Downstream events initiated by expression of FSHD-associated DUX4: Studies of nucleocytoplasmic transport, γH2AX accumulation, and Bax/Bak-dependence

Abstract

Abnormal expression in skeletal muscle of the double homeobox transcription factor DUX4 underlies pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). Though multiple changes are known to be initiated by aberrant DUX4 expression, the downstream events initiated by DUX4 remain incompletely understood. In this study, we examined plausible downstream events initiated by DUX4. First, we found that nucleocytoplasmic protein export appeared to be decreased upon DUX4 expression as indicated by nuclear accumulation of a shuttle-GFP reporter. Second, building on studies from other labs, we showed that phospho(Ser139)-H2AX (γH2AX), an indicator of double-strand DNA breaks, accumulated both in human FSHD1 myotube nuclei upon endogenous DUX4 expression and in Bax-/-;Bak-/- (double knockout), SV40-immortalized mouse embryonic fibroblasts upon exogenous DUX4 expression. In contrast, DUX4-induced caspase 3/7 activation was prevented in Bax-/-;Bak-/- double knockout SV40-MEFs, but not by single knockouts of Bax, Bak, or Bid. Thus, aberrant DUX4 expression appeared to alter nucleocytoplasmic protein transport and generate double-strand DNA breaks in FSHD1 myotube nuclei, and the Bax/Bak pathway is required for DUX4-induced caspase activation but not γH2AX accumulation. These results add to our knowledge of downstream events induced by aberrant DUX4 expression and suggest possibilities for further mechanistic investigation.

Post-transcriptional RNA stabilization of telomere-proximal RNAs FRG2, DBET, D4Z4 at human 4q35 in response to genotoxic stress and D4Z4 macrosatellite repeat length

“…To test whether 4q35 transcription was affected by a wider array of environmental perturbations, we analyzed the effects of genotoxic agents. We treated primary cells with Cisplatin (CIS) [62], Etoposide (ETO) [63] and Doxorubicin (DOXO) [64,65] (Figures 3 and Supplemental_FigS5). The centromere-proximal genes ( ANT1, FAT1 and FRG1 ) were mildly repressed upon genotoxic injury.…”

Proximity ligation assay to detect DUX4 protein in FSHD1 muscle: a pilot study

“…1 B–F). Standard immunofluorescence assays with mAb E5-5 also show inhomogeneity in DUX4 distribution within many individual nuclei [ 13 , 15 , 24 ]. As controls, we noted that nuclear PLA signals were not found when (i) mouse mAb P2G4 was replaced with an anti-GFP antibody or (ii) the myoblasts were incubated with BacMam-GFP instead of BacMam-DUX4-FL-V5.…”

“…ab124699, Abcam, Boston MA), which is a rabbit mAb specific for an epitope in the C-terminal region. Immunostaining for the V5 epitope or GFP were as described [ 13 , 15 , 22 , 24 ].…”

Facioscapulohumeral muscular dystrophy is associated with altered myoblast proteome dynamics

“…Herein, we performed proteomic analysis on previously generated and validated (22) immortalized myoblasts from matched pairs of individuals with FSHD and UASb. To the best of our knowledge, the present work represents the first proteomic analysis of immortalized FSHD and UASb cell lines reported in Homma et al (22), which have been characterized across several previous studies (8,10,(23)(24)(25)(26)(27).…”