An isogenetic myoblast expression screen identifies DUX4-mediated FSHD-associated molecular pathologies
2008; Springer Nature; Volume: 27; Issue: 20 Linguagem: Inglês
10.1038/emboj.2008.201
ISSN1460-2075
AutoresDarko Bosnakovski, Zhaohui Xu, Eun Ji Gang, Cristi L. Galindo, Mingju Liu, Tugba Simsek, Harold R. Garner, Siamak Agha‐Mohammadi, Alexandra Tassin, Frédérique Coppée, Alexandra Belayew, Rita Perlingeiro, Michael Kyba,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle2 October 2008free access An isogenetic myoblast expression screen identifies DUX4-mediated FSHD-associated molecular pathologies Darko Bosnakovski Darko Bosnakovski Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, MN, USA Search for more papers by this author Zhaohui Xu Zhaohui Xu Department of Developmental Biology, UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Eun Ji Gang Eun Ji Gang Department of Developmental Biology, UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Cristi L Galindo Cristi L Galindo Center for Biomedical Invention, UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Mingju Liu Mingju Liu Department of Developmental Biology, UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Tugba Simsek Tugba Simsek Department of Developmental Biology, UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Harold R Garner Harold R Garner Center for Biomedical Invention, UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Siamak Agha-Mohammadi Siamak Agha-Mohammadi Division of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Alexandra Tassin Alexandra Tassin Laboratoire de Biologie Moleculaire, Universite de Mons-Hainaut Pentagone, Mons, Belgium Search for more papers by this author Frédérique Coppée Frédérique Coppée Laboratoire de Biologie Moleculaire, Universite de Mons-Hainaut Pentagone, Mons, Belgium Search for more papers by this author Alexandra Belayew Alexandra Belayew Laboratoire de Biologie Moleculaire, Universite de Mons-Hainaut Pentagone, Mons, Belgium Search for more papers by this author Rita R Perlingeiro Rita R Perlingeiro Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, MN, USA Search for more papers by this author Michael Kyba Corresponding Author Michael Kyba Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, MN, USA Search for more papers by this author Darko Bosnakovski Darko Bosnakovski Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, MN, USA Search for more papers by this author Zhaohui Xu Zhaohui Xu Department of Developmental Biology, UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Eun Ji Gang Eun Ji Gang Department of Developmental Biology, UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Cristi L Galindo Cristi L Galindo Center for Biomedical Invention, UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Mingju Liu Mingju Liu Department of Developmental Biology, UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Tugba Simsek Tugba Simsek Department of Developmental Biology, UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Harold R Garner Harold R Garner Center for Biomedical Invention, UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Siamak Agha-Mohammadi Siamak Agha-Mohammadi Division of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Alexandra Tassin Alexandra Tassin Laboratoire de Biologie Moleculaire, Universite de Mons-Hainaut Pentagone, Mons, Belgium Search for more papers by this author Frédérique Coppée Frédérique Coppée Laboratoire de Biologie Moleculaire, Universite de Mons-Hainaut Pentagone, Mons, Belgium Search for more papers by this author Alexandra Belayew Alexandra Belayew Laboratoire de Biologie Moleculaire, Universite de Mons-Hainaut Pentagone, Mons, Belgium Search for more papers by this author Rita R Perlingeiro Rita R Perlingeiro Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, MN, USA Search for more papers by this author Michael Kyba Corresponding Author Michael Kyba Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, MN, USA Search for more papers by this author Author Information Darko Bosnakovski1, Zhaohui Xu2, Eun Ji Gang2, Cristi L Galindo3, Mingju Liu2, Tugba Simsek2, Harold R Garner3, Siamak Agha-Mohammadi4, Alexandra Tassin5, Frédérique Coppée5, Alexandra Belayew5, Rita R Perlingeiro1 and Michael Kyba 1 1Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, MN, USA 2Department of Developmental Biology, UT Southwestern Medical Center, Dallas, TX, USA 3Center for Biomedical Invention, UT Southwestern Medical Center, Dallas, TX, USA 4Division of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, USA 5Laboratoire de Biologie Moleculaire, Universite de Mons-Hainaut Pentagone, Mons, Belgium *Corresponding author. Lillehei Heart Institute and Department of Pediatrics, 4-126 Nils Hasselmo Hall, 312 Church Street SE, Minneapolis, MN 55455, USA. Tel.: +1 612 626 5869; Fax: +1 612 624 8118; E-mail: [email protected] The EMBO