Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c

Nuclear and cytoplasmic (co-)localization of DUX4 or DUX4c with FUS in myoblasts.

<p>LHCN-M2 cells were transfected with a DUX4c or DUX4 expression vector (<i>p-DUX4</i>, <i>p-DUX4c</i>) or the backbone vector (<i>pCIneo</i>). (<b>A</b>) In the cells transfected with the backbone vector (upper panels), FUS detected by immunofluorescence (green) was localized principally in the nuclei but also in the cytoplasm. In DUX4c- (middle panels) and DUX4- (bottom panels) overexpressing cells (red), nuclear and cytoplasmic FUS was generally observed. (<b>B</b>) Magnified boxed regions in A. Partial co-localization of cytoplasmic DUX4c or DUX4 with FUS are highlighted by triangles or arrowheads and was sometimes observed as emerging from the nuclei (circles). Arrows point to cytoplasmic spots of FUS.</p

Nuclear and cytoplasmic (co-)localization of DUX4 or DUX4c with SFPQ in myoblasts.

<p>LHCN-M2 cells were transfected with a DUX4c or DUX4 expression vector (<i>p-DUX4</i>, <i>p-DUX4c</i>) or the backbone vector (<i>pCIneo</i>). (<b>A</b>) In the cells transfected with the backbone vector (top panels), SFPQ detected by immunofluorescence (green) was localized either in the nuclei, excluding the nucleoli, with sometimes a major presence at the periphery (*) or in the entire cell (circle). In DUX4c-overexpressing cells (red), SFPQ was delocalized inside the nuclei and appeared in approximately 3 large spots (arrows) or seemed present in the entire cell (arrowhead) (middle panels). DUX4-overexpressing cells (red) are shown in the bottom panels with a similar SFPQ delocalization inside the nuclei in one DUX4-overexpressing cell. Boxed regions are magnified in B. (<b>B</b>) Arrows highlight cytoplasmic localization of DUX4c, DUX4 or SFPQ, sometimes observed as emerging from the nuclei.</p

Cytoplasmic detection of endogenous DUX4c in differentiating healthy myoblasts.

<p>DUX4c was detected by immunofluorescence (red) in proliferating immortalized myoblasts and during a differentiation time-course. During proliferation and after one day of differentiation, nuclear staining was observed in almost all myoblasts. However, the few nuclei with intense DAPI staining did not present DUX4c immunofluorescence (asterisks). In myoblasts, DUX4c was detected in the nucleoplasm or as 1 to 4 spots at the nuclear periphery (<b>a</b>). During differentiation, DUX4c began to be detected in the cytoplasm, and its nuclear labeling decreased. However, stronger nuclear DUX4c staining was still observed in myotubes containing at least 3 nuclei at day 3 (<b>b</b>) and in a myoblast presenting a cytoplasmic extension towards a myotube (arrows). In addition, a few muscle cells showed higher cytoplasmic DUX4c staining (the cross in <b>c</b>). At day 6, the nuclear staining was completely lost, and some myotubes (<b>d</b>) or myoblasts (circles) contained strong cytoplasmic staining, mostly on one side of the cell. Some myotube tips were also stained (arrowheads).</p

Partial co-localization of DUX4c or DUX4 and desmin in elongating transfected myoblasts.

<p>Healthy immortalized myoblasts were transfected with <i>pCIneo-DUX4c</i> (<b>A-C</b>) or -<i>DUX4</i> (<b>D-I</b>) expression vectors and switched to the differentiation medium. DUX4c, DUX4 (green), and desmin (red) were immunodetected at day 6, and myoblast nuclei were stained with DAPI. In addition to the strong DUX4/4c nuclear staining, elongating myoblast/myotubes had a few cytoplasmic spots (arrows) with similar or lower intensities; some of these spots were grouped in a myotube tip (<b>G-I</b>). The DUX4c nuclear staining (<b>A</b>) presented a pattern of linear stripes that may reflect interactions with the cytoskeleton and a nuclear bud (circle) (higher magnification is shown in the right panel and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146893#pone.0146893.s009" target="_blank">S9 Fig</a>).</p

Domains involved in the validated and putative interactions with DUX binding partners.

<p>The different domains of the DUX proteins are indicated with their validated protein partners (bold): (i) the double homeodomain can interact with desmin, (ii) the DUX4 double homeodomain (identical to DUX4c) can interact with IPO13 and RNA-binding proteins C1QBP/Splicing factor 2 P32, and (iii) full-length DUX4 can interact with LMCD1, SRSF9, RBM3, FUS/TLS and SFPQ. Question marks indicate putative interactions: (i) the double homeodomain could interact with α-actinins, (ii) the PxLxP motif and proline-rich domain could interact with a subset of MYND proteins (e.g., SMYD1), (iii) the DUX4 terminal (term) region could interact with myosin-related proteins and calmodulin and (iv) the last 32 residues of DUX4c and the homologous DUX4 residues could interact with peroxiredoxin. The interactions with LIM proteins, actin- or tubulin-associated proteins and RNA-binding proteins are supported by Y2H or co-purification experiments and validated for the serine/arginine-rich splicing factor 9, the RNA binding Motif 3 proteins and the LIM and cysteine rich domain 1 protein (LMCD1).</p

Alignment of the DUX1, DUX4 and DUX4c protein sequences.

<p>DUX4c and DUX4 are identical from the NH₂ terminus to residue 342. The two homeodomains highlighted in boxes and are targets of rabbit TAR13 serum [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146893#pone.0146893.ref004" target="_blank">4</a>]. Residues that differ are highlighted in grey. The positions of putative CKII phosphorylation sites (brown) and putative binding regions for tubulin (red) and of a subset of MYND (blue) proteins (putative DUX partners) are indicated. The region recognized by MAb 9A12 (residues 230–303), which cross-reacts with DUX4 and DUX4c, and the peptides used to generate the specific DUX1, DUX4c or DUX4 rabbit antisera are underlined: the SB152 rabbit serum was directed against DUX1 residues 4 to 21, the 314 rabbit serum was directed against DUX4 residues 342 to 356, and the anti-DUX4c rabbit serum was directed against DUX4c residues 351 to 366. The DUX4tail (DUX4-t) and DUX4term (green residues) domains map to residues 172–424 and 349 (alternative initiator methionine)-424, respectively. Two DUX4c polymorphisms are indicated [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146893#pone.0146893.ref019" target="_blank">19</a>].</p

Co-immunoprecipitation of DUX proteins and desmin.

<p>C2C12 cells were transfected with the <i>pCI-neo</i> expression vector for either DUX4 or DUX1 or with the backbone vector. Total cell lysates were prepared 24 h later and incubated with rabbit sera raised against the double homeodomain common to DUX1 and DUX4 (TAR13) or the amino-terminal domains of DUX1 (SB152) or DUX4 (314). Samples of input (total extracts of cells transfected with the backbone vector) and the immunoprecipitates were separated by SDS-PAGE and electrotransferred to a PVDF membrane for Western blotting. Desmin was detected using a specific antiserum (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146893#sec002" target="_blank">Methods</a> for details).</p

Putative DUX4c protein partners based on Y2H analysis.

<p>Putative DUX4c protein partners based on Y2H analysis.</p