Abstract
ABSTRACTAberrant expression of the full-length isoform of DUX4 (DUX4-FL) appears to underlie pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). DUX4-FL is a transcription factor and ectopic expression of DUX4-FL is toxic to most cells. Previous studies showed that DUX4-FL-induced pathology requires intact homeodomains and that transcriptional activation required the C-terminal region. In this study, we further examined the functional domains of DUX4 by generating mutant, deletion, and fusion variants of DUX4. We compared each construct to DUX4-FL for (i) activation of a DUX4 promoter reporter, (ii) expression of the DUX4-FL target gene ZSCAN4, (iii) effect on cell viability, (iv) activation of endogenous caspases, and (v) level of protein ubiquitination. Each construct produced a similarly sized effect (or lack of effect) in each assay. Thus, the ability to activate transcription determined the extent of change in multiple molecular and cellular properties that may be relevant to FSHD pathology. Transcriptional activity was mediated by the C-terminal 80 amino acids of DUX4-FL, with most activity located in the C-terminal 20 amino acids. We also found that non-toxic constructs with both homeodomains intact could act as inhibitors of DUX4-FL transcriptional activation, likely due to competition for promoter sites.This article has an associated First Person interview with the first author of the paper.
Highlights
Aberrant expression of the full-length isoform of the double homeobox protein DUX4 (DUX4-FL), in skeletal muscle, appears to underlie pathogenesis in facioscapulohumeral muscular dystrophy (FSHD)
Aberrant expression of DUX4-FL in FSHD is associated with a decreased number D4Z4 repeats, DNA hypomethylation, and a telomeric sequence that is used as a poly-adenylation signal for the DUX4-FL mRNA (Daxinger et al, 2015; Gatica and Rosa, 2016; Hewitt, 2015; Himeda et al, 2015; Tawil et al, 2014; Wang and Tawil, 2016)
We found that human embryonic kidney line 293 (HEK293) cells had a measureable baseline level of DEVDase activity that was increased ∼3-4× by expression of DUX4-FL (Fig. 4)
Summary
Aberrant expression of the full-length isoform of the double homeobox protein DUX4 (DUX4-FL), in skeletal muscle, appears to underlie pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). In FSHD, the 424 amino acid DUX4FL protein is expressed from an open reading frame in the most telomeric 3.3 kb D4Z4 repeat on chromosome 4q (Lemmers et al, 2010). Aberrant expression of DUX4-FL in FSHD is associated with a decreased number D4Z4 repeats, DNA hypomethylation, and a telomeric sequence that is used as a poly-adenylation signal for the DUX4-FL mRNA (Daxinger et al, 2015; Gatica and Rosa, 2016; Hewitt, 2015; Himeda et al, 2015; Tawil et al, 2014; Wang and Tawil, 2016). A shorter DUX4 isoform (DUX4-S) that consists of just the N-terminal 159 amino acids (including both homeodomains) of DUX4-FL is not toxic (Geng et al, 2011)
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