Intra-chromosomal looping within FSHD locus provides the epigenitic link between D4Z4 array and FRG1 gene expression

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder linked to deletion of D4Z4 repeat within the 4q35 region. ANT1, FRG1 and FRG2 genes, located upstream D4Z4 array (FSHD locus) are found upregulated in patients, and transgenic mice overexpressing FRG1 showed a phenotype resembling the disease. Although the relationship between deletions of D4Z4 and FSHD is established, how this triggers the disease remains unclear. We analysed the higher order structure of FSHD locus finding that FRG1 gene is repressed in myoblasts by H3K27 tri-methylation and Polycomb complex; during differentiation its activation is driven by MyoD and H3K4 tri-methylation. Polycomb complex establishes common chromatin structures on D4Z4 array. In myoblasts these non-contiguous regions physically interact, suggesting a FRG1 repression mediated by intra-chromosomal looping. The FSHD locus regulation is the result of the integration of different level of the epigenome, from DNA methylation and histone code to higher-order structures. This mechanism affects the transcription of FRG1 gene, which in FSHD myoblasts, with fewer D4Z4, shows an aberrant anticipation of its expression. Our results enlighten the epigenetic function for D4Z4 as orchestrator of in cis and in trans chromatin loops, foreseeing a broad effect of these sequences in the coordination of several regulatory elements in the human genome.