Lauren Snider

A Unifying Genetic Model for Facioscapulohumeral Muscular Dystrophy

Addition by Contraction Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common hereditary neuromuscular disorders in Western populations, affecting about 1 in 20,000 people. In most patients, the disorder is associated with contraction of a D4Z4 microsatellite repeat array on chromosome 4q, but this contraction can also occur in the absence of disease, so the underlying genetic mechanisms have remained elusive. Lemmers et al. (p. 1650, published online 19 August; see the Perspective by Mahadevan) now show that FSHD patients carry sequence variants that create a canonical polyadenylation signal for transcripts derived from DUX4, a homeobox gene straddling the last D4Z4 repeat unit and the adjacent sequence. Addition of poly(A) stabilizes the DUX4 transcript, which is likely to be a contributing factor in the disease. Sequence variants shared by patients with a genetically complex form of muscular dystrophy explain how the disease arises. Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy in adults that is foremost characterized by progressive wasting of muscles in the upper body. FSHD is associated with contraction of D4Z4 macrosatellite repeats on chromosome 4q35, but this contraction is pathogenic only in certain “permissive” chromosomal backgrounds. Here, we show that FSHD patients carry specific single-nucleotide polymorphisms in the chromosomal region distal to the last D4Z4 repeat. This FSHD-predisposing configuration creates a canonical polyadenylation signal for transcripts derived from DUX4, a double homeobox gene of unknown function that straddles the last repeat unit and the adjacent sequence. Transfection studies revealed that DUX4 transcripts are efficiently polyadenylated and are more stable when expressed from permissive chromosomes. These findings suggest that FSHD arises through a toxic gain of function attributable to the stabilized distal DUX4 transcript.

Facioscapulohumeral Dystrophy: Incomplete Suppression of a Retrotransposed Gene

Each unit of the D4Z4 macrosatellite repeat contains a retrotransposed gene encoding the DUX4 double-homeobox transcription factor. Facioscapulohumeral dystrophy (FSHD) is caused by deletion of a subset of the D4Z4 units in the subtelomeric region of chromosome 4. Although it has been reported that the deletion of D4Z4 units induces the pathological expression of DUX4 mRNA, the association of DUX4 mRNA expression with FSHD has not been rigorously investigated, nor has any human tissue been identified that normally expresses DUX4 mRNA or protein. We show that FSHD muscle expresses a different splice form of DUX4 mRNA compared to control muscle. Control muscle produces low amounts of a splice form of DUX4 encoding only the amino-terminal portion of DUX4. FSHD muscle produces low amounts of a DUX4 mRNA that encodes the full-length DUX4 protein. The low abundance of full-length DUX4 mRNA in FSHD muscle cells represents a small subset of nuclei producing a relatively high abundance of DUX4 mRNA and protein. In contrast to control skeletal muscle and most other somatic tissues, full-length DUX4 transcript and protein is expressed at relatively abundant levels in human testis, most likely in the germ-line cells. Induced pluripotent (iPS) cells also express full-length DUX4 and differentiation of control iPS cells to embryoid bodies suppresses expression of full-length DUX4, whereas expression of full-length DUX4 persists in differentiated FSHD iPS cells. Together, these findings indicate that full-length DUX4 is normally expressed at specific developmental stages and is suppressed in most somatic tissues. The contraction of the D4Z4 repeat in FSHD results in a less efficient suppression of the full-length DUX4 mRNA in skeletal muscle cells. Therefore, FSHD represents the first human disease to be associated with the incomplete developmental silencing of a retrogene array normally expressed early in development.

