Immunodetection of Human Double Homeobox 4

Expression and purification of DUX4 fusion protein

The sequence encoding the last C-terminal 76 amino acids of DUX4 was amplified by PCR using forward primer 5′-CGCGGATCCCCATGCAAGGCATCCCGGCGC-3′ and reverse primer 5′-CCGGAATTCCTAAAGCTCCTCCAGCAGAGCCCG-3′ and cloned in frame after glutathione-s-transferase in the bacterial expression vector pGEX-3x (Glutagene, Amrad, Kew, Australia). The sequence encoding the first N-terminal 159 amino acids of DUX4 was amplified by PCR using forward primer 5′-CGCGGATCCCCATGGCCCTCCCGACACCCTC-3′ and reverse primer 5′-CCGGAATTCCTGCGCGGGCGCCCTG-3′ and cloned in frame after glutathione-s-transferase in pGEX-3x. The template used for both N- and C-termini cloning was the previously described pCS2+mkgDUX4.⁽⁸⁾ Expression of the fusion protein in E. coli strain BL21DE3pLysS was induced by 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 37°C for 4 h. The expression product was purified using B-Per GST Fusion Protein Purification Kit (Pierce, Rockford, IL), according to the manufacturer's instructions. The purity and size of the fusion protein were determined by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Blue dye.

Antibody production and screening

Purified and concentrated fusion protein was used as immunogen. Antibody production was done in collaboration with Epitomics (Burlingame, CA) for the rabbit monoclonal antibodies (MAbs) and by the Antibody Development Laboratory at the Fred Hutchinson Cancer Research Center (Seattle, WA) for the mouse MAbs. The mouse MAbs will be commercially available. The rabbit MAbs will be available through Epitomics. The antisera from all animals were screened for reactivity by ELISA against the immunogen, and Western blots and immunofluorescence for against transfected DUX4. The best rabbit and mouse were chosen for fusion, and the subsequent hybridoma clones were screened by ELISA for positive reactivity for the fusion protein and negative reactivity for GST protein alone. To narrow the selection of clones, they were further screened by Western blots and immunofluorescence on transfected DUX4. The best clones were subcloned, expanded, and isotyped by the production facilities.

Expression plasmids and ectopic expression

The pCS2+mkgDUX4⁽⁸⁾ and pClneo-DUX4c⁽¹⁰⁾ expression vectors used were previously described. The murine myoblast line C2C12 was maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin (Gibco, Carlsbad, CA) at 37°C in an atmosphere containing 5% CO₂. Transient transfections were performed using SuperFect reagent (Qiagen, Valencia, CA) according to the manufacturer's specifications, and cells were harvested 24 h post-transfection for lysate or direct fixation. pclBABE+DUX4 was constructed by inserting the mkgDUX4 from pCS2 construct into blunted BamHI and EcoRI sites of a pclBABE vector (similar to Addgene Plasmid 20917, in place of MyoD) and transfected into C2C12 myoblasts using SuperFect as described above, and then collected for staining at 42 h post-transfection. Lentiviral constructs were made by inserting the coding sequences of full length and short DUX4 in place of GFP in the plasmid pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene, Cambridge, MA). Full length and short DUX4 are referred to as DUX4-fl and DUX4-s, respectively, in the report by Snider and colleagues.⁽¹⁵⁾

Western blot analysis

Cells were directly lysed in 2x Laemmli sample buffer and sonicated to shear genomic DNA. Equal amounts of untransfected C2C12, transfected DUX4, and transfected DUX4c lysates were loaded onto 10% bis-tris polyacrylamide gels and separated by electrophoresis. Proteins were transferred onto nitrocellulose membranes (Invitrogen, Carlsbad, CA) and blocked with 5% non-fat dry milk in phosphate-buffered saline (PBS) and 0.1% Tween-20 (PBST). The membranes were then separately incubated in appropriate dilutions of E5-5, E14-3, and P4H2 hybridoma supernatants in PBST overnight at 4°C. Blots were washed in PBST and incubated with appropriate secondary antibodies, HRP-conjugated goat-anti-rabbit IgG, or HRP-conjugated goat-anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), and subsequently washed again. Pierce ECL Western Blotting Substrate (Thermo Scientific Pierce, Rockford, IL) was added to membranes, and then blots were exposed to film and developed. Blots were stripped and reprobed with anti-α tubulin antibody (Sigma, St. Louis, MO) as a loading control.

