<em>Rbfox1</em> Downregulation and Altered <em>Calpain 3</em> Splicing by <em>FRG1</em> in a Mouse Model of Facioscapulohumeral Muscular Dystrophy (FSHD)

<div><p>Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (<em>FRG1</em>) in mice, frogs, and worms perturbs muscle development and causes FSHD–like phenotypes. <em>FRG1</em> has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in <em>FRG1</em> mice. We find that splicing perturbations parallel the responses of different muscles to <em>FRG1</em> over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of <em>FRG1</em>-affected splicing events. <em>Rbfox1</em> knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the <em>Rbfox1</em> RNA and decreases its stability. Consistent with this, <em>Rbfox1</em> expression is down-regulated in mice and cells over-expressing <em>FRG1</em> as well as in FSHD patients. Among the genes affected is <em>Calpain 3</em>, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In <em>FRG1</em> mice and FSHD patients, the <em>Calpain 3</em> isoform lacking exon 6 (<em>Capn3 E6–</em>) is increased. Finally, <em>Rbfox1</em> knockdown and over-expression of <em>Capn3 E6-</em> inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of <em>FRG1</em>, reducing <em>Rbfox1</em> levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.</p> </div

Alternative splicing changes are a primary consequence of <i>FRG1</i> overexpression.

<p>(a) Examples of alternative exons (<i>Atl2</i>) or introns (<i>Ttn</i>) spliced normally in the mouse model of Duchenne muscular dystrophy, <i>mdx</i> mice, and example of an alternative exon similarly altered in <i>FRG1</i> and <i>mdx</i> mice (<i>Ktn1</i>). (b) RT-PCR analysis of mRNA splicing variants from proliferating (MB) and differentiating (MT) C2C12 muscle cells over-expressing FRG1. Examples of alternative splicing changes present in both MB and MT (<i>Capn3</i>), only in MB (<i>Ablim1</i>), or only in MT (<i>Nasp</i>). Numbers are the percentage of exon inclusion. Black boxes illustrate constitutive exons, white boxes alternatively spliced exons and double lines represent the affected intron.</p

FRG1 regulates the stability of the <i>Rbfox1</i> mRNA.

<p>(a) ChIP of FRG1 in the promoter region of the <i>Rbfox1</i> gene. (b) The distribution of RNA pol II on C2C12-<i>EV</i> and C2C12-<i>FRG1</i> cells in the promoter region of the <i>Rbfox1</i> gene. (c) Real-time RT-PCR of the kinetics of <i>Rbfox1</i>, <i>c-Myc</i> and <i>Gapdh</i> expression after 8 hours of ActD (Actinomycin D) treatment on C2C12-<i>EV</i> and C2C12-<i>FRG1</i> cells. (d) RNA-IP experiment on samples from (c) using anti-FRG1 antibodies or control IgG antibodies. Immunoprecipitated material was analyzed by real-time RT-PCR, normalized versus the relative input and plotted as fold enrichment versus the IgG. RT-minus control experiments showed the absence of DNA contamination (data not shown).</p

Alternative splicing isoform of <i>Calpain 3</i> increased in <i>FRG1</i> mice and in FSHD patients.

<p>(a) Real-time RT-PCR and immunoblotting analysis confirming that the <i>Capn3</i> alternative splicing isoform lacking exon 6 (<i>Capn3 E6-</i>) is increased in the <i>vastus lateralis</i> muscle from <i>FRG1</i> mice. (b) RT-PCR analysis of <i>CAPN3</i> splicing in human muscle cells derived from three different healthy subjects and three different FSHD patients indicates increased expression of <i>CAPN3 E6-</i> isoform in FSHD patients. Numbers below the image are the percentage of exon skipping. Black boxes illustrate constitutive exons, white boxes alternatively spliced exon. RT-PCR products were quantified using the Typhoon. (c) <i>RBFOX1</i> expression analysis was performed on RNA extracted from the same samples as in (b) by real-time RT-PCR.</p

<i>Rbfox1</i> is selectively down-regulated in mice, in cells over-expressing <i>FRG1</i>, and in FSHD patients.

