Double Homeobox Research Articles

PB1661 CHARACTERIZATION OF A DUX4‐IGH INHIBITOR AS A POSSIBLE TREATMENT FOR ACUTE LYMPHOBLASTIC LEUKEMIA

Background:B cell acute lymphoblastic leukemia (B‐ALL) is the most common pediatric cancer and the major cause of cancer‐related death before the age of 20. In up to 7% of cases, the disease is caused by rearrangements of the Double Homeobox 4 (DUX4) transcription factor to the immunoglobulin heavy chain (IGH) locus, giving rise to the oncogenic fusion protein DUX4‐IGH. While ectopic expression of wild type DUX4 induces apoptosis, DUX4‐IGH has transforming ability in cellular and animal models. These opposite biological activities are mirrored by the ability of DUX4 and DUX4‐IGH to drive the transcription of non‐overlapping sets of target genes, despite the two proteins share the same DNA binding domain (dbd).Aims:Through proteomics, we identified a specific DUX4‐IGH inhibitor. Preliminary data indicate that the inhibitor directly binds to DUX4‐IGH dbd blocking the activation of target genes. Based on this evidence, I aim to test the antileukemic activity of the inhibitor.Methods:Human cell lines and primary murine bone barrow progenitor cells will be lentivirally transduced to induce the expression of DUX4‐IGH alone or in combination with its inhibitor, and the effects on proliferation, transformation, clonogenic potential and self‐renewal ability in B‐cell differentiation conditions will be evaluated.To test the efficacy of DUX4‐IGH inhibition in leukemia development, I will employ murine bone marrow transplantation assays and patient derived xenografts of DUX4‐IGH B‐ALL and assess disease latency in the presence or absence of the DUX4‐IGH inhibitor.Results:Preliminary data indicate that the activity of DUX4‐IGH is restricted to B‐cells, supporting the need of a B‐cell specific co‐factor. Importantly, lentiviral expression of the DUX4‐IGH inhibitor in NALM6 cells that carry a DUX4‐IGH translocation reduce the endogenous levels of DUX4‐IGH targets.I expect to see a significant inhibition of DUX4‐IGH driven transformation in the presence of its inhibitor, associated with reduced proliferation, clonogenic and self‐renewal potential in cell culture systems. I predict that the inhibitor will rescue differentiation potential and block or significantly delay leukemia development in mice injected with DUX4‐IGH expressing pro‐B cells.Summary/Conclusion:Pre‐clinical validation of the DUX4‐IGH inhibitor will help defining effective therapeutic strategies for DUX4‐IGH B‐ALL patients, improving clinical outcome and lowering treatment toxicity.

DUX4 Pathological Expression: Causes and Consequences in Cancer

DUX4, a double homeobox transcription factor, has been mostly studied in facioscapulohumeral dystrophy (FSHD), a pathology linked to a deletion of subtelomeric repeats on chromosome 4q. More recently, however, the gene has been associated with various sarcomas and haematological malignancies. Drugs developed for FSHD could be tested on cancer cells to develop efficient treatment strategies for both pathologies.

Knockdown of pseudogene DUXAP8 expression in glioma suppresses tumor cell proliferation.

A large number of pseudogenes as well as long non-coding RNAs (lncRNAs) have been identified as important regulators in human tumors. However, the clinical role and potential functional effects of the double homeobox A pseudogene 8 (DUXAP8) in glioma remains unknown. In the present study, it was revealed that pseudogene DUXAP8 is significantly upregulated in glioma tissues, compared with adjacent normal tissues. Patients with increased DUXAP8 expression were associated with higher Karnofsky Performance Status, advanced World Health Organization grade, poor disease-free survival and overall survival rates of patients with glioma. Furthermore, in vitro assays, Cell-Counting Kit-8 cell viability and cell colony forming assays demonstrated that reduced DUXAP8 expression significantly suppressed proliferation capacity. Therefore, the results of the present study indicate that pseudogene DUXAP8 is an oncogenic lncRNA and may serve as a potentially prognostic biomarker and novel target of glioma treatment.