Immunofluorescence

Cells were gently washed in PBS and fixed in 2% paraformaldehyde for 7 min at room temperature and then washed twice with PBS. Cells were permeabilized with 1% Triton X-100 (Sigma) in PBS for 10 min at room temperature with gentle rocking. Primary antibodies E5-5, E14-3, and P4H2 were added at appropriate dilutions in PBS overnight at 4°C and then washed with PBS. TRITC- or FITC-conjugated goat anti-rabbit or mouse secondary antibodies (Jackson ImmunoResearch Laboratories) were added for incubation for 1 h at room temperature and then washed with PBS and counterstained for DNA with 4′,6′-diamidino-2-phenylindole (DAPI). Cells were examined under a fluorescence microscope.

Production of antibodies against human DUX4 N- and C-termini

The first 159 amino acid sequence of DUX4 was used as the N-terminus antigen and the last 76 amino acid sequence of DUX4 was used as the C-terminus antigen, which is not shared by DUX4c (Fig. 1A). To generate the recombinant C-terminus antigen for immunization, the last 228 base pairs of DUX4 open reading frame was cloned in frame after a GST tag and expressed in Escherichia coli. Induction of E. coli using 1 mM IPTG resulted in a high level of expression of a protein of the expected size at 36 kDa (Fig. 1B). The expressed product was purified on a glutathione-coupled agarose column and eluted by competition with free glutathione. Peak A280 fractions eluted from the column were pooled and showed a high level of purity by SDS-PAGE (Fig. 1B, lanes 3, 4). The purified GST-DUX4 C-terminus was concentrated and injected into two rabbits and two mice for production of antibodies. Production of the N-terminal antigen was performed in the same manner and yielded similar purity (data not shown), and was injected into two rabbits and two mice for development of antibodies.

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FIG. 1.

Production of recombinant DUX4 antigen. (A) Schematic of DUX4 protein and the 159 amino acid N-terminus and 76 amino acid C-terminus antigens used to raise antibodies. (B) Expression and purification of GST-tagged recombinant C-terminus DUX4 antigen, indicated by black arrowheads. Bacterial lysates and purified fusion protein were separated by SDS-PAGE and stained with Coomassie Blue. Lane 1, uninduced bacterial cells; lane 2, bacterial cells induced with IPTG for 4 h; lanes 3 and 4, purified DUX4 fusion protein.

The antisera from all animals were screened for reactivity by Western blots and immunofluorescence against transfected DUX4 (data not shown). After fusion, the subsequent hybridoma clones were screened by ELISA for positive reactivity for the fusion protein and negative reactivity for GST protein alone. To narrow the selection of clones, they were further screened by Western blots and immunofluorescence on transfected DUX4. The clones with the best reactivity from screening were mouse MAbs P4H2, P2G4, and P2B1 and rabbit MAbs E5-5 and E14-3 (Table 1).

P4H2, P2G4, P2B1, E5-5, and E14-3 specifically recognize DUX4 protein

C2C12 mouse myoblast cells were transfected with plasmid expressing human DUX4. The sequence contained in the expression plasmid includes an upstream alternative initiation site about 60 amino acids away, thus producing two protein species differing by approximately 10 kDa in molecular weight. P4H2, E5-5, and E14-3 were all reactive to transfected DUX4 and none showed reactivity against untransfected C2C12 lysate by Western blot analysis (Fig. 2A). Mouse MAbs P2G4 and P2B1 were less effective on the Western blot method (data not shown). To assess for cross-reactivity against DUX4c, we transfected C2C12 cells with a plasmid expressing human DUX4c. Mouse monoclonal P4H2 and rabbit monoclonal E5-5 displayed no reactivity against DUX4c, whereas E14-3 also recognized DUX4c, which shares its N-terminus antigen sequence (Fig. 2A). Western blot using the previously characterized 9A12 monoclonal antibody confirmed the identity of DUX4 (Fig. 2B). Blots were stripped and reprobed with anti-α-tubulin antibody and showed similar loading of all samples per blot.