<p>(a) Left panel: real-time RT-PCR analysis showing that in <i>FRG1</i> mice <i>Rbfox1</i> expression is preferentially downregulated in <i>vastus lateralis</i> compared to <i>biceps brachii</i>. <i>Gapdh</i> expression was used for sample normalization. Right panel: immunoblotting on <i>vastus lateralis</i> using anti-Rbfox1 antibody. Tubulin was used as loading control and FRG1 antibody as confirmation of the genotype. (b) Real-time RT-PCR analysis showing that <i>Rbfox1</i> expression is normal in <i>mdx</i> mice. (c) Left: real-time RT-PCR analysis showing that <i>Rbfox1</i> expression is down-regulated in proliferating (MB) or differentiating (MT) C2C12 muscle cells over-expressing <i>FRG1</i>. Right: immunoblotting using protein extracted from the same samples as in (c, left) using anti-Rbfox1 antibodies. Tubulin was used as loading control. Note that in proliferating C2C12 cells Rbfox1 protein is almost undetectable, while its levels significantly increase during myogenic differentiation. (d) Left panel: real-time RT-PCR analysis of <i>RBFOX1</i> expression using RNA extracted from muscle cells derived from three different normal individuals and three different FSHD patients. Right panel: immunoblotting using protein extracted from the same samples as in (d, left) using anti-RBFOX1 antibody. Tubulin was used as loading control.</p

RNA map of Rbfox motifs downstream of exons.

<p>Each point represents the average frequency of UGCAUG in the 150 bp downstream of the 260 exons whose inclusion increases (blue triangles), the 273 exons whose inclusion decreases (red squares) or the 821 expressed alternative cassette exons whose splicing did not change in the comparison (gray circles, <i>q</i>>0.2 and |Sepscore|<0.3). Error bars indicate 95% confidence intervals of the mean frequency distribution for this population of background exons.</p

<i>Rbfox1</i> down-regulation is responsible for significant portion of the splicing alterations in <i>FRG1</i> mice.

<p>(a) Specific <i>Rbfox1</i> knockdown was confirmed by real-time RT-PCR and immunoblotting using RNAs and proteins isolated from C2C12 muscle cells expressing a control non-silencing shRNA or an shRNA specific for <i>Rbfox1</i> (shRNA#1). (b) Examples of alternative splicing changes caused by <i>Rbfox1</i> knockdown are showed. Numbers are the percentage of exon inclusion. Black boxes illustrate constitutive exons, white boxes alternatively spliced exons. (c) <i>Rbfox1</i> overexpression causes alternative splicing changes opposite to <i>FRG1</i> over-expression. Specific <i>Rbfox1</i> over-expression was confirmed by real-time RT-PCR and immunoblotting using RNAs and proteins isolated from C2C12 muscle cells expressing an empty vector (<i>EV</i>) or a Myc-tagged Rbfox1 (<i>F1</i>) either in proliferating or differentiating C2C12 muscle cells. (d) Examples of alternative splicing changes caused by <i>Rbfox1</i> over-expression are showed. Black boxes illustrate constitutive exons, white boxes alternatively spliced exons. (e) Selective <i>in vivo</i> association of Rbfox1 to target regions displaying putative Fox binding sites (FBS). RIP experiment on samples from (c) using anti-Myc or control IgG antibodies. Immunoprecipitated material was analyzed by RT-PCR, quantified using the Typhoon, normalized versus the relative input and plotted as fold enrichment versus the IgG. RT-minus control experiments showed the absence of DNA contamination (data not shown).</p

Altered <i>Calpain 3</i> splicing inhibits myogenesis.

<p>(a) C2C12 muscle cells over-expressing <i>FRG1</i>, (b) <i>Rbfox1</i> knockdown with shRNA#1 and (d) over-expressing <i>Capn3 E6-</i> display a reduced myogenic differentiation. (c) C2C12 muscle cells over-expressing <i>FRG1</i> and <i>Rbfox1</i> present increase myogenic differentiation compared to C2C12-<i>FRG1</i>. Left: immunofluorescence using antibodies specific for skeletal muscle myosin heavy chain (MHC), a typical marker of terminally differentiated muscle cells. Right: fusion index defined as the percentage of nuclei belonging to MHC-positive cells with three or more nuclei. The values reported in the graph are the means plus-minus standard deviations from two separate experiments performed in triplicate.</p

Alternative splicing changes in muscles of mice over-expressing FRG1.

<p>Average linkage clustering was applied to alternative cassette exon splicing events with |Sepscore|≥0.3 in at least one of the four comparison datasets (<i>q</i> = 0). Selected nodes are expanded on the right. Exons which shown increased inclusion in FRG1 over-expressing muscles are shown in yellow in the heatmap, while exons with decreased inclusion are represented in blue.</p