Loss of epigenetic silencing of the DUX4 transcription factor gene in facioscapulohumeral muscular dystrophy.

Current genetic and molecular evidence best supports an epigenetic mechanism for facioscapulohumeral muscular dystrophy (FSHD), whereby de-repression of the D4Z4 macrosatellite array leads to aberrant expression of the DUX4 transcription factor in skeletal muscle. This de-repression is triggered by either array contraction or (more rarely) by mutation of the SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) gene. Activation of DUX4 targets, including germline genes and several mammalian retrotransposons, then drives pathogenesis. A direct role for DUX4 mRNA in suppression of nonsense-mediated decay pathways has recently been demonstrated and may also contribute to muscle pathology. Loss of D4Z4 repression in FSHD is observed as hypomethylation of the array accompanied by loss of repressive chromatin marks. The molecular mechanisms of D4Z4 repression are poorly understood, but recent data have identified an Argonaute (AGO)-dependent siRNA pathway. Targeting this pathway by exogenous siRNAs could be a therapeutic strategy for FSHD.

A new monoclonal antibody DAG-6F4 against human alpha-dystroglycan reveals reduced core protein in some, but not all, dystroglycanopathy patients.

We generated a novel monoclonal antibody, DAG-6F4, against alpha-dystroglycan which immunolabels the sarcolemma in human muscle biopsies. Its seven amino-acid epitope, PNQRPEL, was identified using phage-displayed peptides and is located immediately after the highly-glycosylated mucin domain of alpha-dystroglycan. On Western blots of recombinant alpha-dystroglycan, epitope accessibility was reduced, but not entirely prevented, by glycosylation. DAG-6F4 immunolabelling was markedly reduced in muscle biopsies from Duchenne muscular dystrophy patients consistent with disruption of the dystroglycan complex. In a range of dystroglycanopathy patients with reduced/altered glycosylation, staining by DAG-6F4 was often less reduced than staining by IIH6 (antibody against the glycan epitope added by LARGE and commonly used to identify glycosylated alpha-dystroglycan). Whereas IIH6 was reduced in all patients, DAG-6F4 was hardly changed in a LARGE patient, less reduced than IIH6 in limb-girdle muscular dystrophy type 2I, but as reduced as IIH6 in some congenital muscular dystrophy patients. Although absence of the LARGE-dependent laminin-binding site appears not to affect alpha-dystroglycan stability at the sarcolemma, the results suggest that further reduction in aDG glycosylation may reduce its stability. These studies suggest that DAG-6F4 may be a useful addition to the antibody repertoire for evaluating the dystroglycan complex in neuromuscular disorders.

Formation of the embryonic organizer is restricted by the competitive influences of Fgf signaling and the SoxB1 transcription factors.

The organizer is one of the earliest structures to be established during vertebrate development and is crucial to subsequent patterning of the embryo. We have previously shown that the SoxB1 transcription factor, Sox3, plays a central role as a transcriptional repressor of zebrafish organizer gene expression. Recent data suggest that Fgf signaling has a positive influence on organizer formation, but its role remains to be fully elucidated. In order to better understand how Fgf signaling fits into the complex regulatory network that determines when and where the organizer forms, the relationship between the positive effects of Fgf signaling and the repressive effects of the SoxB1 factors must be resolved. This study demonstrates that both fgf3 and fgf8 are required for expression of the organizer genes, gsc and chd, and that SoxB1 factors (Sox3, and the zebrafish specific factors, Sox19a and Sox19b) can repress the expression of both fgf3 and fgf8. However, we also find that these SoxB1 factors inhibit the expression of gsc and chd independently of their repression of fgf expression. We show that ectopic expression of organizer genes induced solely by the inhibition of SoxB1 function is dependent upon the activation of fgf expression. These data allow us to describe a comprehensive signaling network in which the SoxB1 factors restrict organizer formation by inhibiting Fgf, Nodal and Wnt signaling, as well as independently repressing the targets of that signaling. The organizer therefore forms only where Nodal-induced Fgf signaling overlaps with Wnt signaling and the SoxB1 proteins are absent.

Re-evaluation of the role of calcium homeostasis endoplasmic reticulum protein (CHERP) in cellular calcium signaling.

