The role of Sox6 in skeletal muscle fiber type differentiation

描述(申请人提供)：骨骼肌中形成的慢和快抽动纤维是功能成熟的标志，使肌肉能够对复杂的生理需求做出反应。尽管横纹肌被精心设计来执行出生后立即开始的各种物理和新陈代谢任务，但对于骨骼肌发育过程中骨骼肌纤维最初是如何形成的，人们的理解仍然有限。揭示胎儿骨骼肌纤维类型分化的机制是我们的长期目标。我们最近的数据表明，在胎儿骨骼肌发育过程中，Sox6是正常纤维类型特异性基因表达所必需的。在Sox6缺失的骨骼肌中，慢纤维特异基因的表达在胎儿晚期未能发生抑制，而慢纤维特异基因在未来的快速纤维肌肉中保持高水平的表达，即使在出生后阶段也是如此。SOX6是SOX转录因子家族的成员。尽管Sox6基因在骨骼肌中高度表达，但其在肌肉发育中的作用尚不清楚。在这项研究中，我们将研究Sox6在胎儿骨骼肌发育过程中纤维类型特异性基因表达中的作用。我们的中心假设是，Sox6在胎儿骨骼肌未来的快速纤维中作为慢纤维类型特定基因的转录抑制因子发挥作用。为检验我们的假设而提出的三个目标是： 目的：1.验证Sox6在肌肉中的表达是胎儿骨骼肌初始纤维类型分化所必需的假设。目的2.通过检验两个假说：蛋白质-蛋白质相互作用的调节和翻译后修饰，确定调节纤维类型特异性Sox6活性的机制。目的3.验证Sox6通过以下方式协调调节指定纤维类型的多个基因的转录的假设：(1)使用芯片分析确定主要的Sox6靶点；(2)确定Sox6如何抑制MyHC-2基因 这些目标将通过确定骨骼肌特异性Sox6条件性基因敲除小鼠的表型(Aim 1)、通过酵母双杂交分析确定Sox6辅助因子和确定骨骼肌中Sox6蛋白的翻译后修饰(Aim 2)、通过使用芯片分析确定Sox6蛋白的主要靶标和确定Sox6抑制MyHC-2基因转录的分子机制来实现。这些目标的完成将提高我们对胎儿骨骼肌终末分化的认识，并帮助我们获得治疗肌肉退行性疾病的重要信息。项目叙事 在A1的重新提交中，我们研究了Sox6转录因子在胎儿肌肉纤维类型规范中的作用。我们的中心假设是，Sox6在胎儿骨骼肌未来的快速纤维中作为慢纤维类型特定基因的转录抑制因子发挥作用。为了测试这一点，我们将在三个具体目标上提出以下问题：在肌肉发生过程中，Sox6在何时何地发挥作用？(目标1)：SOX6将慢纤维类型特定基因的抑制限制在快纤维上的细胞机制是什么？(目标2)；最后，Sox6如何在分子水平上实现对慢纤维类型特定基因的转录抑制？(目标3)。

DESCRIPTION (provided by applicant): The formation of slow and fast twitch fibers in skeletal muscle is a hallmark of functional maturity, enabling muscles to respond to complex physiological demands. Although striated muscles are exquisitely designed to execute diverse physical and metabolic tasks beginning immediately after birth, an understanding of how slow and fast skeletal muscle fibers initially form during fetal skeletal muscle development is still limited. It is our long-term goal to uncover the mechanisms of fiber type differentiation in the fetal skeletal muscle. Our recent data suggest that during fetal skeletal muscle development, Sox6 is required for normal fiber type-specific gene expression. In Sox6-null skeletal muscle, suppression of slow fiber-specific gene expression fails to occur in late fetal stages and slow fiber-specific genes maintain high levels of expression in the future fast fiber muscles, even in the postnatal stages. Sox6 is a member of the Sox transcription factor family. Although it is highly expressed in skeletal muscle, Sox6's role in muscle development is unknown. In this proposal, we will investigate the role of Sox6 in fiber type-specific gene expression during the fetal skeletal muscle development. Our central hypothesis is that Sox6 functions as a transcriptional repressor of slow fiber type-specific genes in the future fast fibers of the fetal skeletal muscle. The three aims proposed to test our hypothesis are: Aim1. Test the hypothesis that Sox6 expression in muscle is required for the initial fiber type differentiation in fetal skeletal muscle. Aim 2. Determine the mechanisms that regulate fiber type-specific Sox6 activity by testing two hypotheses: modulations by protein-protein interactions and post-translational modifications. Aim 3. Test the hypothesis that Sox6 coordinately regulates transcription of multiple genes which specify fiber types by: (1) identifying primary Sox6 targets using ChIP on chip assays and (2) determining how Sox6 represses the MyHC-2 gene These aims will be achieved by determining the phenotype of skeletal muscle specific Sox6 conditional knockout mice (Aim 1), by identifying Sox6 co-factors using yeast two-hybrid assays and determining post-translational modification of the Sox6 protein in skeletal muscle (Aim 2), and by identifying primary targets of the Sox6 protein using ChIP on chip assays and determining the molecular mechanism of transcriptional suppression of the MyHC-2 gene by Sox6. Completion of the proposed aims will advance our knowledge in the terminal differentiation of fetal skeletal muscle and aid us in gaining important information for treating muscle degenerative diseases. Project Narrative In this A1 resubmission, we investigate the role of the Sox6 transcription factor in the fetal muscle fiber type specification. Our central hypothesis is that Sox6 functions as a transcriptional repressor of slow fiber type-specific genes in the future fast fibers of the fetal skeletal muscle. To test this, we will ask following questions in three specific aims: when and where, during myogenesis, does Sox6 function? (Aim 1); what are the cellular mechanisms to limit the repression of slow fiber type-specific genes by Sox6 to fast fibers? (Aim 2); and finally, how does Sox6 achieve transcriptional suppression of slow fiber type-specific genes at the molecular level? (Aim 3).