The role of BET proteins in the differential regulation of myogenesis: Implications for muscle-wasting disorders.

肌肉质量的丧失是多种疾病的共同特征。例如，在进行性肌肉萎缩的情况下，Duchenne肌营养不良症(DMD)，肌营养不良蛋白基因的丢失使肌肉纤维对收缩损伤敏感。受损的纤维随后会因坏死而丢失，肌肉干细胞通过生成新的肌肉来弥补。这会导致退化和再生的循环，最终导致肌肉干细胞库的功能衰竭。死亡的原因是心脏不能支持血液循环，或者横隔膜不能支持呼吸。因此，肌肉再生可以被认为是DMD病理的重要组成部分，促进再生的策略可能能够减缓在这些患者中观察到的肌肉萎缩。同样，促进肌肉再生也可能逆转与衰老(骨质疏松症)、癌症和艾滋病(恶病质)以及糖尿病/肥胖症等疾病相关的肌肉损耗病理。我们已经确定BET蛋白是肌肉生长和分化的新调节因子。BET蛋白与基因组结合，以协调基因表达。最重要的是观察到BET家族的不同成员对培养细胞中的肌肉细胞分化起相反的作用。具体地说，抑制BET蛋白Brd4阻止分化，而抑制Brd3促进分化。拟议的工作方案旨在详细说明这些调查结果。具体地说，我们将研究这些现象背后的机制，表征BET蛋白的基因组结合位置，在原代人类和小鼠细胞中验证这些发现，并调查BET蛋白在体内转基因和肌肉损伤模型系统中的作用。这项工作计划有可能揭示肌肉功能的基本新见解，对肌肉萎缩障碍的治疗具有重要意义。为了研究选择性BET抑制的治疗潜力，我们将使用DMD的小鼠模型来测试Brd3的中断作为促进肌肉生长的一种手段的可能性。鉴于人们对BET蛋白在癌症领域的浓厚兴趣，研究人员可以使用一些高效的小分子BET抑制剂。然而，这些化合物的一个重要局限性是它们无法区分不同的BET家族成员(例如Brd3和Brd4，我们已经证明它们对肌肉发生具有相反的作用)。因此，我们建议使用反义寡核苷酸通过两种机制中的一种来选择性地针对Brd3进行下调。首先，我们将使用拼接损坏方法，迫使目标Brd3信使RNA被错误处理。我们有丰富的经验使用最先进的反义技术(即多肽-吗啡偶联物)，可以有效地输送到体内营养不良的小鼠肌肉，以这种方式调节基因表达。其次，我们将使用传统的靶向降解方法(即缺口寡核苷酸)直接降低小鼠肌肉中Brd3信使RNA的水平。使用寡核苷酸可以高度特异性地靶向单个BET家族成员(即Brd3)，而不会脱靶抑制其他家族成员(即Brd4和Brd2)--这是现有小分子抑制剂目前无法做到的。因此，该方案的总体目标是利用基础肌肉生物学方面的新发现，以促进新疗法的开发，并改善肌肉衰竭性疾病患者的生活。

Loss of muscle mass is a common feature of a wide variety of diseases. For example, in the progressive muscle wasting condition Duchenne Muscular Dystrophy (DMD), the loss of the dystrophin gene renders muscle fibres sensitive to contractile damage. Damaged fibres are subsequently lost due to necrosis, and muscle stem cells compensate by generating new muscle. This leads to cycles of degeneration and regeneration and ultimately to functional exhaustion of the muscle stem cell pool. Fatality results as a consequence of the failure of the heart to support blood circulation, or the diaphragm to support breathing. As such, muscle regeneration can be considered an important component of DMD pathology, and strategies which promote regeneration may be able to slow the muscle-wasting observed in these patients. Similarly, promoting muscle regeneration may also be able to reverse the muscle-wasting pathologies associated with aging (sarcopenia), diseases such as cancer and AIDS (cachexia), and diabetes/obesity.We have identified BET proteins as novel regulators of muscle growth and differentiation. BET proteins bind to the genome in order to coordinate gene expression. Of key importance is the observation that different members of the BET family exert opposite effects on muscle cell differentiation in cultured cells. Specifically, inhibition of the BET protein Brd4 blocks differentiation, whereas inhibition of Brd3 enhances differentiation. The proposed Programme of work aims to characterise these findings in detail. Specifically, we will investigate the mechanisms behind these phenomena, characterise the genomic binding sites of BET proteins, validate these findings in primary human and mouse cells, and investigate the role of BET proteins in in vivo transgenic and muscle injury model systems. This Programme of work has the potential to reveal fundamental new insights into muscle function with important implications for the treatment of muscle-wasting disorders.To investigate the therapeutic potential of selective BET inhibition, we will test the possibility that disruption of Brd3 could be used as a means of promoting muscle growth using a mouse model of DMD. Given the intense interest in BET proteins in the cancer field, there are a number of highly potent small molecule BET inhibitors available to researchers. However, an important limitation of these compounds is their inability to distinguish between different BET family members (e.g. Brd3 and Brd4, which we have shown have opposite effects on myogenesis). We therefore propose the use of antisense oligonucleotides to selectively target Brd3 for downregulation via one of two mechanisms. Firstly, we will use a splice corruption approach which forces the target Brd3 messenger RNA to be incorrectly processed. We have extensive experience using state-of-the art antisense technology (i.e. peptide-morpholino conjugates) which can be effectively delivered to dystrophic mouse muscle in vivo to modulate gene expression in this manner. Secondly, we will use a conventional target degradation approach (i.e. gapmer oligonucleotides) to directly reduce the levels of Brd3 messenger RNA in mouse muscle. The use of oligonucleotides allows for highly specific targeting of a single BET family member (i.e. Brd3) without off-target inhibition of the other family members (i.e. Brd4 and Brd2) - which is currently not possible with existing small molecule inhibitors. The overall aim of the Programme is therefore to harness new discoveries in basic muscle biology in order to facilitate the development of novel therapeutics and improve the lives of patients with muscle-wasting diseases.