Mice deficient in Six5 develop cataracts : implications for myotonic dystrophy

Expansion of a CTG trinucleotide repeat in the 3′ UTR of the gene DMPK at the DM1 locus on chromosome 19 causes myotonic dystrophy, a dominantly inherited disease characterized by skeletal muscle dystrophy and myotonia, cataracts and cardiac conduction defects. Targeted deletion of Dm15, the mouse orthologue of human DMPK, produced mice with a mild myopathy and cardiac conduction abnormalities, but without other features of myotonic dystrophy, such as myotonia and cataracts. We, and others, have demonstrated that repeat expansion decreases expression of the adjacent gene SIX5 (refs 7,8), which encodes a homeodomain transcription factor. To determine whether SIX5 deficiency contributes to the myotonic dystrophy phenotype, we disrupted mouse Six5 by replacing the first exon with a β-galactosidase reporter. Six5-mutant mice showed reporter expression in multiple tissues, including the developing lens. Homozygous mutant mice had no apparent abnormalities of skeletal muscle function, but developed lenticular opacities at a higher rate than controls. Our results suggest that SIX5 deficiency contributes to the cataract phenotype in myotonic dystrophy, and that myotonic dystrophy represents a multigenic disorder.

DUX4 activates germline genes, retroelements and immune-mediators: Implications for facioscapulohumeral dystrophy

Facioscapulohumeral dystrophy (FSHD) is one of the most common inherited muscular dystrophies. The causative gene remains controversial and the mechanism of pathophysiology unknown. Here we identify genes associated with germline and early stem cell development as targets of the DUX4 transcription factor, a leading candidate gene for FSHD. The genes regulated by DUX4 are reliably detected in FSHD muscle but not in controls, providing direct support for the model that misexpression of DUX4 is a causal factor for FSHD. Additionally, we show that DUX4 binds and activates LTR elements from a class of MaLR endogenous primate retrotransposons and suppresses the innate immune response to viral infection, at least in part through the activation of DEFB103, a human defensin that can inhibit muscle differentiation. These findings suggest specific mechanisms of FSHD pathology and identify candidate biomarkers for disease diagnosis and progression.

NeuroD2 and neuroD3: distinct expression patterns and transcriptional activation potentials within the neuroD gene family.

We have identified two new genes, neuroD2 and neuroD3, on the basis of their similarity to the neurogenic basic-helix-loop-helix (bHLH) gene neuroD. The predicted amino acid sequence of neuroD2 shows a high degree of homology to neuroD and MATH-2/NEX-1 in the bHLH region, whereas neuroD3 is a more distantly related family member. neuroD3 is expressed transiently during embryonic development, with the highest levels of expression between days 10 and 12. neuroD2 is initially expressed at embryonic day 11, with persistent expression in the adult nervous system. In situ and Northern (RNA) analyses demonstrate that different regions of the adult nervous system have different relative amounts of neuroD and neuroD2 RNA. Similar to neuroD, expression of neuroD2 in developing Xenopus laevis embryos results in ectopic neurogenesis, indicating that neuroD2 mediates neuronal differentiation. Transfection of vectors expressing neuroD and neuroD2 into P19 cells shows that both can activate expression through simple E-box-driven reporter constructs and can activate a reporter driven by the neuroD2 promoter region, but the GAP-43 promoter is preferentially activated by neuroD2. The noncongruent expression pattern and target gene specificity of these highly related neurogenic bHLH proteins make them candidates for conferring specific aspects of the neuronal phenotype.

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.

Requirement of the mouse I-mfa gene for placental development and skeletal patterning

The bHLH‐repressor protein I‐mfa binds to MyoD family members, inhibits their activity, and blocks their nuclear import and binding to DNA. In situ hybridization analysis demonstrated that mouse I‐mfa was highly expressed in extraembryonic lineages, in the sclerotome, and subsequently within mesenchymal precursors of the axial and appendicular skeleton, before chondrogenesis occurs. Targeted deletion of I‐mfa in a C57Bl/6 background resulted in embryonic lethality around E10.5, associated with a placental defect and a markedly reduced number of trophoblast giant cells. Overexpression of I‐mfa in rat trophoblast (Rcho‐1) stem cells induced differentiation into trophoblast giant cells. I‐mfa interacted with the bHLH protein Mash2, a negative regulator of trophoblast giant cell formation, and inhibited its transcriptional activity in cell culture. In contrast, I‐mfa did not interfere with the activity of the bHLH protein Hand1, a positive regulator of giant cell differentiation. Interestingly, I‐mfa‐null embryos on a 129/Sv background had no placental defect, generally survived to adulthood, and exhibited delayed caudal neural tube closure and skeletal patterning defects that included fusions of ribs, vertebral bodies and abnormal formation of spinous processes. Our results indicate that I‐mfa plays an important role in trophoblast and chondrogenic differentiation by negatively regulating a subset of lineage‐restricted bHLH proteins.