Changes in cytoplasmic Ca(2+) concentration, resulting from activation of intracellular Ca(2+) channels within the endoplasmic reticulum, regulate several aspects of cellular growth and differentiation. Ca(2+) homeostasis endoplasmic reticulum protein (CHERP) is a ubiquitously expressed protein that has been proposed as a regulator of both major families of endoplasmic reticulum Ca(2+) channels, inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs), with resulting effects on mitotic cycling. However, the manner by which CHERP regulates intracellular Ca(2+) channels to impact cellular growth is unknown. Here, we challenge previous findings that CHERP acts as a direct cytoplasmic regulator of IP(3)Rs and RyRs and propose that CHERP acts in the nucleus to impact cellular proliferation by regulating the function of the U2 snRNA spliceosomal complex. The previously reported effects of CHERP on cellular growth therefore are likely indirect effects of altered spliceosomal function, consistent with prior data showing that loss of function of U2 snRNP components can interfere with cell growth and induce cell cycle arrest.

Defects in glycosylation impair satellite stem cell function and niche composition in the muscles of the dystrophic Large(myd) mouse.

The dystrophin-associated glycoprotein complex (DGC) is found at the muscle fiber sarcolemma and forms an essential structural link between the basal lamina and internal cytoskeleton. In a set of muscular dystrophies known as the dystroglycanopathies, hypoglycosylation of the DGC component α-dystroglycan results in reduced binding to basal lamina components, a loss in structural stability, and repeated cycles of muscle fiber degeneration and regeneration. The satellite cells are the key stem cells responsible for muscle repair and reside between the basal lamina and sarcolemma. In this study, we aimed to determine whether pathological changes associated with the dystroglycanopathies affect satellite cell function. In the Large(myd) mouse dystroglycanopathy model, satellite cells are present in significantly greater numbers but display reduced proliferation on their native muscle fibers in vitro, compared with wild type. However, when removed from their fiber, proliferation in culture is restored to that of wild type. Immunohistochemical analysis of Large(myd) muscle reveals alterations to the basal lamina and interstitium, including marked disorganization of laminin, upregulation of fibronectin and collagens. Proliferation and differentiation of wild-type satellite cells is impaired when cultured on substrates such as collagen and fibronectin, compared with laminins. When engrafted into irradiated tibialis anterior muscles of mdx-nude mice, wild-type satellite cells expanded on laminin contribute significantly more to muscle regeneration than those expanded on fibronectin. These results suggest that defects in α-dystroglycan glycosylation are associated with an alteration in the satellite cell niche, and that regenerative potential in the dystroglycanopathies may be perturbed.

Evolution of DUX gene macrosatellites in placental mammals.

Macrosatellites are large polymorphic tandem arrays. The human subtelomeric macrosatellite D4Z4 has 11-150 repeats, each containing a copy of the intronless DUX4 gene. DUX4 is linked to facioscapulohumeral muscular dystrophy, but its normal function is unknown. The DUX gene family includes DUX4, the intronless Dux macrosatellites in rat and mouse, as well as several intron-containing members (DUXA, DUXB, Duxbl, and DUXC). Here, we report that the genomic organization (though not the syntenic location) of primate DUX4 is conserved in the Afrotheria. In primates and Afrotheria, DUX4 arose by retrotransposition of an ancestral intron-containing DUXC, which is itself not found in these species. Surprisingly, we discovered a similar macrosatellite organization for DUXC in cow and other Laurasiatheria (dog, alpaca, dolphin, pig, and horse), and in Xenarthra (sloth). Therefore, DUX4 and Dux are not the only DUX gene macrosatellites. Our data suggest a new retrotransposition-displacement model for the evolution of intronless DUX macrosatellites.

Diagnosis by sequencing: correction of misdiagnosis from FSHD2 to LGMD2A by whole-exome analysis.

We studied and validated facioscapulohumeral muscular dystrophy (FSHD) samples from patients without a D4Z4 contraction (FSHD2 or 'phenotypic FSHD'). For this, we developed non-radioactive protocols to test D4Z4 allele constitution and DNA methylation, and applied these to samples from the Coriell Institute Cell Repository. The D4Z4 sizing showed two related subjects to have classic chromosome 4 contraction-dependent FSHD1. A third sample (GM17726) did not have a short chromosome 4 fragment, and had been assigned as non-4q FSHD (FSHD2). We tested D4Z4 haplotype and methylation for this individual but found both to be inconsistent with this diagnosis. Using exome sequencing, we identified two known pathogenic mutations in CAPN3 (Arg490Gln and Thr184Argfs(*)36), indicating a case of LGMD2A rather than FSHD. Our study shows how a wrong diagnosis can easily be corrected by whole-exome sequencing by constraining the variant analysis to candidate genes after the data have been generated. This new way of 'diagnosis by sequencing' is likely to become common place in genetic diagnostic laboratories. We also publish a digoxigenin-labeled Southern protocol to test D4Z4 methylation. Our data supports hypomethylation as a good epigenetic predictor for FSHD2. The non-radioactive protocol will help to make this assay more accessible to clinical diagnostic laboratories and the wider FSHD research community.

