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.

Specific loss of histone H3 lysine 9 trimethylation and HP1gamma/cohesin binding at D4Z4 repeats is associated with facioscapulohumeral dystrophy (FSHD).

Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy in which no mutation of pathogenic gene(s) has been identified. Instead, the disease is, in most cases, genetically linked to a contraction in the number of 3.3 kb D4Z4 repeats on chromosome 4q. How contraction of the 4qter D4Z4 repeats causes muscular dystrophy is not understood. In addition, a smaller group of FSHD cases are not associated with D4Z4 repeat contraction (termed "phenotypic" FSHD), and their etiology remains undefined. We carried out chromatin immunoprecipitation analysis using D4Z4-specific PCR primers to examine the D4Z4 chromatin structure in normal and patient cells as well as in small interfering RNA (siRNA)-treated cells. We found that SUV39H1-mediated H3K9 trimethylation at D4Z4 seen in normal cells is lost in FSHD. Furthermore, the loss of this histone modification occurs not only at the contracted 4q D4Z4 allele, but also at the genetically intact D4Z4 alleles on both chromosomes 4q and 10q, providing the first evidence that the genetic change (contraction) of one 4qD4Z4 allele spreads its effect to other genomic regions. Importantly, this epigenetic change was also observed in the phenotypic FSHD cases with no D4Z4 contraction, but not in other types of muscular dystrophies tested. We found that HP1gamma and cohesin are co-recruited to D4Z4 in an H3K9me3-dependent and cell type-specific manner, which is disrupted in FSHD. The results indicate that cohesin plays an active role in HP1 recruitment and is involved in cell type-specific D4Z4 chromatin regulation. Taken together, we identified the loss of both histone H3K9 trimethylation and HP1gamma/cohesin binding at D4Z4 to be a faithful marker for the FSHD phenotype. Based on these results, we propose a new model in which the epigenetic change initiated at 4q D4Z4 spreads its effect to other genomic regions, which compromises muscle-specific gene regulation leading to FSHD pathogenesis.

RNA transcripts, miRNA-sized fragments and proteins produced from D4Z4 units: new candidates for the pathophysiology of facioscapulohumeral dystrophy.

Deletion of a subset of the D4Z4 macrosatellite repeats in the subtelomeric region of chromosome 4q causes facioscapulohumeral muscular dystrophy (FSHD) when occurring on a specific haplotype of 4qter (4qA161). Several genes have been examined as candidates for causing FSHD, including the DUX4 homeobox gene in the D4Z4 repeat, but none have been definitively shown to cause the disease, nor has the full extent of transcripts from the D4Z4 region been carefully characterized. Using strand-specific RT-PCR, we have identified several sense and antisense transcripts originating from the 4q D4Z4 units in wild-type and FSHD muscle cells. Consistent with prior reports, we find that the DUX4 transcript from the last (most telomeric) D4Z4 unit is polyadenylated and has two introns in its 3-prime untranslated region. In addition, we show that this transcript generates (i) small si/miRNA-sized fragments, (ii) uncapped, polyadenylated 3-prime fragments that encode the conserved C-terminal portion of DUX4 and (iii) capped and polyadenylated mRNAs that contain the double-homeobox domain of DUX4 but splice-out the C-terminal portion. Transfection studies demonstrate that translation initiation at an internal methionine can produce the C-terminal polypeptide and developmental studies show that this peptide inhibits myogenesis at a step between MyoD transcription and the activation of MyoD target genes. Together, we have identified new sense and anti-sense RNA transcripts, novel mRNAs and mi/siRNA-sized RNA fragments generated from the D4Z4 units that are new candidates for the pathophysiology of FSHD.

Nuclear envelope dystrophies show a transcriptional fingerprint suggesting disruption of Rb-MyoD pathways in muscle regeneration.

