Department and institution: School of Medicine and Surgery, Department of Health Sciences, University of Milano Bicocca

Research Area(s): Skeletal muscle

Functional genomic approaches to dissect molecular basis of Facio-scapulohumeral Dystrophy (FSHD)
Facioscapulohumeral muscular dystrophy (FSHD) is the third most common form of autosomal dominant muscular dystrophy and it results from deletion of a critical number of D4Z4 repeats on the subtelomeric region of chromosome 4q. Despite extensive searches, no transcription products have been identified from the 3.3 kb D4Z4 sequence. Accordingly, a leading hypothesis of FSHD pathogenesis has been that contractions within the repeat array affect local chromatin structure or function, leading to abnormal expression of genes adjacent to the deletion (ANT1, FRG1 and FRG2). More recently, progressive muscle degeneration was observed in transgenic mice that over-express FRG1, but not ANT1 and FRG2, supporting a model in which FSHD results from the up-regulation of this gene. However, in FSHD patients the increased expression of FRG1 has not been a uniform finding, and other mechanisms of transcriptional de-regulation, such as improper localization of the 4q telomere in the nucleus, have been proposed. In this regard, the observed de-regulation of 4q35 gene expression in FSHD could also be explained by loss of higher order of chromatin organization in the interphase nucleus. These hypotheses should be tested in a dynamic muscular environment, such as human physiological myogenesis, representing the cellular environment in which the FSHD defect more likely occurs. Recently we have found that a dynamic chromatin remodeling of the FSHD locus occurs during human myogenesis and this phenomenon involves the chromatin recruitment of Polycomb repressor complex on D4Z4 repeats. Polycomb group proteins (YY1, SUZ12, Ezh2, EED) are known to mediate transcriptional silencing through chromatin looping of distinct and non-contiguous genomic domains. All these findings set the basis for a co-regulation model in which distinct genomic domains are differently brought together in FSHD patients in respect to healthy
In order to dissect this hypothesis we planned different functional genomic strategies taking advantage of the use of human muscle stem cells (mesoangioblasts and myoblasts) derived from FSHD patients and healthy controls as cellular models of the FSHD disease. The myogenic differentiation conditions of these two stem precursors are already set up in our laboratory and represent the spatial-temporal context to perform the designed experiments:
1) ChIP on chip assays with PcG proteins comparing muscle stem cells derived from FSHD and controls.
2) Chromosome conformation capturing (3C/4C technology) in mesoangioblasts and myoblasts (affected and not affected by FSHD) during muscular differentiation (in collaboration with Prof. Enrico Ginelli lab, at University of Milan).
3) Three-dimensional FISH experiments and quantitative evaluation of 4q nuclear topology of FSHD and controls derived muscle cells (in collaboration with Prof. Stefan Mueller, at Ludwig-Maximilians University of Munchen).

- Lanctot C, Cheutin T, Cremer M, Cavalli G, Cremer T (2007) Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions. Nat Rev Genet. 8:104-15.
- Gabellini D, D'Antona G, Moggio M, Prelle A, Zecca C, Adami R, Angeletti B, Ciscato P, Pellegrino MA, Bottinelli R, Green MR, Tupler R (2006) Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1. Nature 439:973-7.
- Tam R, Smith KP, Lawrence JB (2004) The 4q subtelomere harboring the FSHD locus is specifically anchored with peripheral heterochromatin unlike most human telomeres. J. Cell Biol.167:269-79.
- Gabellini D, Green MR, Tupler R (2002) Inappropriate gene activation in FSHD: a repressor complex binds a chromosomal repeat deleted in dystrophic muscle. Cell 110:339-48.