Journal (2008)27:2766-2779https://doi.org/10.1038/emboj.2008.201 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Facioscapulohumeral muscular dystrophy (FSHD) is caused by an unusual deletion with neomorphic activity. This deletion derepresses genes in cis; however which candidate gene causes the FSHD phenotype, and through what mechanism, is unknown. We describe a novel genetic tool, inducible cassette exchange, enabling rapid generation of isogenetically modified cells with conditional and variable transgene expression. We compare the effects of expressing variable levels of each FSHD candidate gene on myoblasts. This screen identified only one gene with overt toxicity: DUX4 (double homeobox, chromosome 4), a protein with two homeodomains, each similar in sequence to Pax3 and Pax7. DUX4 expression recapitulates key features of the FSHD molecular phenotype, including repression of MyoD and its target genes, diminished myogenic differentiation, repression of glutathione redox pathway components, and sensitivity to oxidative stress. We further demonstrate competition between DUX4 and Pax3/Pax7: when either Pax3 or Pax7 is expressed at high levels, DUX4 is no longer toxic. We propose a hypothesis for FSHD in which DUX4 expression interferes with Pax7 in satellite cells, and inappropriately regulates Pax targets, including myogenic regulatory factors, during regeneration. Introduction Facioscapulohumeral muscular dystrophy (FSHD) is the third most common inherited myopathy, affecting approximately 1/20 000 individuals. It is caused by a deletion within the large tandem D4Z4 repeat sequence near the telomere of 4q. The normal chromosome 4 carries approximately 150 tandem copies of the 3.3 kb D4Z4 repeat, whereas FSHD-associated chromosomes carry 10 or fewer (Wijmenga et al, 1992). Although caused by a deletion, the disease is dominantly inherited. This is not due to haploinsufficiency of 4qter as a large deletion removing all D4Z4 repeats and extending proximally into chromosome 4 does not cause FSHD, implying that at least one copy of D4Z4 or nearby sequence is necessary for disease-related pathology (Gabellini et al, 2002). The D4Z4 repeat binds a YY-1-containing transcriptional repressor complex and it has been proposed that a repeat array of greater than 10 tandem units can silence nearby genes, whereas a deleted array allows inappropriate activation of nearby genes (Gabellini et al, 2002). Consistent with this, 4q35 sequences are hypo-methylated on FSHD-associated chromosome 4 variants compared with controls (van Overveld et al, 2003). The region immediately proximal to the D4Z4 repeats harbours a number of candidate genes, including FRG1 (FSHD-related gene) (van Deutekom et al, 1996), which encodes a nucleolar protein involved in RNA biogenesis (van Koningsbruggen et al, 2004), TUBB4Q, a β-tubulin family member, and FRG2, a predicted transcript of four exons comprising an open reading frame (ORF) with no significant homology to any known protein. The sequence proximal to these three genes is gene poor, with no predicted gene for the next 250 kb (van Geel et al, 1999). Past this proximal region, the gene ANT1 (adenine nucleotide transporter) has attracted attention due to its function in apoptosis (Doerner et al, 1997; Gabellini et al, 2002). The D4Z4 repeat itself was originally cloned in a low-stringency screen for novel homeobox genes (Wijmenga et al, 1992). Remarkably, each D4Z4 repeat contains two homeoboxes within a single predicted ORF (Gabriels et al, 1999). This predicted gene is referred to as DUX4 (double homeobox, chromosome 4). In addition to copies within each unit of the tandem array, there is one copy of DUX4 just proximal of FRG2, embedded within a truncated, inverted D4Z4 repeat (Dellavalle et al, 2007). This gene, referred to as DUX4c, is identical to DUX4 from the N terminus through the homeodomains, but the last 82 amino acids have been substituted for an unrelated 32 amino-acid sequence. Thus, DUX4 and DUX4c are also FSHD candidate genes. As FSHD is a dominant disease resulting from a gain-of-function mutation, modelling disease-related pathology in animals or cells requires testing candidate genes in gain-of-function genetic models. Three candidate genes, FRG1, FRG2, and ANT1, have been tested by overexpression in transgenic mice using the muscle-specific human skeletal actin promoter. Of these three, only FRG1 had a deleterious effect (Gabellini et al, 2002); however, the relevance of this model to FSHD is unclear as the pathology was only seen in mice expressing exceptionally high, non-physiological levels of FRG1. Furthermore, conventional transgenic studies suffer from high variability due to integration site-specific background effects; therefore, it is difficult to conclude that FRG1 is truly more myotoxic when compared with FRG2 or ANT1: expression patterns and absolute levels of expression in