Mechanisms of DUX4 mediated FSHD pathology

描述(申请人提供)：我们建议的实验将确定导致DUX4错误表达并导致面肩肩周肌营养不良(FSHD)发病机制的机制。FSHD是困扰儿童和成人的最常见的肌病，但目前尚无治疗方法。然而，FSHD的研究现在进入了一个重要的新阶段，因为DUX4已经从包括我们自己在内的多个实验室的研究中脱颖而出，成为公认的FSHD候选基因。目前的证据支持一种FSHD病理模型，在该模型中，稳定表达被称为DUX4-FL(FL=全长)的多腺化DUX4剪接变异体，导致产生致病的DUX4-FL蛋白。这一模型与我们的发现是一致的，基于对一大批新的肌源性细胞库和活检组织的分析，DUX4-fl和DUX4-FL蛋白在FSHD患者的肌细胞核中的表达水平和比例都比健康对照肌肉细胞高得多。因此，在FSHD患者中，随着DUX4-FL诱导的病理改变随着时间的推移而积累，临床上可能会出现明显的肌肉无力，而在健康对照肌肉中，极低水平的DUX4-FL显然不足以引发明显的病理变化。尽管FSHD相关的遗传损伤不会明显改变蛋白质编码或mRNA序列，但有强有力的证据表明，异常的表观遗传调控是DUX4错误表达的基础。例如，我们的初步研究表明，DUX4-fl在FSHD肌源性细胞中的表达水平确实可以通过操纵表观遗传状态来改变，例如DNA甲基化。基于这些表观遗传学研究和我们对DUX4-FL表达的互补分析，我们提出并将检验这一假说，即表观遗传失调导致FSHD与未受影响的肌源性细胞中DUX4-f1表达异常增加，从而导致病理学。在特定的目标1中，我们将识别调控骨骼肌细胞DUX4-fl基因表达水平的表观遗传和非表观遗传机制。在特定的目标2中，我们将分析DUX4-FL在活检组织中的表达，并使用单细胞分析来确定DUX4-FL的表达如何改变FSHD与未受影响的细胞的命运。在特定的目标3中，我们将识别受DUX4蛋白异构体差异调控的基因网络，并确定它们在表达DUX4-FL的FSHD细胞中是否被破坏。我们的研究结果将系统地研究DUX4亚型在FSHD和对照肌肉细胞中的表达、调节和功能，将为指导针对DUX4的FSHD疗法的开发提供必要的重要信息。好了！

DESCRIPTION (provided by applicant): Our proposed experiments will identify mechanisms that cause mis-expression of DUX4 and lead to pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). FSHD is the most prevalent myopathy afflicting both children and adults, but no therapy is known. However, FSHD research has now entered an important new stage as DUX4 has emerged from studies in multiple laboratories, including our own, as the consensus FSHD candidate gene. Current evidence supports a model for FSHD pathology in which stable expression of a polyadenylated DUX4 mRNA splicing variant, termed DUX4-fl (fl = full-length), leads to production of a pathogenic DUX4-FL protein. This model is compatible with our finding, based on analysis of a large new library of myogenic cells and biopsies, that DUX4-fl mRNA and DUX4-FL protein are expressed at a much higher level and in a much higher fraction of myonuclei in FSHD than in healthy control muscle cells. Thus, in FSHD patients, clinically apparent muscle weakness may develop as DUX4-FL-induced pathological changes accumulate over time, whereas in healthy control muscles, the extremely low level of DUX4- FL is apparently insufficient to induce overt pathology. Although the FSHD-associated genetic lesion does not obviously alter protein coding or mRNA sequences, there is strong evidence that aberrant epigenetic regulation underlies DUX4 mis-expression. Our preliminary studies, for example, showed that DUX4-fl expression levels in FSHD myogenic cells could indeed be altered by manipulations of epigenetic status, e.g., DNA methlyation. Based on these epigenetic studies and our complementary analyses of DUX4-FL expression, we propose and will test the hypothesis that epigenetic dysregulation leads to aberrantly increased expression of DUX4-fl in FSHD vs. unaffected myogenic cells and thus to pathology. In Specific Aim 1, we wil identify epigenetic and non-epigenetic mechanisms that regulate DUX4-fl mRNA expression levels in skeletal muscle cells. In Specific Aim 2, we will analyze DUX4-FL expression in biopsies and also use single cell analyses to determine how DUX4-FL expression alters the fate of FSHD vs. unaffected cels. In Specific Aim 3, we will identify gene networks that are differentially regulate by the DUX4 protein isoforms and determine if they are disrupted in DUX4-fl-expressing FSHD cells. The results of our studies, in which we will systematically investigate the expression, regulation, and function of DUX4 isoforms in FSHD and control muscle cells, will provide the vital information needed to guide development of FSHD therapies specifically targeted to DUX4. !