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FIG. 2.

Reactivity of monoclonal antibodies on Western blot analysis. Sample 1, C2C12 cells transfected with pCS2+mkgDUX4⁽⁸⁾; sample 2, untransfected C2C12 cells; sample 3, C2C12 cells transfected with pClneo-DUX4c.⁽¹⁰⁾ Blots were probed with the appropriate antibodies and then stripped and reprobed with anti-α-tubulin antibody for loading control. (A) Mouse monoclonal P4H2, rabbit monoclonal E5-5, and rabbit monoclonal E14-3; (B) mouse monoclonal 9A12.⁽⁹⁾

C2C12 cells transfected with DUX4 were also fixed and stained with the antibodies. The immunofluorescence analysis using all five monoclonal antibodies demonstrated strong nuclear staining of cells expressing DUX4 that is not seen in neighboring cells, which were not expressing DUX4 (Fig. 3). The immunostaining indicates that these antibodies are able to detect DUX4 protein expressed in the nuclei of mammalian cells, which agrees with previous studies on DUX4's cellular distribution using epitope tag-based experiments.⁽¹⁴⁾ The nuclear localization of DUX4 is consistent with its function as a transcription factor that is able to bind to DNA sequences via its homeodomains and regulate target gene expression.^(9,10)

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FIG. 3.

Reactivity of monoclonal antibodies on immunofluorescence. C2C12 myoblasts were transfected with human DUX4 and cells were stained with mouse monoclonal anti-DUX4 antibodies P4H2, P2G4, and P2B1 (left), and rabbit monoclonal anti-DUX4 antibodies E5-5 and E14-3 (right). Cells were counterstained with DAPI for nuclei.

Full length DUX4 induces cell death whereas the shorter splice form does not

To examine the effects of DUX4 when expressed at a lower level, a retroviral vector carrying DUX4 was used to introduce DUX4 into C2C12 myoblasts. At 42 h post-infection, cells were fixed, permeabilized, and co-labeled with mouse monoclonal P4H2 and rabbit monoclonal E14-3. Double-positive DUX4 nuclei on immunofluorescence exhibited a punctate, granular pattern suggestive of chromatin condensation and possibly a pre-apoptotic state (Fig. 4A). Kowaljow and colleagues demonstrated that ectopic overexpression of DUX4 in HEp-2 cells, an epidermoid carcinoma line, results in apoptosis via caspase 3/7 activation just 24 h post-transfection, though dramatically more evident at 48 h.⁽¹⁰⁾ The expression of DUX4 from our retroviral construct is likely closer to endogenous levels, and thus the effects on the murine myoblasts are more subtle, but the results are still indicative of cellular changes associated with the early stages of cell death.

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FIG. 4.

Effects of DUX4 in muscle cells. (A) C2C12 cells transfected with pclBABE+DUX4 for 42 h and co-immunostained with E14-3 (a) and P4H2 (b). (B) Equal number of wild-type human primary myoblasts were infected with lentivirus carrying either full length DUX4 (left) or short DUX4 (right) and DAPI stained for nuclei 48 h post-infection. (C) Lentiviral infection of human primary myoblasts with either full length or short DUX4 for 24 h (a, b, e, f) or 48 h (c, d, g, h). Cells were stained with E14-3 (red) and counterstained with DAPI (blue). Cells undergoing apoptosis are indicated by white arrowheads (c, d).

To examine the effects of the different DUX4 splice forms in the context of human muscle cells that would be highly relevant to FSHD, we used a lentiviral delivery system to transduce human primary myoblasts with full length and short DUX4. An equivalent number of myoblasts were infected with either splice form, but 48 h post-infection, more than half the cells were dead on the plates with full length DUX4, whereas there was minimal, if any, cell toxicity was seen on the plates with the short form of DUX4 (Fig. 4B). At 24 h post-infection, both full length and short DUX4 show little effect on cell viability and morphology. However, at 48 h post-transfection, full length DUX4-infected cells exhibited condensation of nuclei and cytoplasm, blebbing of membranes, and fragmentation of cells, features characteristic of apoptosis (Fig. 4B, c,d). The cells infected with short DUX4 remain intact, round and full, and show homogenous staining (Fig. 4B, panels g,h).