Transgenic Drosophila for Investigating DUX4 and FRG1, Two Genes Associated with Facioscapulohumeral Muscular Dystrophy (FSHD)

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is typically an adult onset dominant myopathy. Epigenetic changes in the chromosome 4q35 region linked to both forms of FSHD lead to a relaxation of repression and increased somatic expression of DUX4-fl (DUX4-full length), the pathogenic alternative splicing isoform of the DUX4 gene. DUX4-fl encodes a transcription factor expressed in healthy testis and pluripotent stem cells; however, in FSHD, increased levels of DUX4-fl in myogenic cells lead to aberrant regulation of target genes. DUX4-fl has proven difficult to study in vivo; thus, little is known about its normal and pathogenic roles. The endogenous expression of DUX4-fl in FSHD-derived human muscle and myogenic cells is extremely low, exogenous expression of DUX4-fl in somatic cells rapidly induces cytotoxicity, and, due in part to the lack of conservation beyond primate lineages, viable animal models based on DUX4-fl have been difficult to generate. By contrast, the FRG1 (FSHD region gene 1), which is linked to FSHD, is evolutionarily conserved from invertebrates to humans, and has been studied in several model organisms. FRG1 expression is critical for the development of musculature and vasculature, and overexpression of FRG1 produces a myopathic phenotype, yet the normal and pathological functions of FRG1 are not well understood. Interestingly, DUX4 and FRG1 were recently linked when the latter was identified as a direct transcriptional target of DUX4-FL. To better understand the pathways affected in FSHD by DUX4-fl and FRG1, we generated transgenic lines of Drosophila expressing either gene under control of the UAS/GAL4 binary system. Utilizing these lines, we generated screenable phenotypes recapitulating certain known consequences of DUX4-fl or FRG1 overexpression. These transgenic Drosophila lines provide resources to dissect the pathways affected by DUX4-fl or FRG1 in a genetically tractable organism and may provide insight into both muscle development and pathogenic mechanisms in FSHD.

AAV-mediated follistatin gene therapy improves functional outcomes in the TIC-DUX4 mouse model of FSHD

“…Animal and cell model development has been a primary focus of the FSHD field and our laboratory since the DUX4 gene emerged more than a decade ago (35). During the last decade or so, several DUX4-expressing models have also been reported (6,9,(19)(20)(21)(22)(23)(24)(25)(26)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46). One particularly important study that guided the design of our TIC-DUX4 mice was first reported as a scientific abstract in 2008 and published in 2014 (25,43).…”

ETV transcriptional upregulation is more reliable than RNA sequencing algorithms and FISH in diagnosing round cell sarcomas with CIC gene rearrangements

“…Expression levels of CIC, ETV1, ETV4, ETV5, and WT1 were evaluated on both RNAseq platforms in comparison to other soft tissue tumors in each platform. The mRNA expression of known PEA3 family downstream target genes, including SPRY2, SPRY4, SPRED2, GPR20, DUSP6 GBX2, FGF8, POU5F1, MMP1/2/3/7/9, TWIST1, SNAI1/2, PTGS2, BAX, CCND2, ZEB1, PTK2, PLAU, ICAM1, VEGFA, NOTCH1, NOTCH4, and SPP1 was investigated from the whole transcriptome sequencing platform, while FRG1, PITX1, ZSCAN4, RFPL2, TRIM43, and LEUTX, were evaluated as DUX4 targets …”

“…The mRNA expression of known PEA3 family downstream target genes, including SPRY2, SPRY4, SPRED2, GPR20, DUSP6 GBX2, FGF8, POU5F1, MMP1/2/3/7/9, TWIST1, SNAI1/2, PTGS2, BAX, CCND2, ZEB1, PTK2, PLAU, ICAM1, VEGFA, NOTCH1, NOTCH4, and SPP1 10-19 was investigated from the whole transcriptome sequencing platform, while FRG1, PITX1, ZSCAN4, RFPL2, TRIM43, and LEUTX, were evaluated as DUX4 targets. [20][21][22][23] Cases studied by the whole transcriptome sequencing (n 5 10)…”

Pre-clinical Safety and Off-Target Studies to Support Translation of AAV-Mediated RNAi Therapy for FSHD

“… 8 , 9 , 10 , 11 , 12 , 13 The identification of DUX4 as a target gene provided a platform to finally enable translational research on FSHD, by allowing the development of DUX4 -expressing animal models and DUX4 -targeted molecular therapies. 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21…”