Increased DUX4 expression during muscle differentiation correlates with decreased SMCHD1 protein levels at D4Z4

<p>Facioscapulohumeral muscular dystrophy is caused by incomplete epigenetic repression of the transcription factor DUX4 in skeletal muscle. A copy of <i>DUX4</i> is located within each unit of the D4Z4 macrosatellite repeat array and its derepression in somatic cells is caused by either repeat array contraction (FSHD1) or by mutations in the chromatin repressor SMCHD1 (FSHD2). While DUX4 expression has thus far only been detected in FSHD muscle and muscle cell cultures, and increases with <i>in vitro</i> myogenic differentiation, the D4Z4 chromatin structure has only been studied in proliferating myoblasts or non-myogenic cells. We here show that SMCHD1 protein levels at D4Z4 decline during muscle cell differentiation and correlate with DUX4 derepression. In FSHD2, but not FSHD1, the loss of SMCHD1 repressor activity is partially compensated by increased Polycomb Repressive Complex 2 (PRC2)–mediated H3K27 trimethylation at D4Z4, a situation that can be mimicked by SMCHD1 knockdown in control myotubes. In contrast, moderate overexpression of SMCHD1 results in DUX4 silencing in FSHD1 and FSHD2 myotubes demonstrating that DUX4 derepression in FSHD is reversible. Together, we show that in FSHD1 and FSHD2 the decline in SMCHD1 protein levels during muscle cell differentiation renders skeletal muscle sensitive to DUX4.</p

Analysis of transcriptional activity of DUX4 in a panel of tissues of D4Z4-2.5 and D4Z4-12.5 mice.

<p>DUX4 transcripts measured in 7 weeks old D4Z4-2.5 and D4Z4-12.5 mice (n = 3) in A) muscle tissue: Hea = Heart, Dia = Diaphragm, Pec = Pectoralis Mas = Masseter, Orb = Orbicularis oris, Qua = Quadriceps, TA = Tibialis anterior, Gas = Gastrocnemius, Ton = Tongue; and B) somatic non-muscle and germline tissue: Tes = Testis, Ute = Uterus, Ova = Ovarium, Eye, Cer = Cerebellum, Spl = Spleen, Kid = Kidney, Liv = Liver C) DUX4 transcripts measured in satellite-cell-derived myoblasts, myotubes and interstitial fibroblast extracted from EDL muscle of D4Z4-12.5 and D4Z4-2.5 transgenic mice. D) Quantitative RT-PCR data of DUX4 expression in D4Z4-2.5 myoblasts (n = 2) and myotubes (n = 2) 48 hours after induction of differentiation. Errors indicate SEM of the plotted mean.</p

Integration site and copy number of D4Z4-2.5 and D4Z4-12.5 constructs in the mouse genome.

<p>A) Schematic draw of the L42 <i>Eco</i>RI fragment used to generate the D4Z4-2.5 mouse line B) Metaphase spread of D4Z4-2.5 fibroblasts co-stained with dapi and the CY3 labeled L42 probe shows integration at a single pair of chromosomes C) COBRA-FISH analysis on D4Z4-2.5 fibroblast metaphase spreads probed with biotinylated-L42 fragments shows integration of L42 on chr17; D) Detection of copy number of the integrated fragments in both mouse models by MLPA analysis. The probe mix contained three probes specific for wild type alleles, one probe designed against the human p13E-11 region and one probe against D4Z4 E) Schematic draw of PAC clones used to generate the D4Z4-12.5 mouse; F) COBRA-FISH analysis on D4Z4-12.5 fibroblast metaphase spreads probed with a biotinylated PAC clone shows integration of the PAC clone on chr2; G) Fiber-FISH analysis of D4Z4-12.5 fibroblasts. Both PAC clones, labeled and hybridized to DNA fibers, were shown to be recombined during integration into the mouse genome.</p

Deregulated genes in response to DUX4 expression in C2C12, linked to functional groups shown to be deregulated in human myoblasts upon DUX4 expression.

