FSHD: Chromatin Structure, Looping, & Expression

描述（由申请人提供）：面肩肱骨肌营养不良症（FSHD）是一种独特的疾病，涉及串联3.3 kb重复序列的缩短。未受影响的个体在4号染色体长臂的两个等位亚端粒区域（在4q35处）有11-100个重复D4Z4。患有这种进行性、衰弱性和疼痛性疾病的患者在其4号染色体同源物上只有1-10个重复序列。几乎相同的D4Z4重复序列位于10号染色体长臂的亚端粒末端，嵌入在极其相似的序列中，长度为25-45 kb，但尽管这些序列也可以出现在1-100个拷贝中，但在10号染色体上没有与短D4Z4序列相关的表型。许多证据表明，4号染色体上的短D4Z4阵列通过异常改变4q35上一个相当远的基因的表达而导致FSHD。本研究包括分析D4Z4阵列和4q35基因区域的染色质和染色质蛋白的性质，并寻找候选FSHD基因阵列和启动子区域之间以及阵列末端之间的长距离环相互作用。要分析的细胞将是二倍体成肌细胞，由成肌细胞诱导的肌管，以及异源细胞类型，即淋巴母细胞系和二倍体成纤维细胞。培养物将来自FSHD患者样本（将在本研究期间继续收集）以及疾病对照者；他们的D4Z4阵列的已知大小将与4q35染色质的性质进行比较。体内DNasel和硫酸二甲酯足迹，电泳迁移率转移测定，染色质免疫沉淀测定，免疫细胞化学和两种用于监测远程染色质相互作用的新测定将是本研究中使用的主要技术。提出的研究应阐明基因表达远程控制的新方面，以及为目前难治性疾病提供临床有用的见解。

DESCRIPTION (provided by applicant): Facioscapulohumeral muscular dystrophy (FSHD) is a unique disorder involving shortening of an array of tandem 3.3-kb repeats. Unaffected individuals have 11-100 copies of this repeat, D4Z4, at both allelic subtelomeric regions on the long arm of chromosome 4 (at 4q35). Patients afflicted with this progressive, debilitating and painful disease have only 1-10 copies of the repeat on one of their chromosome 4 homologues. Almost identical arrays of D4Z4 repeats embedded in extremely similar sequences on both sides of the array for 25-45 kb are located at the subtelomeric end of the long arm of chromosome 10 but although these also can be present in 1-100 copies, there is no phenotype associated with short D4Z4 arrays on chromosome 10. Much evidence suggests that a short D4Z4 array on chromosome 4 causes FSHD by abnormally altering expression of a rather distant gene at 4q35. This research involves analyzing the nature of the chromatin and chromatin proteins in the D4Z4 arrays and in 4q35 gene regions and looking for long-distance looping interactions between the array and promoter regions of candidate FSHD genes as well as between the ends of the array. The cells to be analyzed will be diploid myoblasts, myotubes induced from myoblasts, and heterologous cell types, namely, lymphoblastoid cell lines and diploid fibroblasts. The cultures will be derived from FSHD patient samples, which will continue to be collected during this study, as well as from disease-controls; the known sizes of their D4Z4 arrays will be compared to the properties of chromatin at 4q35. In vivo DNasel and dimethyl sulfate footprinting, electrophoretic mobility shift assays, chromatin immunoprecipitation assays, immunocytochemistry, and two new assays developed to monitor long-range chromatin interactions will be the main techniques used in this study. The proposed research should elucidate new aspects of long-distance control of gene expression as well as lending clinically useful insights into this currently intractable disease.