Mechanisms of DUX4 mediated FSHD pathology

DUX4 介导的 FSHD 病理机制

• 批准号: 9457189
• 负责人: Peter L Jones
• 金额: $ 31.06万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9457189
• 关键词: Mechanisms; DUX4; mediated; FSHD; pathology

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.

描述（由申请人提供）：我们提出的实验将鉴定引起DUX 4错误表达并导致面肩肱型肌营养不良症（FSHD）发病的机制。FSHD是困扰儿童和成人的最普遍的肌病，但目前尚无治疗方法。然而，FSHD研究现在已经进入了一个重要的新阶段，因为DUX 4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，成为共识FSHD候选基因。目前的证据支持FSHD病理模型，其中多腺苷酸化DUX 4 mRNA剪接变体（称为DUX 4-fl（fl =全长））的稳定表达导致致病性DUX 4-FL蛋白的产生。该模型与我们的发现相一致，基于对生肌细胞和活检的大型新文库的分析，即DUX 4-fl mRNA和DUX 4-FL蛋白在FSHD中以比健康对照肌细胞高得多的水平和高得多的肌核分数表达。因此，在FSHD患者中，随着DUX 4-FL诱导的病理变化随时间积累，临床上明显的肌无力可能发展，而在健康对照肌肉中，极低水平的DUX 4- FL显然不足以诱导明显的病理。虽然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DUX 4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DUX 4-fl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DUX 4-FL表达的补充分析，我们提出并将检验以下假设：表观遗传学失调导致FSHD中DUX 4-FL的表达相对于未受影响的生肌细胞异常增加，从而导致病理学。在具体目标1中，我们将鉴定调节骨骼肌细胞中DUX 4-fl mRNA表达水平的表观遗传和非表观遗传机制。在特定目标2中，我们将分析活检组织中的DUX 4-FL表达，并使用单细胞分析来确定DUX 4-FL表达如何改变FSHD与未受影响的乳腺癌的命运。在具体目标3中，我们将鉴定由DUX 4蛋白同种型差异调节的基因网络，并确定它们在DUX 4-fl表达的FSHD细胞中是否被破坏。我们的研究结果将系统地研究DUX 4亚型在FSHD和对照肌细胞中的表达、调节和功能，将提供指导开发专门针对DUX 4的FSHD疗法所需的重要信息。