The role of Sox6 in skeletal muscle fiber type differentiation

描述（申请人提供）：骨骼肌中慢肌纤维和快肌纤维的形成是功能成熟的标志，使肌肉能够响应复杂的生理需求。虽然横纹肌是精心设计的，可以在出生后立即执行各种身体和代谢任务，但对胎儿骨骼肌发育过程中骨骼肌纤维最初形成的速度和速度的理解仍然有限。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，Sox 6是正常纤维类型特异性基因表达所必需的。在Sox 6-null骨骼肌中，抑制慢纤维特异性基因表达未能发生在胎儿晚期，慢纤维特异性基因在未来的快纤维肌肉中保持高水平的表达，即使在出生后阶段。Sox 6是Sox转录因子家族的成员。虽然Sox 6在骨骼肌中高度表达，但其在肌肉发育中的作用尚不清楚。在本研究中，我们将探讨Sox 6在胎儿骨骼肌发育过程中纤维类型特异性基因表达的作用。我们的中心假设是，Sox 6功能作为一个转录抑制因子的慢纤维类型特异性基因在未来的快速纤维的胎儿骨骼肌。为检验我们的假设而提出的三个目标是： 目标1.检验Sox 6在肌肉中的表达是胎儿骨骼肌中初始纤维类型分化所必需的假设。目标2.通过测试两个假设来确定调节纤维类型特异性Sox 6活性的机制：蛋白质-蛋白质相互作用和翻译后修饰的调节。目标3。通过以下方法检验Sox 6协同调节指定纤维类型的多个基因的转录的假设：（1）使用ChIP芯片测定法鉴定主要Sox 6靶标，以及（2）确定Sox 6如何抑制MyHC-2基因 这些目的将通过确定骨骼肌特异性Sox 6条件敲除小鼠的表型（Aim 1），通过使用酵母双杂交测定鉴定Sox 6辅因子和确定骨骼肌中Sox 6蛋白的翻译后修饰（Aim 2），并通过使用ChIP芯片测定鉴定Sox 6蛋白的主要靶点，并确定转录抑制的分子机制，MyHC-2基因。所提出的目标的完成将推进我们的知识在胚胎骨骼肌的终末分化，并帮助我们获得重要的信息，治疗肌肉退行性疾病。项目叙述 在这A1重新提交，我们调查的作用，Sox 6转录因子在胎儿肌纤维类型的规范。我们的中心假设是，Sox 6功能作为一个转录抑制因子的慢纤维类型特异性基因在未来的快速纤维的胎儿骨骼肌。为了验证这一点，我们将提出以下三个特定目标的问题：在肌生成过程中，Sox 6在何时何地发挥作用？(Aim 1）;什么是细胞机制，以限制抑制慢纤维类型特异性基因的Sox 6快纤维？(Aim最后，Sox 6如何在分子水平上实现慢纤维类型特异性基因的转录抑制？(Aim 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).