FUNCTIONAL RELEVANCE OF D4Z4-DERIVED ANTISENSE AND CODING SENSE TRANSCRIPTS ON DYSREGULATION OF MYOGENESIS IN FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY

FSHD is the third most common autosomal dominant muscular dystrophy, affecting 1 in 15.000 individuals worldwide. FSHD type I (also called FSHMD1A) represents more than 95% of clinical cases and is associated with the contraction of tandemly repeated 3.3 kb units of the D4Z4 macrosatellite at the subtelomeric q35 region on chromosome 4 (4q35). In particular, D4Z4 repeated array is characterized by a complex bidirectional transcriptional activity, including a protein-conding gene, DUX4, and an antisense long non-coding RNA (which we referred to as DUX4-AS2) overlapping DUX4 5' end. However, their involvement in etiopathogenesis of FSHD is still poorly understood. We then extended findings in altered myogenesis observed in FSHD, investigating the putative role of these D4Z4-deriving sense and antisense transcripts on dysregulation of muscle differentiation of dystrophic cells. By 3'RACE and NGS approaches, we confirmed DUX4-AS2 transcription in both healthy and FSHD muscle cells, during myogenesis. Particularly, DUX4-AS2 was upregulated in early stages of FSHD cell differentation and it was found significantly enriched into the nucleus. Subsequent analysis of its secondary structure and in vitro interaction with DGCR8/DROSHA complex supported hypothesis that it could act as primary microRNA precursor. By RT-qPCR experiments we validated six novel miRNAs deriving from DUX4-AS2 and showed that two out of these (as-miR-276-5p and as-miR-320-3p) was overexpressed in early stages of FSHD myogenesis. Preliminary transfection experiments supported target prediction analysis, resulting in a direct or indirect negative effects of as-miR-276-5p and as-miR-320-3p on expression of genes involved in mTOR signaling pathway, such as TSC1, RAPTOR, GBL and myostatin. The putative impact of overexpression of such D4Z4-deriving miRNAs on mTOR protein network could explain the altered differentiation observed in affected cells, representing a novel molecular mechanism involved in the etiopathogenesis of FSHD. On the other hand, we deepened functional role of DUX4 protein, defining its involvement in muscle differentiation by gene silencing studies. Interestingly, DUX4 downregulation, in early stages of myogenesis, resulted in slowing of myoblast fusion rate and in recovery of normal phenotype. Morover, we inferred DUX4 tridimensional structure and predicted its putative interactors by molecular dynamics simulation and immunofluorescence experiments. In particular, we found that DUX4 c-terminal domain shared homology with ubiquitin-binding CUE domain. Further protein-protein interaction simulation confirmed, in silico, that DUX4 could act as ubiquitin-binding protein via a hydrophobic patch centred on Ile44. Furthermore, by immunofluorescence we observed that DUX4 co-localized with M-cadherins, which appeared widespread along plasma membrane. Since re-cycling and distribution of M-cadherins is mediated by ubiquitin-dependent signaling, we proposed that DUX4, through its predicted putative ubiquitin-binding domain, could interfere with M-cadherins localization, resulting in altered differentiation characterizing FSHD cells.