Genetic and epigenetic mechamisms of FSHD pathogenesis

FSHD发病机制的遗传和表观遗传机制

• 批准号: 10188423
• 负责人: Seyed Ali Mortazavi
• 金额: $ 43.31万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188423
• 关键词: ATAC-seq; Address; Automobile Driving; Binding; Binding Proteins; Binding Sites; Biology; Cell Line; Cell Nucleus; Cell model; Cells; Characteristics; Chromatin; Chromosomes; D4Z4; DNA Binding; Dependence; Development; Disease; Engineering; Epigenetic Process; Facioscapulohumeral; Facioscapulohumeral Muscular Dystrophy; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genome; Heterochromatin; Histone H3; Human; Individual; Lead; Link; Lysine; Mediating; Minor; Modeling; Morphologic artifacts; Mus; Muscle Cells; Muscular Dystrophies; Mutation; Myoblasts; Outcome; Pathogenesis; Pathologic; Patients; Phenotype; Population; Population Analysis; Process; Proteins; Proteomics; Regulation; Sampling; Severities; Small Interfering RNA; Structure; Testing; Therapeutic; Transcription Repressor; Up-Regulation; chromatin immunoprecipitation; genome-wide; genomic locus; histone modification; human model; mouse genome; new therapeutic target; novel diagnostics; small hairpin RNA; transcriptome sequencing

Facioscapulohumeral dystrophy (FSHD) is one of the most prevalent muscular dystrophies. The majority of cases are associated with shortening of the D4Z4 repeat sequences on chromosome 4q (FSHD1) while mutations in the SMCHD1 transcriptional repressor gene are linked to a minor subset of FSHD patients (FSHD2). Mutations in SMCHD1 also greatly exacerbate the phenotype of FSHD1, thus acting as a modifier of the disorder's severity in FSHD1. Abnormal expression of the DUX4 gene present in the D4Z4 repeats is linked to the development of both FSHD1 and FSHD2. However, only a small percentage of patient muscle cells express DUX4 protein, which can also occasionally be observed in muscle cells from unaffected individuals. This suggests that DUX4 expression alone may not be sufficient for FSHD development. Furthermore, exactly how the DUX4 gene is upregulated only in a small number of patient muscle cells and how it contributes to FSHD development and progression are unclear. SMCHD1 is part of the histone H3 lysine 9-trimethylated (H3K9me3) “heterochromatin” structure that normally represses DUX4 expression, which is compromised in both FSHD1 and FSHD2 patient cells. We obtained evidence that H3K9me3 is specifically reduced not only at D4Z4 but also at other parts of the genome in FSHD cells. Furthermore, SMCHD1 mutations may have DUX4-independent effects on FSHD pathogenesis. Since D4Z4 repeats are not present in the mouse genome, patient muscle cells are essential for assessing FSHD-specific cellular changes. However, high-quality patient myoblasts are limited, and variability among samples with only a small subset of cells expressing DUX4 may exacerbate the averaging artifact of population analysis. We plan to take two complementary strategies to circumvent these issues and test our hypothesis: (1) development of clonal FSHD-modeling human myoblast lines, and (2) single-nucleus profiling of primary control and FSHD muscle cells. We propose a hypothesis that FSHD is a heterochromatin abnormality disorder, in which genome-wide changes of H3K9me3 and SMCHD1 function predispose to or initiate FSHD, and a small number of (DUX4- expressing) disease-driving cells dictate the progression of the phenotype. The Specific Aims of this project are (1) to generate “FSHD-modeling” human myoblast lines to establish the FSHD disorder mechanism(s) and to study D4Z4 chromatin regulation, (2) to perform genome-wide epigenetic and expression analyses at the single-nucleus level to understand the consequences of DUX4 upregulation and possibly identify FSHD-driving cells, and (3) to take a proteomics approach to identify the components of D4Z4 heterochromatic structure to further delineate the mechanism and consequence of its dysregulation in FSHD, which will be integrated into the analyses in Aims 1 and 2. The successful outcome of this project may lead to further understanding of the mechanism(s) underlying FSHD pathogenesis and the identification of potential new therapeutic targets and approaches for treatment.

面肩肱营养不良（FSHD）是最常见的肌营养不良症之一。大多数 例与染色体4 q上的D4 Z4重复序列（FSHD 1）缩短有关， SMCHD 1转录抑制基因的突变与FSHD患者的一小部分相关 （FSHD 2）。SMCHD 1中的突变也极大地加剧了FSHD 1的表型，从而作为SMCHD 1的修饰剂。 FSHD 1中疾病的严重程度。D4 Z4重复序列中DUX 4基因的异常表达是 与FSHD 1和FSHD 2的发育有关。然而，只有一小部分病人的肌肉 细胞表达DUX 4蛋白，偶尔也可以在未受影响的肌肉细胞中观察到。 个体这表明DUX 4单独表达可能不足以促进FSHD的发展。 此外，DUX 4基因仅在少数患者肌肉细胞中上调的确切方式， 它如何促进FSHD的发展和进展尚不清楚。SMCHD 1是组蛋白H3的一部分 赖氨酸9-三甲基化（H3 K9 me 3）“异染色质”结构，通常抑制DUX 4表达， 在FSHD 1和FSHD 2患者细胞中均受损。我们获得的证据表明H3 K9 me 3是 在FSHD细胞中，不仅在D4 Z4处减少，而且在基因组的其他部分也减少。此外，SMCHD 1 突变可能对FSHD发病机制具有DUX 4非依赖性作用。由于D4 Z4重复不存在， 在小鼠基因组中，患者肌肉细胞对于评估FSHD特异性细胞变化至关重要。 然而，高质量的患者成肌细胞是有限的，并且仅具有一小部分成肌细胞的样品之间的变异性是有限的。 表达DUX 4的细胞可能加剧群体分析的平均伪像。我们计划带两个 互补策略来规避这些问题，并测试我们的假设：（1）发展克隆 FSHD模拟人成肌细胞系，和（2）初级对照和FSHD肌肉的单核分析 细胞我们提出一个假说，即FSHD是一种异染色质异常疾病，其中全基因组 H3 K9 me 3和SMCHD 1功能的改变易患或启动FSHD，并且少数（DUX 4- 表达）疾病驱动细胞决定表型的进展。本项目的具体目标 （1）产生“FSHD-模拟”人成肌细胞系以建立FSHD病症机制，和 研究D4 Z4染色质调控，（2）在染色体上进行全基因组表观遗传和表达分析， 单核水平，以了解DUX 4上调的后果，并可能确定FSHD驱动 采用蛋白质组学方法鉴定D4 Z4异染色质结构的组分， 进一步阐明其在FSHD中失调的机制和后果，这将被整合到 目标1和2中的分析。该项目的成功结果可能会导致进一步了解 FSHD发病机制的潜在机制和潜在新治疗靶点的鉴定， 治疗方法。