Intrinsic epigenetic regulation of the D4Z4 macrosatellite repeat in a transgenic mouse model for FSHD.

Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy caused by decreased epigenetic repression of the D4Z4 macrosatellite repeats and ectopic expression of DUX4, a retrogene encoding a germline transcription factor encoded in each repeat. Unaffected individuals generally have more than 10 repeats arrayed in the subtelomeric region of chromosome 4, whereas the most common form of FSHD (FSHD1) is caused by a contraction of the array to fewer than 10 repeats, associated with decreased epigenetic repression and variegated expression of DUX4 in skeletal muscle. We have generated transgenic mice carrying D4Z4 arrays from an FSHD1 allele and from a control allele. These mice recapitulate important epigenetic and DUX4 expression attributes seen in patients and controls, respectively, including high DUX4 expression levels in the germline, (incomplete) epigenetic repression in somatic tissue, and FSHD–specific variegated DUX4 expression in sporadic muscle nuclei associated with D4Z4 chromatin relaxation. In addition we show that DUX4 is able to activate similar functional gene groups in mouse muscle cells as it does in human muscle cells. These transgenic mice therefore represent a valuable animal model for FSHD and will be a useful resource to study the molecular mechanisms underlying FSHD and to test new therapeutic intervention strategies.

DUX4 binding to retroelements creates promoters that are active in FSHD muscle and testis.

The human double-homeodomain retrogene DUX4 is expressed in the testis and epigenetically repressed in somatic tissues. Facioscapulohumeral muscular dystrophy (FSHD) is caused by mutations that decrease the epigenetic repression of DUX4 in somatic tissues and result in mis-expression of this transcription factor in skeletal muscle. DUX4 binds sites in the human genome that contain a double-homeobox sequence motif, including sites in unique regions of the genome as well as many sites in repetitive elements. Using ChIP-seq and RNA-seq on myoblasts transduced with DUX4 we show that DUX4 binds and activates transcription of mammalian apparent LTR-retrotransposons (MaLRs), endogenous retrovirus (ERVL and ERVK) elements, and pericentromeric satellite HSATII sequences. Some DUX4-activated MaLR and ERV elements create novel promoters for genes, long non-coding RNAs, and antisense transcripts. Many of these novel transcripts are expressed in FSHD muscle cells but not control cells, and thus might contribute to FSHD pathology. For example, HEY1, a repressor of myogenesis, is activated by DUX4 through a MaLR promoter. DUX4-bound motifs, including those in repetitive elements, show evolutionary conservation and some repeat-initiated transcripts are expressed in healthy testis, the normal expression site of DUX4, but more rarely in other somatic tissues. Testis expression patterns are known to have evolved rapidly in mammals, but the mechanisms behind this rapid change have not yet been identified: our results suggest that mobilization of MaLR and ERV elements during mammalian evolution altered germline gene expression patterns through transcriptional activation by DUX4. Our findings demonstrate a role for DUX4 and repetitive elements in mammalian germline evolution and in FSHD muscular dystrophy.

DUX4-induced gene expression is the major molecular signature in FSHD skeletal muscle

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 the expression of DUX4 target genes. This individual has a sibling with FSHD and also without a known FSHD-causing mutation, suggesting the presence of an unidentified modifier locus for DUX4 expression and FSHD. These findings demonstrate that the expression of DUX4 accounts for the majority of the gene expression changes in FSHD skeletal muscle together with an immune cell infiltration.