Mutations of lamin A/C (LMNA) cause a wide range of human disorders, including progeria, lipodystrophy, neuropathies and autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD). EDMD is also caused by X-linked recessive loss-of-function mutations of emerin, another component of the inner nuclear lamina that directly interacts with LMNA. One model for disease pathogenesis of LMNA and emerin mutations is cell-specific perturbations of the mRNA transcriptome in terminally differentiated cells. To test this model, we studied 125 human muscle biopsies from 13 diagnostic groups (125 U133A, 125 U133B microarrays), including EDMD patients with LMNA and emerin mutations. A Visual and Statistical Data Analyzer (VISDA) algorithm was used to statistically model cluster hierarchy, resulting in a tree of phenotypic classifications. Validations of the diagnostic tree included permutations of U133A and U133B arrays, and use of two probe set algorithms (MAS5.0 and MBEI). This showed that the two nuclear envelope defects (EDMD LMNA, EDMD emerin) were highly related disorders and were also related to fascioscapulohumeral muscular dystrophy (FSHD). FSHD has recently been hypothesized to involve abnormal interactions of chromatin with the nuclear envelope. To identify disease-specific transcripts for EDMD, we applied a leave-one-out (LOO) cross-validation approach using LMNA patient muscle as a test data set, with reverse transcription-polymerase chain reaction (RT-PCR) validations in both LMNA and emerin patient muscle. A high proportion of top-ranked and validated transcripts were components of the same transcriptional regulatory pathway involving Rb1 and MyoD during muscle regeneration (CRI-1, CREBBP, Nap1L1, ECREBBP/p300), where each was specifically upregulated in EDMD. Using a muscle regeneration time series (27 time points) we develop a transcriptional model for downstream consequences of LMNA and emerin mutations. We propose that key interactions between the nuclear envelope and Rb and MyoD fail in EDMD at the point of myoblast exit from the cell cycle, leading to poorly coordinated phosphorylation and acetylation steps. Our data is consistent with mutations of nuclear lamina components leading to destabilization of the transcriptome in differentiated cells.

Variable hypomethylation of D4Z4 in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) progressively affects the facial, shoulder, and upper arm muscles and is associated with contractions of the polymorphic D4Z4 repeat array in 4q35. Recently, we demonstrated that FSHD alleles are hypomethylated at D4Z4. To study potential relationships between D4Z4 hypomethylation and both residual repeat size and clinical severity, we compared the clinical severity score with D4Z4 methylation in unrelated FSHD patients. Correcting the clinical severity score for age at examination improves the parameter to define clinical severity and provides further support for hypomethylation of FSHD alleles. However, a linear relationship between repeat size and clinical severity of the disease cannot be established. Interestingly, FSHD can be separated in two clinical severity classes: patients with residual repeat sizes of 10 to 20 kb are severely affected and show pronounced D4Z4 hypomethylation. In contrast, patients with repeat sizes of 20 to 31kb show large interindividual variation in clinical severity and D4Z4 hypomethylation. Because the majority of familial FSHD cases are represented in this interval and considering the overt variation in clinical severity in these familial cases, it thus is imperative to develop comprehensive allele-specific assays monitoring total D4Z4 methylation to investigate whether interindividual variation in D4Z4 methylation can be translated into a prognostic factor for clinical severity.

Localization of 4q35.2 to the nuclear periphery: is FSHD a nuclear envelope disease?

Facioscapulohumeral muscular dystrophy (FSHD) may be a new member of the class of neuromuscular diseases (NMD) due to defects in the nuclear envelope. Unlike other NMDs with primary defects in nuclear envelope proteins, however, FSHD may result from inappropriate chromatin interactions at the envelope. 3D Immuno-FISH and a novel method of 3D by 2D analysis using NucProfile were developed to examine nuclear organization of the FSHD genomic region. In contrast to most other telomeres, the FSHD region at 4q35.2 localizes to the nuclear periphery. This localization is consistent in normal myoblasts, myotubes, fibroblasts and lymphoblasts, does not vary significantly throughout the cell cycle, and is independent of chromosome territory effects. The nuclear lamina protein lamin A/C is required for FSHD region chromatin localization to the nuclear envelope, as the association is lost in lamin A/C null fibroblasts. As both normal and affected alleles (deleted for the subtelomeric repeat D4Z4) localize to the nuclear periphery, FSHD likely arises instead from improper interactions with transcription factors or chromatin modifiers at the nuclear envelope. Interestingly, it is not D4Z4 itself that mediates interaction with the envelope, as sequences proximal to D4Z4 (i.e. D4S139) localize closer to the nuclear periphery, perhaps accounting for the chromosome 4 specificity of the disease.

Hypomethylation of D4Z4 in 4q-linked and non-4q-linked facioscapulohumeral muscular dystrophy.