LARGE enzyme activity deciphered: a new therapeutic target for muscular dystrophies.

A significant proportion of severe, inherited congenital muscular dystrophies are due to aberrant glycosylation of the extracellular matrix receptor α-dystroglycan and a consequent lack of ligand-binding activity. A key member of this glycosylation pathway is the LARGE protein, which was originally identified through genome sequencing and genetic studies. Until recently, the biochemical activity of this enzyme proved frustratingly elusive, but a recent study shows that LARGE encodes a bifunctional glycosyltransferase that synthesizes a novel polysaccharide structure, which is required for functional dystroglycan. Identification of this structure should lead to development of new diagnostic tools and therapeutic strategies for these dystrophies.

Glycoproteomic characterization of recombinant mouse α-dystroglycan.

α-Dystroglycan (DG) is a key component of the dystrophin-glycoprotein complex. Aberrant glycosylation of the protein has been linked to various forms of congenital muscular dystrophy. Unusually α-DG has previously been demonstrated to be modified with both O-N-acetylgalactosamine and O-mannose initiated glycans. In the present study, Fc-tagged recombinant mouse α-DG was expressed and purified from human embryonic kidney 293T cells. α-DG glycopeptides were characterized by glycoproteomic strategies using both nano-liquid chromatography matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry. A total of 14 different peptide sequences and 38 glycopeptides were identified which displayed heterogeneous O-glycosylation. These data provide new insights into the complex domain-specific O-glycosylation of α-DG.

Glycomarkers for muscular dystrophy.

During the last 10 years it has become apparent that a significant subset of inherited muscular dystrophy is caused by errors in the glycosylation of α-dystroglycan. Many of these dystrophies are also associated with abnormalities of the central nervous system. Dystroglycan has to be fully glycosylated in order bind to its ligands. To date, six genes have been shown to be essential for functional dystroglycan glycosylation and most, if not all, of these genes act in the formation of O-mannosyl glycans. Genetic heterogeneity indicates that other genes are involved in this pathway. Identification of these additional genes would increase our understanding of this specific and essential glycosylation pathway.

A family history of DUX4: phylogenetic analysis of DUXA, B, C and Duxbl reveals the ancestral DUX gene.

BACKGROUND: DUX4 is causally involved in the molecular pathogenesis of the neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD). It has previously been proposed to have arisen by retrotransposition of DUXC, one of four known intron-containing DUX genes. Here, we investigate the evolutionary history of this multi-member double-homeobox gene family in eutherian mammals. RESULTS: Our analysis of the DUX family shows the distribution of different homologues across the mammalian class, including events of secondary loss. Phylogenetic comparison, analysis of gene structures and information from syntenic regions confirm the paralogous relationship of Duxbl and DUXB and characterize their relationship with DUXA and DUXC. We further identify Duxbl pseudogene orthologues in primates. A survey of non-mammalian genomes identified a single-homeobox gene (sDUX) as a likely representative homologue of the mammalian DUX ancestor before the homeobox duplication. Based on the gene structure maps, we suggest a possible mechanism for the generation of the DUX gene structure. CONCLUSIONS: Our study underlines how secondary loss of orthologues can obscure the true ancestry of individual gene family members. Their relationships should be considered when interpreting the relevance of functional data from DUX4 homologues such as Dux and Duxbl to FSHD.

Investigating the functions of LARGE: lessons from mutant mice.

The Large gene encodes a predicted glycosyltransferase of undefined biological activity. However, one important target of the protein is known, alpha-dystroglycan. This protein is a key component of the dystrophin-associated glycoprotein in skeletal muscle, which links cytoskeletal actin to the extracellular matrix (ECM), stabilizing the muscle sarcolemmal membrane. alpha-Dystroglycan binds to extracellular proteins such as laminin through a heavily glycosylated mucin-like domain. Functional Large protein is required for full glycosylation and ligand-binding activity of dystroglycan. The role of Large in this pathway was identified by positional cloning of the mutation in the myodystrophy mouse, an animal model of muscular dystrophy that also has defects in the central and peripheral nervous system and retinal abnormalities. Mice deficient in Large are models for a group of human disorders that have defective alpha-dystroglycan glycosylation.

Analysis of allele-specific RNA transcription in FSHD by RNA-DNA FISH in single myonuclei.

