The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

Precise epigenetic gene and genome regulation is critical for normal development and epigenetic dysregulation is an underlying factor in many human disorders. One striking example of the failure to properly epigenetically regulate gene expression is FacioScapuloHumeral muscular dystrophy (FSHD), an autosomal dominant myopathy characterized by progressive atrophy of the facial, shoulder, and upper arm muscles. The FSHD genetic lesion is not a mutation in a protein-encoding gene, but a deletion at chromosome 4q35 within a large tandem array of noncoding DNA repeats (D4Z4) below a threshold number of 11 repeats (11-150 repeat units at 4q35 in the unaffected population). Although D4Z4 repeats are dispersed throughout the human genome, FSHD is strictly associated with deletions in 4q35 array. Complicating the issue, the FSHD is exclusively associated with one of two equally prevalent subtelomere variants, 4qA, (keeping in mind that the majority of people with 4q35A do not have the repeat deletion or FSHD) and never with 4qB. A deletion of the entire 4q35 region does not present FSHD, arguing against a loss of function model for FSHD. Thus, to exhibit FSHD pathology requires a deletion in the 4q35 D4Z4 array resulting in between 1 and 10 repeats in cis with subtelomere type 4qA, and requires additional 4q35 localized sequences. Overall, the circumstances resulting in FSHD strongly implicate an epigenetic misregulation of gene expression resulting in the increased expression of a 4q35-localized gene. There is evidence in the literature supporting each of several 4q35 candidate genes including FRG1, DUX4, and DUX4C, as well as genes located elsewhere including μ-crystallin and PITX1, and there is still no agreement in the field as to which gene(s) leads to FSHD and microarray expression data has been inconclusive.

We are attacking FSHD on two fronts; 1) understanding how the D4Z4 array and the 4q35A and B subtelomeres regulate gene expression (the cause of the epigenetic dysregulation), and 2) how increased 4q35 candidate gene expression leads to FSHD pathology (the effect of the dysregulation). For the epigenetic regulation studies, we use the highly efficient Xenopus transgenic technology to generate engineered telomeres in transgenic Xenopus, recapitulating the human 4q35 subtelomere and array in a controlled, manipulatable context and assay their affects on candidate gene promoter expression. For the studies on the developmental effects of FSHD candidate gene expression, their associated functions, and induced pathology, we use multiple model systems (Xenopus, Drosophila, C. elegans, and mouse), exploiting the benefits of each model organism, then confirming or extending our work to the more relevant vertebrate muscle cells.

Multiple FSHD candidate genes are being investigated in the lab. Based on our work and others, the strongest candidate gene for which misexpression leads to FSHD-like pathology is FRG1 (FSHD region gene 1). Overexpression of FRG1 (but no other candidate gene alone) in Xenopus leads to muscular and vascular phenotypes consistent with FSHD-like pathology. However, the precise function of FRG1 is not known making it difficult to understand its role in FSHD pathology. Fortunately, FRG1 is one of the most highly conserved proteins across its entire sequence from C. elegans to humans making it particularly amenable to a cross-species approach to a functional analysis. As such, a main thrust of the lab is determining the normal function of FRG1 protein in lower organisms to provide insight on how increasing FRG1 expression in humans leads to FSHD pathology.



Fig. 1. FRG1 overexpression leads to skeletal muscle disruption and disorganization. This tadpole is half-transgenic, overexpressing FRG1-EGFP from the human skeletal actin promoter in only one side (*right half). Thus, this tadpole is internally controlled for the effects of FRG1 overexpression on development. A) bright-field and B) EGFP fluorescence show the FRG1-EGFP fusion protein expression is confined to the right side of the animal and generally restricted to the skeletal muscle. Growth is partially inhibited or slowed on the FRG1 overexpressing side (*) compared with the control, non-transgenic side of the animal as indicated by the curvature of the body towards the transgenic side. In panels C-J, the tadpole was immunostained for myosin to show the musculature. C) Dorsal view of the head (with the transgenic* side being the top). D) Zoomed in view of the musculature of the eye on the FRG1 overexpressing side shows disorganized muscle architecture and misaligned muscle fibers compared with E) the non-transgenic side that has organized muscles with properly aligned fibers. F) Ventral view of the head (with the transgenic* side now on the bottom). The FRG1-mediated impairment of growth is readily apparent in the musculature under the mouth. These muscle fibers are also disorganized. G) Magnified view of the chest muscles on the FRG1 overexpressing side again show muscle disorganization and even broken fibers compared with H, the non transgenic side. The abdomen muscle fibers in J) the FRG1 overexpressing animals are misaligned and malformed compared with I) the non-transgenic side. Not shown, numerous tadpoles expressing EGFP alone were analyzed and absolutely none exhibited any of the phenotypes seen in FRG1-EGFP expressing animals confirming that the FRG1 overexpression was responsible for the disrupted musculature.