Crystal Structure of the Double Homeodomain of DUX4 in Complex with DNA

Double homeobox (DUX) transcription factors are unique to eutherian mammals. DUX4 regulates expression of repetitive elements during early embryogenesis, but misexpression of DUX4 causes facioscapulohumeral muscular dystrophy (FSHD) and translocations overexpressing the DUX4 double homeodomain cause B cell leukemia. Here, we report the crystal structure of the tandem homeodomains of DUX4 bound to DNA. The homeodomains bind DNA in a head-to-head fashion, with the linker making anchoring DNA minor-groove interactions and unique protein contacts. Remarkably, despite being tandem duplicates, the DUX4 homeodomains recognize different core sequences. This results from an arginine-to-glutamate mutation, unique to primates, causing alternative positioning of a key arginine side chain in the recognition helix. Mutational studies demonstrate that this primate-specificchange is responsible for the divergence in sequence recognition that likely drove coevolution of embryonically regulated repeats in primates. Our work provides a framework for understanding the endogenous function of DUX4 and its role in FSHD and cancer.

Transgenic zebrafish model of DUX4 misexpression reveals a developmental role in FSHD pathogenesis

Facioscapulohumeral dystrophy type 1 (FSHD-1) is the most common autosomal dominant form of muscular dystrophy with a prevalence of ∼1 in 8000 individuals. It is considered a late-onset form of muscular dystrophy and leads to asymmetric muscle weakness in the facial, scapular, trunk and lower extremities. The prevalent hypothesis on disease pathogenesis is explained by misexpression of a germ line, primate-specific transcription factor DUX4-fl (double homeobox 4, full-length isoform) linked to the chromosome 4q35. In vitro and in vivo studies have demonstrated that very low levels of DUX4-fl expression are sufficient to induce an apoptotic and/or lethal phenotype, and therefore modeling of the disease has proved challenging. In this study, we expand upon our previously established injection model of DUX4 misexpression in zebrafish and describe a DUX4-inducible transgenic zebrafish model that better recapitulates the expression pattern and late onset phenotype characteristic of FSHD patients. We show that an induced burst of DUX4 expression during early development results in the onset of FSHD-like phenotypes in adulthood, even when DUX4 is no longer detectable. We also utilize our injection model to study long-term consequences of DUX4 expression in those that fail to show a developmental phenotype. Herein, we introduce a hypothesis that DUX4 expression during developmental stages is sufficient to induce FSHD-like phenotypes in later adulthood. Our findings point to a developmental role of DUX4 misexpression in the pathogenesis of FSHD and should be factored into the design of future therapies.

A patient-derived iPSC model revealed oxidative stress increases facioscapulohumeral muscular dystrophy-causative DUX4.

Double homeobox 4 (DUX4), the causative gene of facioscapulohumeral muscular dystrophy (FSHD), is ectopically expressed in the skeletal muscle cells of FSHD patients because of chromatin relaxation at 4q35. The diminished heterochromatic state at 4q35 is caused by either large genome contractions [FSHD type 1 (FSHD1)] or mutations in genes encoding chromatin regulators, such as SMCHD1 [FSHD type 2 (FSHD2)]. However, the mechanism by which DUX4 expression is regulated remains largely unknown. Here, using a myocyte model developed from patient-derived induced pluripotent stem cells, we determined that DUX4 expression was increased by oxidative stress (OS), a common environmental stress in skeletal muscle, in both FSHD1 and FSHD2 myocytes. We generated FSHD2-derived isogenic control clones with SMCHD1 mutation corrected by clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) and homologous recombination and found in the myocytes obtained from these clones that DUX4 basal expression and the OS-induced upregulation were markedly suppressed due to an increase in the heterochromatic state at 4q35. We further found that DNA damage response (DDR) was involved in OS-induced DUX4 increase and identified ataxia-telangiectasia mutated, a DDR regulator, as a mediator of this effect. Our results suggest that the relaxed chromatin state in FSHD muscle cells permits aberrant access of OS-induced DDR signaling, thus increasing DUX4 expression. These results suggest OS could represent an environmental risk factor that promotes FSHD progression.