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is likely caused by epigenetic alterations in chromatin involving contraction of the D4Z4 repeat array near the telomere of chromosome 4q. The precise mechanism by which deletions of D4Z4 influence gene expression in FSHD is not yet resolved. Regulatory models include a cis effect on proximal gene transcription (position effect), DNA looping, non-coding RNA, nuclear localization and trans-effects. To directly test whether deletions of D4Z4 affect gene expression in cis, nascent RNA was examined in single myonuclei so that transcription from each allele could be measured independently. FSHD and control myotubes (differentiated myoblasts) were subjected to sequential RNA-DNA FISH. A total of 16 genes in the FSHD region (FRG2, TUBB4Q, FRG1, FAT1, F11, KLKB1, CYP4V2, TLR3, SORBS2, PDLIM3 (ALP), LRP2BP, ING2, SNX25, SLC25A4 (ANT1), HELT and IRF2) were examined for interallelic variation in RNA expression within individual myonuclei. Sequential DNA hybridization with a unique 4q35 chromosome probe was then applied to confirm the localization of nascent RNA to 4q. A D4Z4 probe, labeled with a third fluorochrome, distinguished between the deleted and normal allele in FSHD nuclei. Our data do not support an FSHD model in which contracted D4Z4 arrays induce altered transcription in cis from 4q35 genes, even for those genes (FRG1, FRG2 and SLC25A4 (ANT1)) for which such an effect has been proposed.

Abnormal glycosylation of dystroglycan in human genetic disease.

The dystroglycanopathies are a group of inherited muscular dystrophies that have a common underlying mechanism, hypoglycosylation of the extracellular receptor alpha-dystroglycan. Many of these disorders are also associated with defects in the central nervous system and the eye. Defects in alpha-dystroglycan may also play a role in cancer progression. This review discusses the six dystroglycanopathy genes identified so far, their known or proposed roles in dystroglycan glycosylation and their relevance to human disease, and some of animal models now available for the study of the dystroglycanopathies.

A laminin-2, dystroglycan, utrophin axis is required for compartmentalization and elongation of myelin segments.

Animal and plant cells compartmentalize to perform morphogenetic functions. Compartmentalization of myelin-forming Schwann cells may favor elongation of myelin segments to the size required for efficient conduction of nerve impulses. Compartments in myelinated fibers were described by Ramón y Cajal and depend on periaxin, mutated in the hereditary neuropathy Charcot-Marie-Tooth disease type 4F (Charcot-Marie-Tooth 4F). Lack of periaxin in mice causes loss of compartments, formation of short myelin segments (internodes) and reduced nerve conduction velocity. How compartments are formed and maintained, and their relevance to human neuropathies is largely unknown. Here we show that formation of compartments around myelin is driven by the actin cytoskeleton, and maintained by actin and tubulin fences through linkage to the dystroglycan complex. Compartmentalization and establishment of correct internodal length requires the presence of glycosylated dystroglycan, utrophin and extracellular laminin-2/211. A neuropathic patient with reduced internodal length and nerve conduction velocity because of absence of laminin-2/211 (congenital muscular dystrophy 1A) also shows abnormal compartmentalization. These data link formation of compartments through a laminin2, dystroglycan, utrophin, actin axis to internodal length, and provide a common pathogenetic mechanism for two inherited human neuropathies. Other cell types may exploit dystroglycan complexes in similar fashions to create barriers and compartments.

Dystroglycan glycosylation and muscular dystrophy.

Dystroglycan is an integral member of the skeletal muscle dystrophin glycoprotein complex, which links dystrophin to proteins in the extracellular matrix. Recently, a group of human muscular dystrophy disorders have been demonstrated to result from defective glycosylation of the alpha-dystroglycan subunit. Genetic studies of these diseases have identified six genes that encode proteins required for the synthesis of essential carbohydrate structures on dystroglycan. Here we highlight their known or postulated functions. This glycosylation pathway appears to be highly specific (dystroglycan is the only substrate identified thus far) and to be highly conserved during evolution.

Genes required for functional glycosylation of dystroglycan are conserved in zebrafish.

