Research ArticleCELL BIOLOGY

Identification of the hyaluronic acid pathway as a therapeutic target for facioscapulohumeral muscular dystrophy

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Science Advances  11 Dec 2019:
Vol. 5, no. 12, eaaw7099
DOI: 10.1126/sciadv.aaw7099
  • Fig. 1 DUX4 expression causes HA accumulation.

    (A) MB135-DUX4i myoblasts were left untreated or treated with DOX (2 μg/ml) for 24 hours and stained with biotinylated HA-binding protein (bHABP) and Hoechst dye. Control samples were treated with hyaluronidase (HAase) to show specificity of the biotinylated HA-binding protein stain. (B) MB135-DUX4i myoblasts were treated as in (A), and the expression of HAS3 was measured by RT-PCR. The mean ± SEM are indicated. Significance was determined using t tests. n = 3. (C) MB135-DUX4i myoblasts were left untreated or treated as in (A) and immunostained with C1QBP and DUX4 antibodies and Hoechst dye. The arrow indicates examples of overlapping staining. (D) Myoblasts were treated as in (A) and stained with C1QBP antibodies and MitoSpy and Hoechst dyes. Arrows indicate examples of overlapping staining. (E) Myoblasts were either left uninduced or induced with DOX, treated with 30 μM Z-DEVD-FMK or dimethyl sulfoxide (DMSO) vehicle, and immunostained with C1QBP and DUX4 antibodies and Hoechst dye. Scale bar, 35 μm. Inset: 75 μm. All experiments were performed a minimum of three times. ***P < 0.001.

  • Fig. 2 C1QBP and mitochondrial pathology depend on HA synthesis.

    (A) MB135-DUX4i myoblasts were left uninduced or induced with DOX, simultaneously treated with 1 mM 4MU or 1% DMSO vehicle for 24 hours, and stained with biotinylated HA-binding protein and Hoechst dye as in Fig. 1. (B) HAS3 expression was measured by RT-PCR in uninduced or 24 hour–induced myoblasts treated with 2 mM 4MU or an equal volume of DMSO vehicle. The means ± SEM are indicated. Significance was determined using t tests with multiple hypothesis correction using the Bonferroni-Dunn method. n = 3. (C) Myoblasts were induced, treated with 4MU or DMSO vehicle as in (A), and stained for C1QBP and DUX4 as in Fig. 1C. The arrow indicates examples of overlapping staining. (D) Cells were treated as in (C) and stained for C1QBP and mitochondria as in Fig. 1B. Arrows indicate examples of overlapping staining. Scale bar, 35 μm. Inset: 75 μm. (E) MB135-DUX4i myoblasts were either left uninduced or induced and treated with the indicated compounds as above. Immunoprecipitations (IP) were performed from nuclear lysates using either DUX4 antibodies or isotype-matched control antibodies, and DUX4 and C1QBP were analyzed by immunoblotting (IB). All experiments were performed a minimum of three times. ***P < 0.001, n.s., not significant.

  • Fig. 3 DUX4-induced toxicity requires HA.

    (A) Myoblasts were left untreated, treated with DOX (2 μg/ml) alone, 1 mM 4MU alone, or DOX (2 μg/ml) plus the indicated concentration of 4MU or DMSO for 24 hours and were imaged by phase-contrast microscopy. Experiments were performed three times. (B) Representative scatterplots of flow cytometry experiments. Cells were treated as in (A) and were then stained for caspase-3/7 activation and cell death using the CellEvent Assay Kit. (C) Left: The total fraction of caspase-3/7–activated cells (quadrants 1 and 2) from three independent experiments was plotted against the concentration of 4MU used and fitted to a linear trend line. Data points from individual experiments are indicated with dots. n = 3. Right: The fraction of caspase-3/7–activated cells from induced myoblasts treated with 1 mM 4MU plotted against induced myoblasts treated with an equal volume (1%) of DMSO. Values from individual experiments are indicated with dots. The means ± SEM are indicated. n = 3. (D) Left: As in (C), with the total fraction of live cells (quadrant 4) plotted against the concentration of 4MU and fitted to a second-degree polynomial trend line. Right: The fraction of live cells from induced cultures treated with 1 mM 4MU plotted against induced cultures treated with an equal volume (1%) of DMSO. Data points, mean, and SEM are indicated as in (C). Significance was determined using Welch’s t tests. n = 3. Scale bar, 100 μm *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 4 Nuclear DUX4-induced pathologies are dependent on HA.