Protein kinase A activation inhibits DUX4 gene expression in myotubes from patients with facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent of the adult-onset muscular dystrophies. FSHD causes a loss of muscle mass and function, resulting in severe debilitation and reduction in quality of life. Currently, only the symptoms of FSHD can be treated, and such treatments have minimal benefit. The available options are not curative, and none of the treatments address the underlying cause of FSHD. The genetic, epigenetic, and molecular mechanisms triggering FSHD are now quite well-understood, and it has been shown that expression of the transcriptional regulator double homeobox 4 (DUX4) is necessary for disease onset and is largely thought to be the causative factor in FSHD. Therefore, we sought to identify compounds suppressing DUX4 expression in a phenotypic screen using FSHD patient-derived muscle cells, a zinc finger and SCAN domain-containing 4 (ZSCAN4)-based reporter gene assay for measuring DUX4 activity, and ∼3,000 small molecules. This effort identified molecules that reduce DUX4 gene expression and hence DUX4 activity. Among those, β2-adrenergic receptor agonists and phosphodiesterase inhibitors, both leading to increased cellular cAMP, effectively decreased DUX4 expression by >75% in cells from individuals with FSHD. Of note, we found that cAMP production reduces DUX4 expression through a protein kinase A-dependent mode of action in FSHD patient myotubes. These findings increase our understanding of how DUX4 expression is regulated in FSHD and point to potential areas of therapeutic intervention.

Facioscapulohumeral dystrophy: activating an early embryonic transcriptional program in human skeletal muscle.

Facioscapulohumeral dystrophy (FSHD) is the third most prevalent muscular dystrophy. A progressive disease, it presents clinically as weakness and wasting of the face, shoulder and upper arm muscles, with later involvement of the trunk and lower extremities. FSHD develops through complex genetic and epigenetic events that converge on a common mechanism of toxicity with mis-expression of the transcription factor double homeobox 4 (DUX4). There is currently no treatment available for FSHD. However, the consensus that ectopic DUX4 expression in skeletal muscle is the root cause of FSHD pathophysiology has allowed research efforts to turn toward cultivating a deeper understanding of DUX4 biology and the pathways that underlie FSHD muscle pathology, and to translational studies aimed at developing targeted therapeutics using ever more sophisticated cell and animal-based models of FSHD. This review summarizes recent advances in our understanding of FSHD, including the regulation and activity of DUX4 in its normal developmental roles as well as its pathological contexts. We highlight how these advances raise new questions and challenges for the field as it moves into the next decade of FSHD research.

Structural basis of DUX4/IGH-driven transactivation

Oncogenic fusions are major drivers in leukemogenesis and may serve as potent targets for treatment. DUX4/IGHs have been shown to trigger the abnormal expression of ERGalt through binding to DUX4-Responsive-Element (DRE), which leads to B-cell differentiation arrest and a full-fledged B-ALL. Here, we determined the crystal structures of Apo- and DNADRE-bound DUX4HD2 and revealed a clamp-like transactivation mechanism via the double homeobox domain. Biophysical characterization showed that mutations in the interacting interfaces significantly impaired the DNA binding affinity of DUX4 homeobox. These mutations, when introduced into DUX4/IGH, abrogated its transactivation activity in Reh cells. More importantly, the structure-based mutants significantly impaired the inhibitory effects of DUX4/IGH upon B-cell differentiation in mouse progenitor cells. All these results help to define a key DUX4/IGH-DRE recognition/step in B-ALL.

A cre-inducible DUX4 transgenic mouse model for investigating facioscapulohumeral muscular dystrophy.

