Probing the myopathic activity of DUX4 using isogenic telomere-edited iPS cells

项目摘要 面肩肱型肌营养不良症（FSHD）是一种显性退行性肌肉疾病， 治愈或治疗。FSHD 4 q35亚端粒D4 Z4重复拷贝数减少的遗传原因 编码DUX 4同源框蛋白，DUX 4同源框蛋白是对细胞有毒的有效转录因子。收缩 导致重复诱导沉默的丧失，从而允许DUX 4的转录。有趣的是， D4 Z4重复序列模拟WT等位基因，因为没有病理后果。因为D4 Z4是最后一个 在端粒之前的chr 4元件，删除chr 4的末端有效地纠正了突变等位基因。在这 研究中，我建议使用基因组工程的方法介导的位点特异性核酸酶删除4 q35 在一些实施方案中，D4 Z4是含有D4 Z4重复序列的端粒区域，因此特异性消除DUX 4。有针对性地整合 人工端粒产生末端染色体缺失以前从未被证明， FSHD是一种显性疾病，其基因是4号染色体上的末端基因。 FSHD的常见诊断方法显示使用血细胞的gDNA的D4 Z4的去甲基化，并且 我们的实验室已经表明，D4 Z4在FSHD iPS细胞中是去甲基化的，这意味着FSHD中的任何细胞类型都可以在FSHD iPS细胞中发生去甲基化。 患者可能会受到DUX 4的失调。我们之前已经证明DUX 4招募p300， 诱导组蛋白乙酰化整体变化，大规模破坏基因表达，因此病理学不 明显是肌肉特异性的为什么这种疾病是肌肉特异性的，肌肉中的哪些细胞可能失调 DUX 4是未知数。因为没有证据表明DUX 4表达是成肌细胞或肌纤维特异性的， 我推测DUX 4可能在非肌源性细胞中错误表达，而这些细胞参与肌源性细胞分化。 支持，这将导致FSHD致病进展。这些细胞类型在很大程度上被忽视， 外地因此，我将产生一组校正/患病iPS系的等基因对，并研究 DUX 4在肌原性细胞和支持性细胞类型中表达的存在和结果（通过 分化iPS细胞）。目的1将集中在肌源性谱系和利用异种移植模型 由我们的合作者Perlingdom实验室开创。目标2将集中在支持细胞类型和评估 细胞对肌生成的非自主作用。这些研究将开发一种独特的创新方法， 基因组工程和阐明FSHD肌肉变性的病理生理机制。

Project Summary Facioscapulohumeral Muscular Dystrophy (FSHD) is a dominant degenerative muscle disease with no cure or treatment. The genetic cause of FSHD reduction of copy number of subtelomeric D4Z4 repeats at 4q35 encoding DUX4 homeobox protein, which is a potent transcription factor that is toxic to the cell. Contractions lead to loss of repeat-induced silencing, allowing for transcription of DUX4. Interestingly, the complete absence of D4Z4 repeats mimics the WT allele in that there is no pathologic consequence. Since D4Z4 is the last element on chr4 before the telomere, deleting the end of chr4 effectively corrects the mutant allele. In this study, I propose to use a genome engineering approach mediated by site-specific nucleases to delete the 4q35 telomeric region containing D4Z4 repeats, thus specifically eliminating DUX4. Targeted integration of an artificial telomere to generate terminal chromosomal deletions has never been demonstrated before, and has a unique application in FSHD, a dominant disease whose gene is the terminal gene on chromosome 4. A common diagnostic method for FSHD shows demethylation of D4Z4 using gDNA of blood cells, and our lab has shown that D4Z4 is demethylated in FSHD iPS cells, meaning that any cell type in an FSHD patient could be subject to deregulation by DUX4. We have previously shown that DUX4 recruits p300 to induce global changes in histone acetylation, massively disrupting gene expression, thus pathology is not obviously muscle-specific. Why the disease is muscle-specific and which cells in muscle may be deregulated by DUX4 are unknowns. Because there is no evidence that DUX4 expression is myoblast or myofiber-specific, I hypothesize that DUX4 might be misexpressed in non-myogenenic cells that are involved in myogenic support, which would lead to FSHD pathogenic progression. These cell types have been largely overlooked in the field. I will therefore generate a panel of isogenic pairs of corrected/diseased iPS lines, and study the presence and consequence of DUX4 expression in myogenic as well as supportive cell types (obtained by differentiating the iPS cells). Aim 1 will focus on the myogenic lineage and make use of xenograft models pioneered by our collaborators, the Perlingeiro Lab. Aim 2 will focus on the supportive cell types and evaluate cell non-autonomous effects on myogenesis. These studies will develop a unique and innovative method of genome engineering and shed light on the pathophysiological mechanism of muscle degeneration in FSHD.