each transgenic strain differ. For such comparative studies, the ideal expression system would express each candidate gene from precisely the same genetic locus, and would be conditional and variable, so that low as well as high levels of expression could be tested. With this application in mind, we have therefore developed a novel, portable, cre-mediated gene targeting system, referred to as inducible cassette exchange (ICE). We have used this system to derive a murine myoblast ICE acceptor cell line, targeted this cell line with each FSHD candidate gene, and directly compared the effect of each on proliferating and differentiated myoblasts. We identify disease-related pathological changes from only one candidate, DUX4, and perform a detailed molecular analysis of the downstream effects of its expression during myoblast proliferation and differentiation. Results Generation of ICE myoblasts We have described earlier an inducible gene expression system for ES cells (Kyba et al, 2002) in which a circular plasmid carrying a gene of interest is targeted to a doxycycline (dox)-regulated locus. Cre-mediated recombination places a promoter and initiation codon in frame upstream of a G418 resistance gene, ensuring correct targeting (Fukushige and Sauer, 1992) while simultaneously placing the gene of interest under the control of the dox-regulated promoter. To enable site-directed integration in any cell type, we have modified this system to improve efficiency and adapted it for lentiviral transduction. First, to improve recombination efficiency, we have inserted a mutant version of loxP (lox2272 which self-recombines but does not recombine with loxP (Canales et al, 2006), referred to as loxM in Figure 1A) upstream of loxP, which now allows cassette exchange recombination. As the DNA in between loxM and loxP is replaced by the integrating plasmid, we have placed cre recombinase followed by ires–GFP into this space (Figure 1A). Finally, we have inserted the second-generation tetracycline response element (sgTRE, mutated to eliminate basal leakiness; Agha-Mohammadi et al, 2004) upstream of the floxed cre and placed the entire construct onto a self-inactivating lentiviral vector. A companion lentiviral vector carrying the ubiquitin C promoter (Lois et al, 2002) was used to express the reverse tetracycline transactivator (rtTA2(s)-m2; Urlinger et al, 2000). In cells transduced with both constructs, cre is induced with dox, and catalyses its own removal through cassette exchange recombination, placing the gene of interest under the control of dox (Figure 1A). Derivative cell lines can be made in parallel, each with a different gene inserted into the same locus. We refer to this system as ICE. Figure 1.Generation of ICE-recipient cell lines. (A) Schematic representation of the lentiviral constructs carrying the components of the ICE system, and the proviral ICE locus before and after recombination. (B) Flow cytometry of iC2C12 cells, which carry an inducible cre–ires–GFP proviral locus before recombination, and derivative cell lines carrying DsRed2 or luciferase, after recombination. (C) Southern blot analyses with GFP-specific probe DNA to detect copy number and recombination status of the ICE locus in iC2C12, iC2C12-DUX4 and i3T3 cell lines. Note that a single band hybridized with the GFP probe in iC2C12 and i3T3 cells but was missing following recombination (in iC2C12-DUX4 cells, in which GFP was replaced with DUX4). DNA from a spermatogonial cell line carrying GFP was used as a positive control. (D) Dose–response and time course (500 ng/ml) of luciferase gene expression in response to doxycycline. Download figure Download PowerPoint As the principle virtue of an ICE cell line is that multiple genes of interest can be compared directly at various levels of expression, it is essential that such a cell line carry only a single copy of the ICE locus. We transduced murine C2C12 myoblasts sequentially, first with rtTA at high titre, then with the 2Lox.cre–ires–GFP construct over a titration series. After these two transductions, a transient dose of dox allowed cotransduced cells to be identified, enumerated, and sorted through GFP fluorescence. To derive a single-copy integration clone, we single cell sorted from a transduction in which <1% of cells were GFP+, expanded multiple clones, and retested each for efficiency of GFP expression. We derived several clones in which GFP was detectable in 100% of cells after 24 h of dox, and undetectable by flow cytometry in the absence of dox. Each clone was tested for ICE by dox treatment to induce cre, followed by transfection of an exchange plasmid carrying DsRed2 and selection in G418. Single-copy integrants show dox-inducible green fluorescence converting to dox-inducible red fluorescence (Figure 1B), whereas multi-copy integrants acquire inducible red fluorescence but do not lose green fluorescence (not