The Double homeobox 4 (DUX4) gene is an important regulator of early human development and its aberrant expression is causal for facioscapulohumeral muscular dystrophy (FSHD). The DUX4-full length (DUX4-fl) mRNA splice isoform encodes a transcriptional activator; however, DUX4 and its unique DNA binding preferences are specific to old-world primates. Regardless, the somatic cytotoxicity caused by DUX4 expression is conserved when expressed in cells and animals ranging from fly to mouse. Thus, viable animal models based on DUX4-fl expression have been difficult to generate due in large part to overt developmental toxicity of low DUX4-fl expression from leaky transgenes. We have overcome this obstacle and here we report the generation and initial characterization of a line of conditional floxed DUX4-fl transgenic mice, FLExDUX4, that is viable and fertile. In the absence of cre, these mice express a very low level of DUX4-fl mRNA from the transgene, resulting in mild phenotypes. However, when crossed with appropriate cre-driver lines of mice, the double transgenic offspring readily express DUX4-fl mRNA, protein, and target genes with the spatiotemporal pattern of nuclear cre expression dictated by the chosen system. When cre is expressed from the ACTA1 skeletal muscle-specific promoter, the double transgenic animals exhibit a developmental myopathy. When crossed with tamoxifen-inducible cre lines, DUX4-mediated pathology can be induced in adult animals. Thus, the appearance and progression of pathology can be controlled to provide readily screenable phenotypes useful for assessing therapeutic approaches targeting DUX4-fl mRNA and protein. Overall, the FLExDUX4 line of mice is quite versatile and will allow new investigations into mechanisms of DUX4-mediated pathophysiology as well as much-needed pre-clinical testing of DUX4-targeted FSHD interventions in vivo.

Abstract B24: Molecular and functional dissection of the CIC-DUX4 fusion in undifferentiated round cell sarcoma

Abstract CIC-DUX4 soft-tissue tumors are an aggressive subset of undifferentiated round cell sarcomas that arise in children and young adults. The cytogenetic hallmark, (t(4;19) or t(10;19)), of this tumor is a chromosomal translocation that fuses the transcription factor, Capicua (CIC), with the double homeobox 4 protein, DUX4. Despite morphologic similarities to Ewing sarcoma, CIC-DUX4 sarcomas are clinically distinct with rapid development of lethal metastatic disease and chemoinsensitivity. The unique cytogenetic and clinical features that distinguish CIC-DUX4 sarcomas from other small round cell tumors provide a unique opportunity to identify and rationally target this population to improve clinical outcomes. To meet this need, we are taking a systematic functional approach to identify critical CIC-DUX4 targets that promote tumor progression and metastasis. Leveraging a comparative transcriptional analysis between cytogenetically distinct small round cell sarcomas, we reveal that the CIC-DUX4 fusion activates highly conserved molecular networks to control tumor growth and metastatic capacity. Using an orthotopic soft-tissue sarcoma model, we show that CIC-DUX4 induces ETV4 expression to drive invasion and metastasis. Genetic silencing of ETV4 in CIC-DUX4 expressing tumors limits tumor dissemination without a profound impact on tumor cell growth. These findings suggest that CIC-DUX4 enhances metastasis and tumor growth through distinct transcriptional repertoires. Consistent with this, we identify multiple components of the cell cycle machinery that are transcriptionally regulated by CIC-DUX4 expression. Specifically, the CIC-DUX4 fusion co-opts native CIC-binding specificity to transcriptionally activate these cell cycle target genes. Altogether, our molecular and functional dissection of the CIC-DUX4 fusion reveals highly conserved CIC-regulated transcriptional targets that promote tumor growth and metastasis. We aim to pharmacologically exploit these key CIC-DUX4-dependent transcriptional nodes to improve outcomes for this population with few therapeutic options. Citation Format: Ross A. Okimoto, Wei Wu, Trever G. Bivona. Molecular and functional dissection of the CIC-DUX4 fusion in undifferentiated round cell sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B24.

