Project Summary. Facioscapulohumeral muscular dystrophy (FSHD) is an adult onset myopathy caused by epigenetic changes that most commonly occur as a consequence of a reduction in the number of 3.3 kb D4Z4 units arrayed on chromosome 4 to less than 11 units. The contraction-induced epigenetic profile results in the possibility of transcription of the normally repressed DUX4 retrogene when contractions occur on arrays with permissive D4Z4 haplotypes. Additional unknown events result in DUX4 transcription in < 5% of nuclei and it is generally accepted that DUX4 produces myopathic changes that manifest as muscle weakness. The small percentage of DUX4-expressing cells suggests that even in the diseased state there is significant (> 95%) suppression of DUX4 transcription. FSHD appears to be driven by the occasional nucleus that overcomes suppressive mechanisms and begins to produce DUX4 that spreads to adjacent nuclei within the same myofiber. We have developed novel techniques for purification of FSHD-expressing myocytes from non-expressing cells in the same cultures, and techniques for precise and exclusive analysis of DUX4-transcribing arrays in these cells. This approach increases the sensitivity of detection of FSHD-related events and allows us to ask new questions regarding the regulation of DUX4 transcription and the chromatin modifications associated with DUX4-activated cells. We propose three aims to determine the chromatin differences between DUX4- expressing and DUX4-silenced cells within the same FSHD-derived cultures, to perform genetic and chemical screens to identify mechanisms of DUX4 activation and suppression, and to identify small interfering RNAs that regulate DUX4 transcription through D4Z4 chromatin modifications.

Project Narrative (Relevance to public health) Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating genetic condition manifest by weakness of facial and upper extremity musculature that presents in the 2nd decade of life. There is no known treatment for FSHD and its symptoms can be disfiguring, debilitating, and can shorten the life of affected individuals. This research proposal describes three scientific aims designed to understand cellular pathways that determine how FSHD-associated DNA is packaged and potentially identify new reagents for therapeutic development.