shown). We selected one clone, referred to as iC2C12, and confirmed that it has a single ICE site by Southern blot analysis. Using a GFP-specific probe, we detected a single band in iC2C12 cells but not in iC2C12-DUX4 where cre–ires–GFP was substituted with DUX4 (Figure 1C). To evaluate dox sensitivity and gene expression kinetics, we targeted iC2C12 with luciferase. Luciferase expression could be titrated across a three-log range by varying the dose of dox, and abundant gene expression was observed within 2 h of dox addition (Figure 1D). These kinetics and dynamic range are sufficient in this context for both a comprehensive comparative analysis of FSHD candidate genes at different levels of expression, and for the molecular evaluation of cell physiological responses to the expression of particular genes of interest. Head-to-head comparison of FSHD candidate genes We targeted iC2C12 cells with each FSHD candidate gene: FRG1, FRG2, TUBB4q, ANT1, DUX4, and DUX4c. The derivative cell lines were then exposed to various doses of dox and effects on morphology and viability were evaluated. After 24 h of expression, viability, as measured by ATP content, was reduced by over 80% at 100 ng/ml and over 90% at 500 ng/ml dox in the iC2C12-DUX4 cells, whereas no changes in viability were observed for any other candidate gene (Figure 2A). At this time point, iC2C12-DUX4 cells appeared dead and began lifting from the plate (Figure 2B), whereas cells expressing other candidate genes remained unchanged (not shown). As antibodies are not available to the majority of FSHD candidate genes, to show that these could also be regulated by dox, we generated FLAG tag fusions of several other candidates, and determined by western blotting that they were expressed strictly in response to dox (Figure 2C). As the amount of accumulated protein depends on factors in addition to transcription (RNA and protein stability), absolute protein levels are likely to vary between cell lines; however, at the maximum level possible for each gene, overt toxicity was seen only with DUX4. Figure 2.Identification of DUX4-specific cell pathological phenotypes. (A) Cell viability (ATP content), after 24 h in 100 or 500 ng/ml doxycycline-treated cells. DUX4 has a unique dose-dependent effect on viability. (B) Morphology of iC2C12 myoblasts 24 h after DUX4 induction with 500 ng/ml dox. (C) Western blotting analysis of FLAG tag fusion protein expression in FLAG–DUX4, FLAG–ANT1, FLAG–TUBB4q and FRG1-3xFLAG cell lines. Cells were induced with 125 and 500 ng/ml doxycycline for 14 h. (D) Immunofluorescence showing DUX4 (red) accumulation in the nucleus and cell morphological changes (lamin, green) in iC2C12-DUX4 cells over a 16 h time course (500 ng/ml). (E) Dose–response at 20 h (upper panels) and time course (in 500 ng/ml doxycycline, lower panels) of DUX4 expression in iC2C12-DUX4 cells. (F) Western blot analyses of DUX4 expression in differentiated and proliferating cells. iC2C12-DUX4 cells were differentiated into myotubes for 4 days and DUX4 was induced for 14 h for comparison to proliferating cells. (G) Western blots for p21 and cyclin E during a time course of DUX4 induction at 500 ng/ml dox. (H) FACS analyses of DUX4-induced apoptosis and cell death using annexin V (x axis) and 7AAD (y axis) staining. Early apoptotic cells are annexin V+, dead cells are annexin V+/7AAD+. (I) Western blots for activated caspases 6, 8 and 9 in DUX4-expressing myoblasts over a time course. (J) Morphology of cells expressing DUX4 at low levels while proliferating, and viability (K) of these cells by ATP assay. (L) Morphology of DUX4-expressing myotubes and their viability (M). Download figure Download PowerPoint DUX4 toxicity The inducible system provides rapid and synchronous gene expression (Figure 2E), which enabled us to visualize the temporal phenotypic progression from normal to non-viable. After varying periods of induction, cells were stained with a monoclonal antibody to DUX4 and counterstained with laminin to visualize morphology. Intranuclear localization of DUX4 protein was apparent in virtually all cells within 2 h after high-level (500 ng/ml) induction (Figure 2D). By 4 h (500 ng/ml), morphological changes were detectable, as cells began to stretch and nuclei acquired an ovoid shape, a change that became more extreme with time. A dose-dependent decrease of Ki67 was also observed (Supplementary Figure S1A). Expression of p21 was markedly elevated as soon as 2 h after induction, and cyclin E levels were reduced from 6 h (Figure 2G). Markers of apoptosis, including activated caspases 6, 8, and 9 were apparent by 6 h, and cleaved caspase 3 and annexin V staining cells by 12 h, and increased thereafter (Figure 2H and I; Supplementary Figure S1C). At lower levels of expression, DUX4 reduced proliferation and induced morphological changes, but did cause obvious cell death (Figure 2J and