FSHD: Chromatin Structure, Looping, & Expression

描述(由申请人提供)：面肩肩周型肌营养不良症(FSHD)是一种独特的疾病，涉及串联的3.3-kb重复序列的缩短。在4号染色体长臂的两个等位亚端粒区域(位于4q35)，未受感染的个体有11-100个重复的D4Z4拷贝。患有这种进行性、衰弱和痛苦的疾病的患者在他们的4号染色体同源物中的一个上只有1-10个重复的拷贝。几乎完全相同的D4Z4重复序列嵌入在阵列两侧极其相似的序列中，25-45kb位于10号染色体长臂的亚端粒末端，尽管这些重复序列也可以存在1-100个拷贝，但10号染色体上没有与短D4Z4阵列相关的表型。许多证据表明，4号染色体上的短D4Z4阵列通过异常改变4q35上一个相当遥远的基因的表达而导致FSHD。这项研究包括分析D4Z4阵列和4q35基因区域中染色质和染色质蛋白质的性质，并寻找阵列与候选FSHD基因的启动子区域以及阵列末端之间的长距离环状相互作用。将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种细胞类型，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，这些样本将在本研究期间继续收集，以及来自疾病对照组；他们D4Z4阵列的已知大小将与4q35的染色质属性进行比较。在体内DNasel和二甲基硫酸酯足迹、凝胶迁移率改变分析、染色质免疫沉淀分析、免疫细胞化学和两种新开发的用于监测染色质远程相互作用的方法将是本研究中使用的主要技术。这项拟议的研究应该会阐明基因表达的远程控制的新方面，并为这种目前难以治愈的疾病提供临床上有用的见解。

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.