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.
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