K). At 30 ng/ml dox, the elongated shape and ovoid nuclei were apparent, but cells were viable for as long as 8 days (not shown). A 1-h pulse of DUX4 expression at 500 ng/ml followed by a 19 h chase resulted in a morphology similar to that of cells induced with a low dose of dox for 24 h (Supplementary Figure S2A). To analyse the effect of DUX4 on myotubes, we differentiated myoblasts into myotubes for 4–6 days using conventional differentiation medium (switch to 2% horse serum). When induced at high levels for 24 h in terminally differentiated myotubes, loss of myotubes was evident; however, large numbers of differentiated cells were still viable (Figure 2L), and by ATP assay we did not detect severe cell death even at relatively high levels of expression (Figure 2M). We confirmed that DUX4 protein is expressed at relatively similar levels in differentiated myotubes and proliferating myoblasts by western blotting (Figure 2F). To determine whether DUX4 is specifically toxic for myoblasts, we generated DUX4-inducible fibroblasts i3T3-DUX4 from ICE-modified 3T3 cells (manuscript in preparation) and DUX4-inducible embryonic stem cells iES-DUX4 (manuscript in preparation). In 3T3 fibroblasts, low-dose induction of DUX4 resulted in similar cell morphological changes as seen in C2C12 myoblasts, and at high levels DUX4 induced rapid cell death (Supplementary Figure S3A). DUX4 impaired cell viability in a dose-dependent manner, as confirmed by the ATP assay (Supplementary Figure S3B and C). Similar toxic effects were observed when DUX4 was induced in mouse ES cells (Supplementary Figure S4B); however, cell death was not as rapid as observed in C2C12 and 3T3 cells. Interestingly, ES cells progressively lost their characteristic morphology and colony structure and acquired a fibroblastic cell shape on induction with DUX4 (Supplementary Figure S4B). To determine whether DUX4 is toxic for all cell types, we induced it in embryoid bodies (EBs, which contain cell types representing all three germ layers) derived from these ES cells. The toxic effect of DUX4 during EB differentiation was distinguished by a decrease in EB size, increased cell death and apoptosis seen by annexin V staining, and decreased outgrowth of the cells when plated in monolayer (Supplementary Figure S4D–F). However, all cell types were not equally sensitive to the DUX4 expression, as some cells remained viable even after 5 days of induction (500 ng/ml) (Supplementary Figure S4D–F). In support of the in vitro assay, we measured teratoma formation in immunodeficient mice and found that mice injected with iES-DUX4 cells and treated with dox from the first day of transplantation did not develop tumors; however, when dox was initiated at day 14 post-injection, tumors regressed but did not disappear (Supplementary Figure S4G). We conclude that different cell types have different sensitivities to DUX4. Gene expression changes provoked by DUX4 Because DUX4 is a homeodomain protein and rapidly transits to the nucleus after expression, we reasoned that its toxic effects were mediated through changes in gene expression. We therefore performed microarray transcriptional profiling experiments comparing uninduced cells to those expressing DUX4 for 4 or 12 h. We identified 156 genes differentially expressed at 4 h (107 upregulated and 49 downregulated) and 1011 genes at 12 h (576 upregulated and 435 downregulated). The majority of the genes (more than two-third) that were altered at 4 h were also changed in the 12 h sample. Representatives of a variety of functional gene ontology classes were identified (Figure 3A). Moreover, these alterations were consistent and reproducible across replicates, as demonstrated by hierarchical clustering (Figure 3B). For both time points, approximately one-third of the altered genes were uncharacterized cDNAs or genes with unknown functions (Supplementary Tables S1 and S2). Of the genes with known or suspected functions, the greatest numbers of genes at both 4 and 12 h were involved in cell cycle control or regulation of growth/development. Signal transduction and stress response were the next largest functional categories. Genes identified as differentially expressed at 12 h exhibited a similar ontological profile, with the addition of DNA packaging/processing, intracellular protein transport, and energy production categories represented. The gene lists in their entirety are shown in Supplementary Tables S1 and S2. Figure 3.DUX4 target genes. (A) DUX4 target genes (left chart, 4 h post-induction, right chart 12 h) represented by gene ontology. (B) Hierarchical clustering. Signal values for genes differentially expressed at 4 or 12 h were normalized by Z score and clustered using Spotfire DecisionSite software. Bright green represents low signal values, bright red indicates very high signal values, and black represents median signal values. Experiments are