Antisense Oligonucleotide Targeting of 3'-UTR of mRNA for Expression Knockdown.

With the recent conditional approval of an antisense oligonucleotide (AON) that restores the reading frame of DMD transcript in a subset of Duchenne muscular dystrophy patients, it has been established that AONs sharing similar chemistry have clear clinical potential. Genetic diseases, such as facioscapulohumeral dystrophy (FSHD), can be the result of gain-of-function mutations. Since mRNA processing in terms of termination of transcription, its transport from the nucleus to the cytoplasm, its stability and translation efficiency are dependent on key 3'UTR elements, it follows that targeting theseelements with AONs have the potential to induce gene silencing. Aberrant expression of the Double homeobox 4 (DUX4) transcription factor and the downstream consequences of such expression is the hallmark of FSHD. Here we describe the bioinformatic strategies behind the design of AONs targeting polyadenylation signals and the methodologies relevant to their in vitro screening for efficacy and safety, including analysis of expression at the transcript and protein level of the specific target and downstream genes, and measurement of the effect on the fusion index of myotubes. The targeting of permissive DUX4 and MSTN are used as examples. MSTN encodes for myostatin, a negative regulator of myogenesis; the downregulation of MSTN expression has the potential to address the muscular atrophy associated with muscular dystrophies, sarcopenia, cancer and acquired immunodeficiency syndrome.

BET bromodomain inhibitors and agonists of the beta-2 adrenergic receptor identified in screens for compounds that inhibit DUX4 expression in FSHD muscle cells

BackgroundFacioscapulohumeral dystrophy (FSHD) is a progressive muscle disease caused by mutations that lead to epigenetic derepression and inappropriate transcription of the double homeobox 4 (DUX4) gene in skeletal muscle. Drugs that enhance the repression of DUX4 and prevent its expression in skeletal muscle cells therefore represent candidate therapies for FSHD.MethodsWe screened an aggregated chemical library enriched for compounds with epigenetic activities and the Pharmakon 1600 library composed of compounds that have reached clinical testing to identify molecules that decrease DUX4 expression as monitored by the levels of DUX4 target genes in FSHD patient-derived skeletal muscle cell cultures.ResultsOur screens identified several classes of molecules that include inhibitors of the bromodomain and extra-terminal (BET) family of proteins and agonists of the beta-2 adrenergic receptor. Further studies showed that compounds from these two classes suppress the expression of DUX4 messenger RNA (mRNA) by blocking the activity of bromodomain-containing protein 4 (BRD4) or by increasing cyclic adenosine monophosphate (cAMP) levels, respectively.ConclusionsThese data uncover pathways involved in the regulation of DUX4 expression in somatic cells, provide potential candidate classes of compounds for FSHD therapeutic development, and create an important opportunity for mechanistic studies that may uncover additional therapeutic targets.

A distal auxiliary element facilitates cleavage and polyadenylation of Dux4 mRNA in the pathogenic haplotype of FSHD.

The degenerative muscle disorder facioscapulohumeral dystrophy (FSHD) is thought to be caused by the inappropriate expression of the Double Homeobox 4 (Dux4) protein in muscle cells leading to apoptosis. Expression of Dux4 in the major form of FSHD is a function of two contributing molecular changes: contractions in the D4Z4 microsatellite repeat region where Dux4 is located and an SNP present within a region downstream of the D4Z4. This SNP provides a functional, yet non-consensus polyadenylation signal (PAS) is used for the Dux4 mRNA 3' end processing. Surprisingly, the sequences flanking the Dux4 PAS do not resemble a typical cleavage and polyadenylation landscape with no recognizable downstream sequence element and a suboptimal cleavage site. Here, we conducted a systematic analysis of the cis-acting elements that govern Dux4 cleavage and polyadenylation. Using a transcriptional read-through reporter, we determined that sequences downstream of the SNP located within the β-satellite region are critical for Dux4 cleavage and polyadenylation. We also demonstrate the feasibility of using antisense oligonucleotides to target these sequences as a means to reduce Dux4 expression. Our results underscore the complexity of the region immediately downstream of the D4Z4 and uncover a previously unknown function for the β-satellite region in Dux4 cleavage and polyadenylation.