    (A) MB135-DUX4i myoblasts were treated with the indicated compounds for 24 hours and stained with antibodies against dsRNA and FUS and with Hoechst dye. (B) Myoblasts were transfected with 0.25 μg of poly(I:C) followed by exposure to the indicated compounds for 13 hours and were then stained with dsRNA antibodies and Hoechst dye. Experiments were performed four times. (C) The fraction of nuclei containing at least one dsRNA focus was quantified. Significance was determined using Welch’s t tests with multiple hypothesis correction using the Bonferroni-Dunn method. n = 3. (D) The fraction of dsRNA foci colocalizing with FUS aggregates was quantified. Values from individual experiments are indicated with dots, and bars indicate the mean of experiments ± SDs. A total of 718 uninduced, 650 induced, 859 induced, 4MU-treated, and 696 induced, ethanol-treated cells were counted among three independent experiments. A total of 290 and 188 dsRNA foci were counted in induced and induced, ethanol-treated cells, respectively. Scale bar, 35 μm. Inset: 75 μm. *P < 0.05.

  • Fig. 5 dsDNA breaks and dsRNA foci appear in response to DUX4.

    (A) Myoblasts were treated as indicated for 24 hours and stained with dsRNA and γH2A.X antibodies and Hoechst dye. Arrows indicate examples of overlapping staining. (B and C) dsRNA/γH2A.X colocalization was quantitated as in Fig. 3 (C and D). A total of 786 uninduced, 700 induced, 677 induced, 4MU-treated, and 821 induced, ethanol-treated cells were counted among three independent experiments. A total of 381 and 243 dsRNA foci were counted in induced and induced, ethanol-treated cells, respectively. No dsRNA foci were detected in uninduced or induced, 4MU-treated cells. (D and E) MB135-DUX4i myoblasts were treated with 500 μM H2O2 or 20 μM etoposide for 1 hour and stained as in (A). (F) Myoblasts were left uninduced, induced for 24 hours or induced and treated with either 1 mM 4MU or 1% EtOH for 24 hours, fixed, labeled with fluorescein isothiocyanate–conjugated control or γH2A.X antibodies, and analyzed by flow cytometry. All experiments were performed at least three times. For (B) and (F), values from individual experiments are indicated with dots, and means ± SEM are indicated with bars. For (C), means and SDs are shown. Significance was determined using Welch’s t tests. n = 3. All experiments were performed at least three times. Scale bar, 35 μm. Inset: 75 μm. *P < 0.05 and **P < 0.01.

  • Fig. 6 Effects of HA depletion on DUX4 transcriptional Activity.

    Uninduced or induced MB135-DUX4i myoblasts were treated with 2 mM 4MU or 2% DMSO for 24 hours, and the expression of C1QBP and seven validated DUX4 target genes was measured by RT-PCR. Values from six independent experiments are indicated with dots and boxes. Means ± SEM are indicated with bars but are concealed by the individual data points for many samples. Significance was determined using t tests and multiple hypothesis correction was performed with the Bonferroni-Dunn method. n = 6. ***P < 0.001 and ****P ≤ 0.0001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/12/eaaw7099/DC1

    Fig. S1. DOX does not cause pathology in parental myoblasts.

    Fig. S2. Inhibition of caspase-3/7 or ROS does not prevent C1QBP mislocalization.

    Fig. S3. Compounds can provide resistance to DUX4-induced toxicity.

    Fig. S4. DOX does not cause nuclear pathologies in parental myoblasts.

    Fig. S5. FUS aggregates localize to sites of dsDNA breaks and are inhibited by 4MU.

    Fig. S6. DNA damage does not cause accumulation of dsRNA after 24 hours and depends on 4MU.

    Fig. S7. βME does not prevent DUX4-induced appearance of dsRNA foci.

    Table S1. Cells used in this study.

    Table S2. Primers used in this study.

    Data S1. Identification of DUX4 copurifying proteins by mass spectrometry.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. DOX does not cause pathology in parental myoblasts.
    • Fig. S2. Inhibition of caspase-3/7 or ROS does not prevent C1QBP mislocalization.
    • Fig. S3. Compounds can provide resistance to DUX4-induced toxicity.
    • Fig. S4. DOX does not cause nuclear pathologies in parental myoblasts.
    • Fig. S5. FUS aggregates localize to sites of dsDNA breaks and are inhibited by 4MU.
    • Fig. S6. DNA damage does not cause accumulation of dsRNA after 24 hours and depends on 4MU.
    • Fig. S7. βME does not prevent DUX4-induced appearance of dsRNA foci.
    • Table S1. Cells used in this study.
    • Table S2. Primers used in this study.
    • Legend for data S1

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    Other Supplementary Material for this manuscript includes the following:

    • Data S1 (Microsoft Excel format). Identification of DUX4 copurifying proteins by mass spectrometry.

    Files in this Data Supplement:

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