Mutations in human genes encoding proteins involved in alpha-dystroglycan glycosylation result in dystroglycanopathies: severe congenital muscular dystrophy phenotypes often accompanied by CNS abnormalities and ocular defects. We have identified the zebrafish orthologues of the seven known genes in this pathway and examined their expression during embryonic development. Zebrafish Large, POMT1, POMT2, POMGnT1, Fukutin, and FKRP show in situ hybridization patterns similar to those of dystroglycan, with broad expression throughout early development. By 30 h postfertilization (hpf), transcripts of all these genes are most prominent in the CNS, eye, and muscle, tissues that are predominantly affected in the dystroglycanopathies. In contrast, Large2 expression is more restricted and by 30 hpf is confined to the lens, cerebellum, and pronephric duct. We show that the monoclonal antibody IIH6, which recognizes a glycoform of dystroglycan, also detects the zebrafish protein. Injection of morpholino oligonucleotides against zebrafish Large2 resulted in loss of IIH6 immunostaining. These data indicate that the dystroglycan glycosylation pathway is conserved in zebrafish and suggest this organism is likely to be a useful model system for functional studies.

Evolutionary conservation of a coding function for D4Z4, the tandem DNA repeat mutated in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions within the polymorphic DNA tandem array D4Z4. Each D4Z4 repeat unit has an open reading frame (ORF), termed "DUX4," containing two homeobox sequences. Because there has been no evidence of a transcript from the array, these deletions are thought to cause FSHD by a position effect on other genes. Here, we identify D4Z4 homologues in the genomes of rodents, Afrotheria (superorder of elephants and related species), and other species and show that the DUX4 ORF is conserved. Phylogenetic analysis suggests that primate and Afrotherian D4Z4 arrays are orthologous and originated from a retrotransposed copy of an intron-containing DUX gene, DUXC. Reverse-transcriptase polymerase chain reaction and RNA fluorescence and tissue in situ hybridization data indicate transcription of the mouse array. Together with the conservation of the DUX4 ORF for >100 million years, this strongly supports a coding function for D4Z4 and necessitates re-examination of current models of the FSHD disease mechanism.

Intragenic deletion in the LARGE gene causes Walker-Warburg syndrome.

Intragenic homozygous deletions in the Large gene are associated with a severe neuromuscular phenotype in the myodystrophy (myd) mouse. These mutations result in a virtual lack of glycosylation of alpha-dystroglycan. Compound heterozygous LARGE mutations have been reported in a single human patient, manifesting with mild congenital muscular dystrophy (CMD) and severe mental retardation. These mutations are likely to retain some residual LARGE glycosyltransferase activity as indicated by residual alpha-dystroglycan glycosylation in patient cells. We hypothesized that more severe LARGE mutations are associated with a more severe CMD phenotype in humans. Here we report a 63-kb intragenic LARGE deletion in a family with Walker-Warburg syndrome (WWS), which is characterized by CMD, and severe structural brain and eye malformations. This finding demonstrates that LARGE gene mutations can give rise to a wide clinical spectrum, similar as for other genes that have a role in the post-translational modification of the alpha-dystroglycan protein.

Characterization of the LARGE family of putative glycosyltransferases associated with dystroglycanopathies.

The Large(myd) mouse has a loss-of-function mutation in the putative glycosyltransferase gene Large. Mutations in the human homolog (LARGE) have been described in a form of congenital muscular dystrophy (MDC1D). Other genes (POMT1, POMGnT1, fukutin, and FKRP) that encode known or putative glycosylation enzymes are also causally associated with human congenital muscular dystrophies. All these diseases are associated with hypoglycosylation of the membrane protein alpha-dystroglycan (alpha-DG) and consequent loss of extracellular ligand binding. Hence, they are termed dystroglycanopathies. A paralogous gene for LARGE (LARGE2 or GYLTL1B) may also have a role in DG glycosylation. Using database interrogation and reverse-transcriptase polymerase chain reaction (RT-PCR), we identified vertebrate orthologs of each of these LARGE genes in many vertebrates, including human, mouse, dog, chicken, zebrafish, and pufferfish. However, within invertebrate genomes, we were able to identify only single homologs. We suggest that vertebrate LARGE orthologs be referred to as LARGE1. RT-PCR, dot-blot, and northern analysis indicated that LARGE2 has a more restricted tissue-expression profile than LARGE1. Using epitope-tagged proteins, we show that both LARGE1 and LARGE2 localize to the Golgi apparatus. The high similarity between the LARGE paralogs suggests that LARGE2 may also act on DG. Overexpression of LARGE2 in mouse C2C12 myoblasts results in increased glycosylation of alpha-DG accompanied by an increase in laminin binding. Thus, there may be functional redundancy between LARGE1 and LARGE2. Consistent with this idea, we show that alpha-DG is still fully glycosylated in kidney (a tissue that expresses a high level of LARGE2 mRNA) of Large(myd) mutant mice.