*<p>Genes in cluster, possible duplications: >97% sequence homology.</p

Bursts of DUX4 protein expression in differentiating D4Z4-2.5 muscle cells.

<p>Satellite-cell-derived myoblasts extracted from single EDL fibers of D4Z4-2.5 mice were differentiated for 12, 24 and 48 hrs and co-stained for DUX4 and Myog or for DUX4 and Myosin heavy chain. A) Representative DUX4 and Myog IF staining images of D4Z4-2.5 myotubes, 24 hrs after induction of differentiation, indicate absence of Myog in DUX4 expressing cells. B) Representative DUX4 and Myosin HC IF staining images of D4Z4-2.5 myotubes, 24 hrs after induction of differentiation, indicate exclusion of DUX4 positive cells from newly formed myotubes. Both DUX4 (panel C) and Myog (panel D) positive nuclei in relation to total amount of nuclei (DAPI staining) were counted during the differentiation process. C) Approximately 2∶1000 nuclei showed nuclear DUX4 staining. D) The percentage of Myog positive nuclei revealed an increase in differentiation committed cells with time. After 48 hours of differentiation almost all myoblasts are committed to differentiation. Error bars indicate stdev of the plotted mean (n = 7); *p<0,05 compared to t = 12 hrs; ^#p<0,05 compared to t = 24 hrs.</p

Validation of expression levels of DUX4 deregulated genes in C2C12 myoblasts.

<p>A set of deregulated genes obtained from expression array analysis was confirmed by qRT-PCR. Expression analysis of A) DUX4 and genes that are switched on by DUX4 in C2C12 cells, B) genes that respond to DUX4 in humans and mice, C) germ line and early development associated genes, D) innate immunity genes, untr = untransfected control, transfection activates innate immunity which is dampened by DUX4 expression, E) genes directly regulated by DUX4 which were identified by ChIP-seq and F) activated L1 and MaLR retrotransposons. For panel A and Mte2b in panel F, DUX4- values refer to the DUX4 depleted FACS sorted fraction, enabling proper normalization of genes switched on upon DUX4 expression. In all other panels DUX4- refers to pCS2 transfected cells. All expression levels are relative to Cyclophillin-B and normalized to DUX4- or wt conditions. Error bars indicate SEM of at least triplicate measurements, asterisks indicate p-values<0.05 based on a student's t-test (panels A, B, C, E & F) or one way ANOVA (panel D) analysis.</p

Epigenetic structure of D4Z4 in D4Z4-2.5 and D4Z4-12.5 mice.

<p>A) Schematic draw of the regions within D4Z4 where CpG and histone methylation were interrogated. B) Representative figure of a methylation sensitive Southern blot assay to quantify DNA methylation levels. Upon <i>Bsa</i>AI digestion, gel separation and blotting, two distinct bands representing the unmethylated and methylated fragment are visualized and quantified; C) Southern Blot analysis was done using two different methylation sensitive restriction enzymes, <i>Bsa</i>AI and <i>Fsp</i>I, in adult gastrocnemius muscle tissue of D4Z4-12.5 and D4Z4-2.5 mice. Both probes p13E-11 and D4Z4 were used to measure CpG methylation levels in the most proximal unit and all units, respectively. The methylation percentages of the two different CpG sites are plotted. Error bars indicate stdev of the plotted mean (n = 4 D4Z4-12.5 vs n = 5 D4Z4-2.5, *p<0.001). D) Histone methylation levels of D4Z4 in transgenic D4Z4-12.5 and D4Z4-2.5 embryonic (MEFs) and adult fibroblasts. Chromatin was precipitated with H3K4me2, H3K9me3 and control IgG antibodies. Precipitated DNA was amplified with qPCR primers amplifying the transcription start site of DUX4. Levels of H3K9me3 in relation to H3K4me2 have been plotted as the chromatin compaction score (ChCS).</p