Potential role of ZEB1 as a DNA repair regulator in colorectal cancer cells revealed by cancer-associated promoter profiling.

Besides being a key contributor to epithelial-to-mesenchymal transition (EMT) activation and stemness maintenance, zinc finger E-box binding homeobox 1 (ZEB1) is also a crucial inducer of chemoresistance and radioresistance. Unlike the clear mechanism that mediates its effect on EMT and dedifferentiation, the mechanism of how ZEB1 promotes chemo- and radio-resistance remains to be elucidated. It has been previously reported that ZEB1 promotes DNA double-strand break clearance by enhancing the deubiquitylating activity of ubiquitin-specific peptidase (USP)7 on checkpoint kinase 1, which is an important step during DNA repair. It was hypothesized that as a transcriptional suppressor, ZEB1 may be involved in an unbalanced DNA damage response (DDR) by affecting other key components. Therefore, in the present study, the target gene occupancy of ZEB1 was mapped in colorectal cancer cells using the ChIP-on-chip method, revealing positive intervals enriched along the three DDR-associated genes: USP17, chromodomain helicase DNA-binding protein 1-like and double homeobox 4. The E-boxes identified in the binding regions and the enhanced mRNA expression of the three genes following the knockdown of ZEB1 supported the identification of these three genes as downstream target genes of ZEB1. Furthermore, ZEB1 knockdown initiated a chemosensitization effect, induced G1/S arrest and increased apoptosis, which functionally validated the three ZEB1 downstream targets. In summary, the present study identified three DDR-associated genes as ZEB1 downstream targets, and demonstrated that their suppression by ZEB1 contributes to ZEB1-mediated chemoresistance.

Estrogens enhance myoblast differentiation in facioscapulohumeral muscular dystrophy by antagonizing DUX4 activity.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is characterized by extreme variability in symptoms, with females being less severely affected than males and presenting a higher proportion of asymptomatic carriers. The sex-related factors involved in the disease are not known. Here, we have utilized myoblasts isolated from FSHD patients (FSHD myoblasts) to investigate the effect of estrogens on muscle properties. Our results demonstrated that estrogens counteract the differentiation impairment of FSHD myoblasts without affecting cell proliferation or survival. Estrogen effects are mediated by estrogen receptor β (ERβ), which reduces chromatin occupancy and transcriptional activity of double homeobox 4 (DUX4), a protein whose aberrant expression has been implicated in FSHD pathogenesis. During myoblast differentiation, we observed that the levels and activity of DUX4 increased progressively and were associated with its enhanced recruitment in the nucleus. ERβ interfered with this recruitment by relocalizing DUX4 in the cytoplasm. This work identifies estrogens as a potential disease modifier that underlie sex-related differences in FSHD by protecting against myoblast differentiation impairments in this disease.

Are Antioxidants a Potential Therapy for FSHD? A Review of the Literature.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy affecting approximately 1 in 7500 individuals worldwide. It is a progressive disease characterised by skeletal muscle weakness and wasting. A genetic mutation on the 4q35 chromosome results in the expression of the double homeobox 4 gene (DUX4) which drives oxidative stress, inflammation, toxicity, and atrophy within the skeletal muscle. FSHD is characterised by oxidative stress, and there is currently no cure and a lack of therapies for the disease. Antioxidants have been researched for many years, with investigators aiming to use antioxidants therapeutically for oxidative stress-associated diseases. This has included both natural and synthetic antioxidants. The use of antioxidants in preclinical or clinical models has been largely successful with a plethora of research reporting positive results. However, when translated to clinical trials, the use of antioxidants as a therapeutic intervention for a variety of disease has been largely unsuccessful. Moreover, specifically focusing on FSHD, limited research has been conducted on the use of antioxidants as a therapy in either preclinical or clinical models. This review summarises the current state of antioxidant use in the treatment of FSHD and discusses their potential avenue for therapeutic use for FSHD patients.

