Generation of Isogenic D4Z4 Contracted and Noncontracted Immortal Muscle Cell Clones from a Mosaic Patient: A Cellular Model for FSHD.

Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
American Journal Of Pathology (Impact Factor: 4.6). 08/2012; 181(4):1387-401. DOI: 10.1016/j.ajpath.2012.07.007
Source: PubMed

ABSTRACT In most cases facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of the D4Z4 repeat in the 4q subtelomere. This contraction is associated with local chromatin decondensation and derepression of the DUX4 retrogene. Its complex genetic and epigenetic cause and high clinical variability in disease severity complicate investigations on the pathogenic mechanism underlying FSHD. A validated cellular model bypassing the considerable heterogeneity would facilitate mechanistic and therapeutic studies of FSHD. Taking advantage of the high incidence of somatic mosaicism for D4Z4 repeat contraction in de novo FSHD, we have established a clonal myogenic cell model from a mosaic patient. Individual clones are genetically identical except for the size of the D4Z4 repeat array, being either normal or FSHD sized. These clones retain their myogenic characteristics, and D4Z4 contracted clones differ from the noncontracted clones by the bursts of expression of DUX4 in sporadic nuclei, showing that this burst-like phenomenon is a locus-intrinsic feature. Consequently, downstream effects of DUX4 expression can be observed in D4Z4 contracted clones, like differential expression of DUX4 target genes. We also show their participation to in vivo regeneration with immunodeficient mice, further expanding the potential of these clones for mechanistic and therapeutic studies. These cell lines will facilitate pairwise comparisons to identify FSHD-specific differences and are expected to create new opportunities for high-throughput drug screens.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Facioscapulohumeral dystrophy (FSHD) is caused by decreased epigenetic repression of the D4Z4 macrosatellite array and recent studies have shown that this results in the expression of low levels of the DUX4 mRNA in skeletal muscle. Several other mechanisms have been suggested for FSHD pathophysiology and it remains unknown whether DUX4 expression can account for most of the molecular changes seen in FSHD. Since DUX4 is a transcription factor, we used RNA-seq to measure gene expression in muscle cells transduced with DUX4, and in muscle cells and biopsies from control and FSHD individuals. We show that DUX4 target gene expression is the major molecular signature in FSHD muscle together with a gene expression signature consistent with an immune cell infiltration. In addition, one unaffected individual without a known FSHD-causing mutation showed expression of DUX4 target genes. This individual has a sibling with FSHD and also without a known FSHD-causing mutation, suggesting the presence of yet unidentified modifier locus for DUX4 expression and FSHD. These findings demonstrate that expression of DUX4 accounts for the majority of the gene expression changes in FSHD skeletal muscle together with an immune cell infiltration.
    Human Molecular Genetics 05/2014; · 6.68 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Although the pathophysiology of facioscapulohumeral dystrophy (FSHD) has been controversial over the last decades, progress in recent years has led to a model that incorporates these decades of findings and is gaining general acceptance in the FSHD research community. Here we review how the contributions from many labs over many years led to an understanding of a fundamentally new mechanism of human disease. FSHD is caused by inefficient repeat-mediated epigenetic repression of the D4Z4 macrosatellite repeat array on chromosome 4, resulting in the variegated expression of the DUX4 retrogene, encoding a double-homeobox transcription factor, in skeletal muscle. Normally expressed in the testis and epigenetically repressed in somatic tissues, DUX4 expression in skeletal muscle induces expression of many germline, stem cell, and other genes that might account for the pathophysiology of FSHD. Although some disagreements regarding the details of mechanisms remain in the field, the coalescing agreement on a central model of pathophysiology represents a pivot-point in FSHD research, transitioning the field from discovery-oriented studies to translational studies aimed at developing therapies based on a sound model of disease pathophysiology.
    Skeletal muscle. 01/2014; 4:12.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: While transfer of a protein encoded by a single nucleus to nearby nuclei in multinucleated cells has been known for almost 25 years, the biological consequences for gain-of-function diseases have not been considered. Here, we have investigated nuclear protein spreading and its potential consequences in 2 of the 3 most prevalent neuromuscular diseases. By performing co-cultures between diseased or control human myoblasts and murine C2C12 myoblasts, we demonstrate that in FacioScapuloHumeral Dystrophy (FSHD), although the transcription of the toxic protein DUX4 occurs in only a limited number of nuclei, the resulting protein diffuses into nearby nuclei within the myotubes, thus spreading aberrant gene expression. In Myotonic Dystrophy type 1 (DM1), we observed that in human-mouse heterokaryons, the expression of a mutated DMPK from a human nuclei titrates splicing factors produced by neighbouring nuclei, inducing the mis-splicing of several pre-mRNAs in murine nuclei. In both cases, the spreading of the pathological phenotypes from one nucleus to another is observed, highlighting an additional mechanism that contributes to the dissemination and worsening of the muscle pathogenesis. These results indicate that nuclear protein spreading may be an important component of pathophysiology of gain of function muscular diseases which should be taken into consideration in the design of new therapeutic approaches.
    Human Molecular Genetics 03/2014; · 6.68 Impact Factor

Full-text (2 Sources)

Available from
May 20, 2014