Polycomb complex shapes the higher order of D4Z4 chromatin structure during differentiation of normal and FSHD muscle stem cells

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscolar disorder. FSHD involves a complex cascade of epigenetic events following contraction of a D4Z4 repeat located on chromosome 4q35.2 (FSHD locus). Previous work has indicated that transgenic mice overexpressing FRG1, a gene proximal to the deletion, showed a phenotype resembling the FSHD disease. However, increased expression of FRG1 in FSHD patients has not been a uniform finding and up to now, several studies have failed in identifying the molecular mechanism affecting the FSHD locus functionality. We took advantage of ChIP/MeDIP and 3D immuno-FISH assays as complementary approaches to depict the higher order of chromatin organization of 4q35.2 region during myogenic differentiation of healthy and FSHD myoblast and mesoangioblast stem cells. We found that FRG1 undergoes to muscle specific regulation through a two-step activation mechanism, whereby removal of H3-K27 methylation and Polycomb complex components precedes MyoD recruitment on the FRG1 promoter; intriguingly, the same chromatin structure and PcG recruitment were contemporaneously found on D4Z4 array, rendering the Polycomb complex the first molecular player that link s FSHD locus to myogenic differentiation. Moreover, D4Z4 H3- mK27 signals were strongly reduced in FSHD myoblasts in respect to controls, suggesting the severe impairment of the PcG complex recruitment. Nevertheless, molecular alterations of the D4Z4 array do not have in FSHD myoblasts an effect in cis on FRG1 gene expression. These observations evidence a role of 4q35 D4Z4 in muscle differentiation, probably through inter-chromosomal interactions.