Integrating clinical and genetic observations in facioscapulohumeral muscular dystrophy

This review gives an overview of the currently known key clinical and (epi)genetic aspects of facioscapulohumeral muscular dystrophy (FSHD) and provides perspectives to facilitate future research. Clinically, imaging studies have contributed to a detailed characterization of the FSHD phenotype, and a model is proposed with five stages of disease progression. A number of clinical trials have been conducted regarding exercise and diet aiming to reduce symptoms. Genetically, at least two different mechanisms (FSHD1 and FSHD2) lead to double homeobox 4 (DUX4) expression in skeletal myocytes, which is expected to be necessary for the disease. Disease severity is most likely determined by a combination of the D4Z4 repeat size and its epigenetic state. FSHD is one of the most common muscular dystrophies and is characterized by a typical distribution of muscle weakness. Progress has been made on clinical as well as on (epi)genetic aspects of the disease. Currently, there is no cure available for FSHD. For successful development of new treatments targeting the disease process, integration of clinical and pathogenetic knowledge is essential. A clinical trial toolbox that consists of patient registries, biomarkers and clinical outcome measures will be required to effectively conduct future clinical trials.

Dux4 controls migration of mesenchymal stem cells through the Cxcr4-Sdf1 axis.

We performed transcriptome profiling of human immortalized myoblasts (MB) transiently expressing double homeobox transcription factor 4 (DUX4) and double homeobox transcription factor 4 centromeric (DUX4c) and identified 114 and 70 genes differentially expressed in DUX4- and DUX4c-transfected myoblasts, respectively. A significant number of differentially expressed genes were involved in inflammation, cellular migration and chemotaxis suggesting a role for DUX4 and DUX4c in these processes. DUX4 but not DUX4c overexpression resulted in upregulation of the CXCR4 (C-X-C motif Receptor 4) and CXCL12 (C-X-C motif ligand 12 also known as SDF1) expression in human immortalized myoblasts. In a Transwell cell migration assay, human bone marrow-derived mesenchymal stem cells (BMSCs) were migrating more efficiently towards human immortalized myoblasts overexpressing DUX4 as compared to controls; the migration efficiency of DUX4-transfected BMSCs was also increased. DUX4c overexpression in myoblasts or in BMSCs had no impact on the rate of BMSC migration. Antibodies against SDF1 and CXCR4 blocked the positive effect of DUX4 overexpression on BMSC migration. We propose that DUX4 controls the cellular migration of mesenchymal stem cells through the CXCR4 receptor.

DUX4-induced constitutive DNA damage and oxidative stress contribute to aberrant differentiation of myoblasts from FSHD patients

Facioscapulohumeral dystrophy (FSHD) is one of the three most common muscular dystrophies in the Western world, however, its etiology remains only partially understood. Here, we provide evidence of constitutive DNA damage in in vitro cultured myoblasts isolated from FSHD patients and demonstrate oxidative DNA damage implication in the differentiation of these cells into phenotypically-aberrant myotubes. Double homeobox 4 (DUX4), the major actor in FSHD pathology induced DNA damage accumulation when overexpressed in normal human myoblasts, and RNAi-mediated DUX4 inhibition reduced the level of DNA damage in FSHD myoblasts. Addition of tempol, a powerful antioxidant, to the culture medium of proliferating DUX4-transfected myoblasts and FSHD myoblasts reduced the level of DNA damage, suggesting that DNA alterations are mainly due to oxidative stress. Antioxidant treatment during the myogenic differentiation of FSHD myoblasts significantly reduced morphological defects in myotube formation. We propose that the induction of DNA damage is a novel function of the DUX4 protein affecting myogenic differentiation of FSHD myoblasts.