clustered by columns, and individual genes by rows. C1 through C3 represent 0 h controls, and 4 and 12 h represent samples treated with doxycycline for the indicated times. As shown, all controls clustered together as did the 4 and 12 h replicates. The majority of changes in gene expression increased over time (i.e. became brighter red by 12 h, the latest time point test. (C–F) Real-time PCR confirmation of expression changes for key FSHD-related genes: (C) glutathione redox pathway components, (D) heat-shock proteins, (E) Lamin A and p21, and (F) MyoD, which made the cutoff only in one replicate due to sensitivity of the microarray. Download figure Download PowerPoint Previous microarray studies on FSHD patient biopsies have identified two classes of genes uniquely altered in FSHD when compared with other muscular dystrophies: MyoD target genes and oxidative stress response genes (Winokur et al, 2003b; Celegato et al, 2006). A proteomics study on patient biopsies has confirmed that oxidative stress response proteins, several proteins regulated by MyoD, and MyoD itself, are present at much reduced levels in FSHD biopsies when compared with unaffected controls (Celegato et al, 2006). We therefore evaluated the expression of these genes in our data and observed numerous examples of oxidative stress response gene downregulation. These changes were significant only in the 12-h sample, suggesting that they may be secondary targets of DUX4. The genes we identified, similar to the studies on FSHD patient biopsies, are primarily enzymes involved in glutathione redox metabolism. We confirmed these results independently by real-time PCR (Figure 3C). In addition to oxidative stress/glutathione redox genes, a number of heat-shock genes were downregulated, including Hspb1, Hsp12a, Serpinin, confirmed by real-time PCR (Figure 3D). We also noted repression of LaminA, the protein product of which is altered in several other muscular dystrophies (Figure 3E). Many upregulated genes were also observed, for example p21 (Figure 3E). Although MyoD was downregulated in one microarray, it was excluded from the final data set as its signal was undetectable from the other two replicates. Transcription factors are often expressed at mRNA levels that are not detectable with microarrays (Canales et al, 2006), so we performed real-time PCR to evaluate MyoD expression. This clearly demonstrated that MyoD was rapidly downregulated by DUX4 (Figure 3F). The fact that the two unique classes of genes altered in FSHD are downstream targets of DUX4 suggests a potential role for this candidate gene in FSHD pathology. DUX4 and oxidative stress As genes involved in the glutathione redox cycle were repressed, we assumed that DUX4-expressing cells would have lower capacity to buffer oxidative stress. Additionally, a previous study has found that myoblasts from FSHD patients are more sensitive to oxidative stress than are control myoblasts from unaffected individuals (Winokur et al, 2003a). We therefore tested the sensitivity of DUX4-expressing cells to a variety of stress-inducing reagents at different concentrations. We found in all cases that DUX4 expression, even at levels only weakly detectable by western blot (10 ng/ml dox), enhanced the sensitivity of stress-inducing compounds (Figure 4A). In the case of tBHP and Paraquat, compounds that specifically induce oxidative stress, this enhancement was highly synergistic. Concentrations of these compounds, which have no effect or a weak effect on viability in the absence of DUX4, have a significant effect on viability when DUX4 is expressed (tBHP at 1 μM or Paraquat at 1 mM, for example). Concentrations that reduce but do not eliminate viability, combined with DUX4 expression, completely abolish viability with low levels of DUX4 expression. On the other hand, the dose–response curves for staurosporine and tunicamycin, non-oxidative stressors, although shifted, do not show threshold changes when DUX4 is expressed. Figure 4.Effect of antioxidants on DUX4-expressing myoblasts. (A) ATP assay for cell survival of DUX4-expressing myoblasts exposed to different stress-inducing agents. Oxidative stressors (tBHP and Paraquat) show a synergistic interaction with DUX4 at low levels. (B) Cell rescue with various concentrations of antioxidants demonstrated by ATP assay after 24 h of DUX4 induction (500 ng/ml dox). (C) Cell morphology of iC1C12-DUX4 myoblasts induced with 500 ng/ml dox for 24 h and treated with various antioxidants. (D) Western blot demonstrating that DUX4 was still expressed in the rescued cells (24 h post-induction). (E) qRT–PCR analyses of MyoD and Myf5 in antioxidant-rescued, DUX4-expressing cells. Data represent the fold difference compared with the level of GAPDH, error bars are STDEV (n=3). Antioxidants have no effect on the expression of these downstream genes.
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