Fig 2: The DUX4 protein. (A) Ordered and disordered regions in the DUX4-FL protein as predicted by RaptorX Structure Prediction (raptorx.uchicago.edu). The two DNA-binding homeodomains and a C-terminal were predicted to have defined tertiary structures, whereas the ‘Mid’ region between homeodomain 2 and the C-terminal was predicted to be disordered. Shown is the most likely of the many similar structures returned by RaptorX. Similar predictions of ordered and disordered domains were generated by other prediction sites (not shown) as described in the Materials and Methods. In addition, there is a potential nine-amino acid transcription-activating domain (9aaTAD) at amino acids 371-379 as predicted by the online Nine Amino Acids Transactivation Domain Prediction Tool (http://www.med.muni.cz/9aaTAD/). (B) Linear representation of the DUX4 protein and sites of modification for this study. The diagram shows the two homeodomains, the predicted disordered Mid region, and sub-regions of the C-terminal domain as used to generate the DUX4 deletion and fusion cDNA constructs that are listed in Table 1. Each construct was modified by addition to the C-terminus of a seven-amino acid linker (gray unlabeled box) and the 17-amino acid V5 epitope. (C) Amino acid sequence of the full-length DUX4-FL-V5 protein as expressed in this study. The first 159 amino acids that compose the DUX4-S isoform are shown in blue with the two homeodomains underlined. The remaining amino acids (160-424) of endogenous DUX4-FL are shown in green, the linker sequence is in black, and the V5 epitope is in red.

Fig 3: FRG1 Peak1 region is sufficient for specific DUX4 transactivation. (A) Schematic representation of the constructs employed in luciferase assays. Genomic fragments of the FRG1 gene containing DUX4 putative binding sites were cloned upstream of the SV40 promoter, which controlled the expression of the Luciferase reporter gene. DUX4 core binding motifs within FRG1 genomic regions are reported. The three nucleotides of FRG1 Peak1 region that were mutated to generate FRG1 Peak1-mutated region are underlined. (B) FRG1 Peak1 region is able to transactivate the Luciferase reporter gene in the presence of DUX4, while the mutation in key DUX4 consensus sequence nucleotides abolishes the transactivation. DUX4 is not able to transactivate the Luciferase gene through FRG1 Peak2 region (paired t-test, *P < 0.05, n = 3, mean ± SEM).

Fig 4: Expression of SDS-resistant, potential multimers upon transfection of DUX4 construct plasmids. (A) SDS-PAGE and immunoblotting were used to analyze proteins expressed in HeLa cells at 24 h after transfection with the indicated DUX4 pCS2(+)-V5 plasmids. On the upper blot, proteins were detected with a mAb that reacted with the V5 epitope on the C-terminus of each construct. In this figure and all following figures, each band marked with a single asterisk is the size predicted for a monomeric protein expressed from the corresponding cDNA construct, whereas bands marked with double and triple asterisks are the sizes expected for potential dimers and trimers, respectively. The potential dimer band was most noticeable for DUX4-S (lane 2). Numbers to left of blot indicate molecular mass in kDa. (B) Densitometry of immunoblots was used to determine the percentage of protein expressed from each construct that was the size of a potential dimer. Linear curve-fitting was used to derive the solid line (y = 73.6-0.053x, R = 0.90). The percentage of potential dimers decreased as the cDNA insert became longer. (C) In a separate experiment, HEK293 cells were transfected with the DUX4-FL plasmid for 48 h and the resulting proteins were detected by immunoblotting either with mAb E55, which is specific to an epitope in the C-terminal portion of DUX4-FL22, or with anti-V5 as indicated. Two major bands reacted with both antibodies, one the size of the expected monomer (single asterisk) and a second the size of a potential dimer (double asterisk). Also seen were potential higher order multimers, as well as intermediate and smaller bands that could be proteolytic products. This result shows that the SDS-resistant, potential multimers included the E55 (endogenous DUX4-FL) and V5 (tag) epitopes. MW = markers of protein molecular mass shown in kDa.

Fig 5: Identification of trans-spliced mRNAs produced from pCS2(+)-DUX4-S-V5. (A) Sequence of PCR band “B” from Fig. 3C determined with reverse primer #269 included (in order from 5' to 3'): (i) the C-terminal portion of a DUX4-S open reading frames (DUX4-S ORF1, light blue box); (ii) the first linker sequence (light green box); (iii) a single G nucleotide from the V5 epitope (light red box) which, at the indicated trans-splicing site, was joined to; (iv) a sequence from the 5' region just upstream of DUX4-S cDNA in the original plasmid (light orange box); (v) a second complete DUX4-S (ORF2, light blue box, initial ATG in bold); (vi) a second linker (light green box); (vii) a complete V5 epitope sequence (light red box); and (viii) a TAG stop codon (shown in red). The indicated trans-splicing site and surrounding sequences were also found in bands “D” and “F” in Fig. 3C. (B) The upper diagram shows how “Band B” was generated by trans-splicing of two mRNAs (mRNA1 x mRNA2) using a donor site at the beginning of the V5 epitope and an acceptor site in the 5' region upstream of the DUX4-S cDNA. The lower diagram shows the covalent, head-to-tail, DUX4-S-V5 dimer that would be generated from the trans-spiced mRNA. (C) Diagram structures of PCR bands “C” and “E” in Fig. 3C as determined from sequencing. In this case, two splicing events occurred. One splice (Splice 1) joined the donor site in the V5 epitope (as also used in band “B”) to an acceptor site in the downstream ampicillin resistance (AmpR) gene. This first splice could have arisen either by splicing within a single mRNA or by trans-splicing. The second splice (Splice 2) joined a donor site in AmpR to the acceptor site (as also used in band “B”) in the 5' region upstream of the DUX4-S cDNA and thus must have arisen by trans-splicing. The 206 nucleotide (nt) sequence of the AmpR gene was in the reverse coding orientation (as indicated by the upside down and reversed AmpR symbol in the yellow box) and contained two stop codons in the DUX4-S reading frame as indicated by the asterisks. As seen in the diagram, the only intact V5 epitope sequence in this mRNA was downstream (3') of the stop codons in AmpR, so no V5-tagged protein would have been produced from this mRNA. (D) Diagram structure of PCR band “F” in Fig. 3C as determined from sequencing. In this case, three splicing events occurred. The sites labeled Splice 1 and Splice 2 were the same as in panel C. The site labeled Splice 3 resulted in the inclusion of a third DUX4-s open reading frame in the predicted mRNA.