，例如MyF5。此外，我们发现10％的卫星细胞从未表达MYF5（基于MyF5-CRE谱系标记），并且该细胞（称为卫星干细胞）的该子集能够重新填充卫星细胞生态位。使用质谱法，我们将Carm1鉴定为PAX7结合蛋白。 Carm1直接在N末端精氨酸上甲基化PAX7。这些位点的突变导致PAX7上调MYF5转录的能力明显降低，但不会影响DNA结合。重要的是，我们的实验表明PAX7的CARM1甲基化是PAX7结合MLL2所必需的。因此，我们假设CARM1通过调节PAX7结合MLL2（以及募集WDR5/ASH2L/MLL2组蛋白甲基转移酶）来积极地控制PAX7的转录激活，从而激活PAX7-target基因。在此应用中，我们提出了一项研究计划，以分子定义CARM1在调节PAX7功能中的作用。为了研究PAX7通过表观遗传机制介导进入肌源程序的假说，我们将对卫星干细胞和肌源性细胞之间的PAX7，CARM1和ASH2L结合位点进行比较分析，并将修饰的组蛋白作为基因表达的功能绘制。 CARM1的酶促活性受丝氨酸229的磷酸化调节。因此，我们将在卫星干细胞中表达Carm1的激活突变版本，以询问这是否足以诱导MyF5转录。为了调查卫星细胞发育程序的水平CARM1正在调节PAX7功能，我们将研究静态卫星干细胞和肌生成细胞中PAX7-MLL2相互作用的调节，在激活的卫星细胞中，在细胞周期中的肌细胞进展过程中，在肌细胞增殖的肌细胞和终末分化过程中。我们将使用重组CARM1蛋白作为底物进行多重体外激酶筛选，以鉴定能够磷酸化CARM1的激酶。我们将进行质谱分析，以识别CARM1的其他调节剂。最后，我们将在小鼠中采用siRNA敲低和有条件的功能丧失分析，以表征CARM1介导的PAX7功能调节的生物学意义。这些研究将提供有关卫星细胞功能的分子调节的重要新信息。可能，这种见解会导致肌肉浪费疾病（如肌肉营养不良和肌肉减少症）的新型治疗干预措施。公共卫生相关性：了解调节基因如何控制骨骼肌的生长和修复，尤其是肌原干细胞的形成，激活，增殖和终末分化与了解肌肉浪费肌肉疾病（如肌肉疾病或年龄降低的肌肉症患者）中发生的再生过程高度相关。我们认为，我们提出的研究将提供对肌肉再生生物学的新见解。可能，这种见解将导致治疗干预的新方式。