Genetic and epigenetic mechamisms of FSHD pathogenesis

面肩肱骨营养不良(FSHD)是最常见的肌营养不良症之一。大多数人 4例与染色体4Q上的D4Z4重复序列缩短(FSHD1)有关，而 Smchd1转录抑制基因突变与FSHD患者的一小部分有关 (FSHD2)。Smchd1的突变也极大地加剧了FSHD1的表型，从而充当了 在FSHD1中，这种疾病的严重性。D4Z4重复序列中DUX4基因的异常表达 与FSHD1和FSHD2的发展相联系。然而，只有一小部分患者的肌肉 细胞表达DUX4蛋白，这种蛋白偶尔也可以在未受影响的肌肉细胞中观察到 个人。这表明仅有DUX4的表达可能不足以促进FSHD的发生。 此外，DUX4基因到底是如何在少数患者肌肉细胞中上调的，以及 它对FSHD的发展和进展的贡献尚不清楚。Smchd1是组蛋白H3的一部分 赖氨酸9-三甲基化(H3K9me3)“异染色质”结构，通常抑制DUX4的表达， 在FSHD1和FSHD2患者细胞中都受到影响。我们获得了H3K9me3特定的证据 在FSHD细胞中，不仅在D4Z4，而且在基因组的其他部分也是如此。此外，Smchd1 突变可能在FSHD的发病机制中具有DUX4非依赖的作用。因为D4Z4重复序列不存在 在小鼠基因组中，患者肌肉细胞是评估FSHD特异性细胞变化的关键。 然而，高质量的患者成肌细胞是有限的，而且样本之间的变异性只有一小部分。 表达DUX4的细胞可能会加剧群体分析的平均伪影。我们计划带两个人 绕过这些问题并检验我们的假设的补充策略：(1)克隆的发展 FSHD-模拟人成肌细胞系，以及(2)初级对照和FSHD肌肉的单核图谱 细胞。我们提出了一种假设，即FSHD是一种异染色质异常疾病，在这种疾病中，全基因组 H3K9me3和Smchd1功能改变易患或引发FSHD，少量(DUX4- 表达)疾病驱动细胞决定表型的进展。这个项目的具体目标是 目的：(1)建立人成肌细胞FSHD模型，建立FSHD紊乱机制(S)； 研究D4Z4染色质调节，(2)进行全基因组表观遗传和表达分析 单核水平，以了解DUX4上调的后果，并可能识别FSHD驱动 细胞，以及(3)采用蛋白质组学方法鉴定D4Z4异染色质结构的成分 进一步描述其在FSHD中失调的机制和后果，这将被整合到 目标1和目标2中的分析。这一项目的成功结果可能有助于进一步了解 FSHD发病机制及潜在治疗新靶点的确定(S) 治疗方法。

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.