The autosomal dominant myopathy facioscapulohumeral muscular dystrophy (FSHD1, OMIM 158900) is caused by contraction of the D4Z4 repeat array on 4qter. We show that this contraction causes marked hypomethylation of the contracted D4Z4 allele in individuals with FSHD1. Individuals with phenotypic FSHD1, who are clinically identical to FSHD1 but have an unaltered D4Z4, also have hypomethylation of D4Z4. These results strongly suggest that hypomethylation of D4Z4 is a key event in the cascade of epigenetic events causing FSHD1.

Expression profiling of FSHD muscle supports a defect in specific stages of myogenic differentiation.

The neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) results from integral deletions of the subtelomeric repeat D4Z4 on chromosome 4q. A disruption of chromatin structure affecting gene expression is thought to underlie the pathophysiology. The global gene expression profiling of mature muscle tissue presented here provides the first insight into an FSHD-specific defect in myogenic differentiation. FSHD expression profiles generated by oligonucleotide microarrays were compared with those from normal muscle as well as other types of muscular dystrophies (DMD, aSGD) in order to determine FSHD-specific changes. In addition, matched biopsies (affected and unaffected muscle) from individuals with FSHD served to monitor expression changes during the progression of the disease as well as to diminish non-specific changes resulting from individual variability. Among genes altered in an FSHD-specific and highly significant manner, many are involved in myogenic differentiation and suggest a partial block in the normal differentiation program. Indeed, many of the transcripts affected in FSHD represent direct targets of the transcription factor MyoD. Additional mis-expressed genes confirm a diminished capacity to buffer oxidative stress, as demonstrated in FSHD myoblasts. This enhanced vulnerability of proliferative stage myoblasts to reactive oxygen species is also disease-specific, further implicating a defect in FSHD muscle satellite cells. Importantly, none of the genes localizing to the FSHD region at 4q35 were found to exhibit a significantly altered pattern of expression in FSHD muscle. This finding was corroborated by expression analysis of FSHD muscle using a custom cDNA microarray containing 51 genes and ESTs from the 4q35 region. Disruptions in FSHD myogenesis and oxidative capacity may therefore not arise from a position effect mechanism as has been previously suggested, but rather from a global effect on gene regulation. Improper nuclear localization of 4qter is discussed as an alternative model for FSHD gene regulation and pathogenesis.

Facioscapulohumeral muscular dystrophy (FSHD) myoblasts demonstrate increased susceptibility to oxidative stress.

Facioscapulohumeral muscular dystrophy is an autosomal dominant disorder resulting from an unusual genetic mechanism. The mutation, a deletion of 3.3 kb subtelomeric repeats, appears to disrupt the regional regulation of 4q35 g ene expression. The specific gene(s)responsible for facioscapulohumeral muscular dystrophy have not been identified. However, the 'vacuolar/necrotic' phenotype exhibited by facioscapulohumeral muscular dystrophy myoblasts suggests that aberrant gene expression occurs early in facioscapulohumeral muscular dystrophy muscle development. In order to test this hypothesis, global gene expression profiling and in vitro characterization of facioscapulohumeral muscular dystrophy and control myoblasts were carried out. Genes involved in several cellular processes such as oxidative stress were found to be dysregulated. In vitro studies confirmed this susceptibility to oxidative stress, as proliferative stage facioscapulohumeral muscular dystrophy myoblasts exhibit greatly reduced viability when exposed to the oxidative stressor paraquat. This effect was not seen in either normal or disease control myoblasts, or in any of the cell lines upon differentiation to multinucleated myotubes. Immunocytochemical studies of the cyclin dependent kinase inhibitor p21 demonstrated increased expression in facioscapulohumeral muscular dystrophy myoblasts, suggesting an early cell cycle arrest. Another process distinguishing facioscapulohumeral muscular dystrophy from controls involves the transcription of extracellular matrix components. Expression of elastin, decorin, lumican and the extracellular matrix remodeling factor TIMP3 were reduced in facioscapulohumeral muscular dystrophy myoblasts. These studies suggest that facioscapulohumeral muscular dystrophy muscular dystrophy results from a defect in early myogenesis, manifested as increased susceptibility to oxidative stress, morphological aberrations and early cell cycle arrest.