FSHD: Chromatin Structure, Looping, & Expression

描述（由申请人提供）：面肩肱型肌营养不良症（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号染色体长臂上的两个等位基因亚端粒区域（在4 q35处）具有11-100个该重复D4 Z4的拷贝。患有这种进行性的、使人衰弱的和痛苦的疾病的患者在他们的4号染色体同源物之一上只有1-10个重复拷贝。几乎相同的D4 Z4重复序列阵列嵌入在阵列两侧25-45 kb的极其相似的序列中，位于10号染色体长臂的亚端粒末端，但尽管这些也可以以1-100个拷贝存在，但没有与10号染色体上的短D4 Z4阵列相关的表型。许多证据表明，4号染色体上的短D4 Z4阵列通过异常改变4 q35处相当远的基因的表达而引起FSHD。这项研究涉及分析D4 Z4阵列和4 q35基因区域中染色质和染色质蛋白的性质，并寻找阵列和候选FSHD基因的启动子区域之间以及阵列末端之间的长距离循环相互作用。待分析的细胞将是二倍体成肌细胞、从成肌细胞诱导的肌管和异源细胞类型，即淋巴母细胞样细胞系和二倍体成纤维细胞。培养物将来自FSHD患者样本，这些样本将在本研究期间继续收集，以及来自疾病对照;将其D4 Z4阵列的已知大小与4 q35染色质的性质进行比较。在体内DNase 1和硫酸二甲酯足迹，电泳迁移率变动分析，染色质免疫沉淀分析，免疫细胞化学，和两个新的检测开发，以监测长距离染色质相互作用将在这项研究中使用的主要技术。这项拟议中的研究应该阐明基因表达远程控制的新方面，并为这种目前难以治愈的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.