RESUMO
Facioscapulohumeral muscular dystrophy is a dominantly inherited myopathy associated with chromatin relaxation of the D4Z4 macrosatellite array on chromosome 4. DUX4 is encoded within each unit of the D4Z4 array where it is normally transcriptionally silenced and packaged as constitutive heterochromatin. Truncation of the array to less than 11 D4Z4 units (FSHD1) or mutations in SMCHD1 (FSHD2) results in chromatin relaxation and a small percentage of cultured myoblasts from these individuals exhibit infrequent bursts of DUX4 expression. There are no cellular or animal models to determine the trigger of the DUX4 producing transcriptional bursts and there has been a failure to date to detect the protein in significant numbers of cells from FSHD-affected individuals. Here, we demonstrate for the first time that myotubes generated from FSHD patients express sufficient amounts of DUX4 to undergo DUX4-dependent apoptosis. We show that activation of the Wnt/ß-catenin signaling pathway suppresses DUX4 transcription in FSHD1 and FSHD2 myotubes and can rescue DUX4-mediated myotube apoptosis. In addition, reduction of mRNA transcripts from Wnt pathway genes ß-catenin, Wnt3A and Wnt9B results in DUX4 activation. We propose that Wnt/ß-catenin signaling is important for transcriptional repression of DUX4 and identify a novel group of therapeutic targets for the treatment of FSHD.
Assuntos
Apoptose , Proteínas de Homeodomínio/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Distrofia Muscular Facioescapuloumeral/genética , Via de Sinalização Wnt , Animais , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Camundongos , RNA Interferente Pequeno/genéticaRESUMO
Facioscapulohumeral dystrophy (FSHD) is characterized by chromatin relaxation of the D4Z4 macrosatellite array on chromosome 4 and expression of the D4Z4-encoded DUX4 gene in skeletal muscle. The more common form, autosomal dominant FSHD1, is caused by contraction of the D4Z4 array, whereas the genetic determinants and inheritance of D4Z4 array contraction-independent FSHD2 are unclear. Here, we show that mutations in SMCHD1 (encoding structural maintenance of chromosomes flexible hinge domain containing 1) on chromosome 18 reduce SMCHD1 protein levels and segregate with genome-wide D4Z4 CpG hypomethylation in human kindreds. FSHD2 occurs in individuals who inherited both the SMCHD1 mutation and a normal-sized D4Z4 array on a chromosome 4 haplotype permissive for DUX4 expression. Reducing SMCHD1 levels in skeletal muscle results in D4Z4 contraction-independent DUX4 expression. Our study identifies SMCHD1 as an epigenetic modifier of the D4Z4 metastable epiallele and as a causal genetic determinant of FSHD2 and possibly other human diseases subject to epigenetic regulation.
Assuntos
Proteínas Cromossômicas não Histona/genética , Hereditariedade/genética , Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/genética , Mutação , Adulto , Idoso , Cromossomos Humanos Par 18/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Epigênese Genética , Feminino , Haplótipos , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Facioscapulohumeral Disease (FSHD) is a dominantly inherited progressive myopathy associated with aberrant production of the transcription factor, Double Homeobox Protein 4 (DUX4). The expression of DUX4 depends on an open chromatin conformation of the D4Z4 macrosatellite array and a specific haplotype on chromosome 4. Even when these requirements are met, DUX4 transcripts and protein are only detectable in a subset of cells indicating that additional constraints govern DUX4 production. Since the direction of transcription, along with the production of non-coding antisense transcripts is an important regulatory feature of other macrosatellite repeats, we developed constructs that contain the non-coding region of a single D4Z4 unit flanked by genes that report transcriptional activity in the sense and antisense directions. We found that D4Z4 contains two promoters that initiate sense and antisense transcription within the array, and that antisense transcription predominates. Transcriptional start sites for the antisense transcripts, as well as D4Z4 regions that regulate the balance of sense and antisense transcripts were identified. We show that the choice of transcriptional direction is reversible but not mutually exclusive, since sense and antisense reporter activity was often present in the same cell and simultaneously upregulated during myotube formation. Similarly, levels of endogenous sense and antisense D4Z4 transcripts were upregulated in FSHD myotubes. These studies offer insight into the autonomous distribution of muscle weakness that is characteristic of FSHD.