Mechanisms of DUX4 mediated FSHD pathology

描述（由申请人提供）：我们提出的实验将确定导致DUX4错误表达并导致面肩肱骨肌营养不良（FSHD）发病的机制。FSHD是儿童和成人中最常见的肌病，但目前尚无治疗方法。然而，随着DUX4从包括我们自己的多个实验室的研究中脱颖而出，作为FSHD的共识候选基因，FSHD的研究现在进入了一个重要的新阶段。目前的证据支持FSHD病理模型，在该模型中，被称为DUX4-fl （fl =全长）的多腺苷化DUX4 mRNA剪接变体的稳定表达导致致病性DUX4-fl蛋白的产生。该模型与我们的发现是一致的，基于对一个新的大型肌源性细胞文库的分析和活组织检查，DUX4-fl mRNA和DUX4-fl蛋白在FSHD中的表达水平和在肌核中的比例比在健康对照肌细胞中要高得多。因此，在FSHD患者中，临床明显的肌肉无力可能是由于DUX4-FL引起的病理变化随着时间的推移而积累，而在健康对照肌肉中，极低水平的DUX4-FL显然不足以诱导明显的病理。尽管fshd相关的遗传病变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，FSHD肌源性细胞中的DUX4-fl表达水平确实可以通过操纵表观遗传状态（例如DNA甲基化）来改变。基于这些表观遗传学研究和我们对DUX4-FL表达的补充分析，我们提出并将检验表观遗传失调导致FSHD中DUX4-FL表达异常增加的假设，而不是未受影响的肌源性细胞，从而导致病理。在Specific Aim 1中，我们将确定调控骨骼肌细胞中DUX4-fl mRNA表达水平的表观遗传和非表观遗传机制。在Specific Aim 2中，我们将分析活组织检查中的DUX4-FL表达，并使用单细胞分析来确定DUX4-FL表达如何改变FSHD与未受影响细胞的命运。在Specific Aim 3中，我们将鉴定受DUX4蛋白异构体差异调节的基因网络，并确定它们是否在表达DUX4-fl的FSHD细胞中被破坏。我们的研究结果，我们将系统地研究DUX4亚型在FSHD和控制肌肉细胞中的表达、调节和功能，将提供指导开发专门针对DUX4的FSHD治疗所需的重要信息。！

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. !