Research ArticleHUMAN GENETICS

A novel P300 inhibitor reverses DUX4-mediated global histone H3 hyperacetylation, target gene expression, and cell death

See allHide authors and affiliations

Science Advances  11 Sep 2019:
Vol. 5, no. 9, eaaw7781
DOI: 10.1126/sciadv.aaw7781
  • Fig. 1 iP300w diminishes DUX4-induced toxicity.

    (A) Chemical structure of iP300w. (B) Cell viability assay [adenosine triphosphate (ATP) assay] on LHCN-iDUX4 myoblasts at 48 hours. Red series (Dox-continual): Cells were continually exposed to dox (200 ng/ml) for the full 48 hours and treated with serial dilutions of iP300w. Blue series (Dox-2-hr. pulse): Cells were induced for 2 hours with dox (200 ng/ml) and then washed, and medium was replaced with dox-free medium containing different concentrations of iP300w. Green series (Control): Noninduced LHCN-iDUX4 cells were treated only with different concentrations of iP300w. Data are presented as fold difference compared to the control untreated cells (n = 8). (C) Morphology of LHCN-iDUX4 cells after 48 hours with continual dox (200 ng/ml) induction and treatment with iP300w (1 μM). (D) ATP assay on iC2C12-DUX4 at 48 hours of induction with dox (500 ng/ml) and treatment with various concentrations of iP300w, as in (B). (E) Morphology of iC2C12-DUX4 cells at 48 hours of continual dox (500 ng/ml) induction and iP300w (1 μM) treatment. Concentrations of dox for both cell lines were determined to provide rapid cell death within 48 hours of induction.

  • Fig. 2 iP300w disables transcription of DUX4-induced target genes.

    (A) Western blot for DUX4 and RT-qPCR for DUX4 target genes in LHCN-iDUX4 cells pulse induced for 2 hours with dox (200 ng/ml) and then treated with 0.1 μM iP300w. Protein and RNA samples were harvested at 1, 2, 4, and 8 hours of treatment with iP300w. Data represent means ± SEM; ****P < 0.0001, **P < 0.01, and *P < 0.05 by two-way analysis of variance (ANOVA) with Tukey’s post hoc test. Results are presented as fold difference compared to GAPDH (n = 3). (B) RNA-seq in LHCN-iDUX4 cells induced for 12 hours with dox (200 ng/ml) and treated with 0.25 μM iP300w. Left: Total gene expression given as log2 fold change of dox-treated versus control cells on the x axis and iP300w + dox–treated cells versus controls on the y axis. Middle: Same analysis showing only presumed DUX4 direct targets, defined as at least eight-fold up-regulated by 6 hours (from the Choi et al., 2016 dataset) and with a DUX4 chromatin immunoprecipitation sequencing (ChIP-seq) peak within 10 kb of the transcription start site. Right: Effect of iP300w on all strongly up-regulated DUX4 target genes (defined as all genes up-regulated by dox at least eight-fold in the current 12-hour dataset), organized by magnitude of the iP300w effect. The great majority of these are down-regulated by iP300w. In all panels, color indicates the level of gene expression. (C) RT-qPCR for DUX4 and DUX4 target genes in myotubes from three different FSHD myoblast clonal cell lines (2, A2, and 12) after 12 hours of treatment with 0.25 μM iP300w (n = 9). ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05 by two-way analysis of variance (ANOVA) with Tukey’s post hoc test.

  • Fig. 3 Effect of iP300w on DUX4-induced alterations in vivo.

    (A) Muscle mass of tibialis anterior (TA), quadriceps, gastrocnemius and soleus, and pectoralis, normalized to the body weight (BW) at day 12. iDUX4pA;HSA female mice were daily injected with dox (5 mg/kg, intraperitoneally) with or without iP300w (0.3 mg/kg) (n = 8). Wild-type (WT) siblings were used as a control (n = 4). (B) Representative FACS analyses for PDGFRα in pooled sample from quadriceps, gastrocnemius and soleus, pectoralis, and triceps at day 12. (C) Quantification of FACS analyses presented in (B). Data are presented as means ± SEM; *P < 0.05 and ***P < 0.001 by one-way ANOVA with Tukey’s post hoc test (n = 3). (D) RT-qPCR on RNA isolated from gastrocnemius at day 12. Note suppression of DUX4 target genes in the iP300w-treated group and reduction of expression of markers related to fibrosis. Data are presented as means ± SEM; ****P < 0.001 by one-way ANOVA with Tukey’s post hoc test. Results are presented as relative expression to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (n = 3).

  • Fig. 4 DUX4 induces acetylation on H3 that is reversible by iP300w.

    (A) Western blot analyses for acetylation of H3K27, H3K18, and total lysine in LHCN-iDUX4 cells induced for 12 hours with various concentrations of dox. Note that increased acetylation appears at levels of dox at which DUX4 is still not detectable. (B) Western blot analyses for the markers of H3 acetylation in LHCN-iDUX4ΔC cells continually induced with dox (200 ng/ml) and treated with 0.25 μM iP300w for 12 hours. DUX4ΔC is the C-terminal deletion construct of DUX4. (C) Western blot for acetylation and methylation of H3 and H4 in LHCN-iDUX4 cells that were continually induced with dox (20 and 200 ng/ml) for 12 hours. Cells were continually treated with 0.25 μM iP300w. Acetylation in LHCN cells was evaluated as an additional control.

  • Fig. 5 mDUX induces H3 acetylation that is p300 dependent.

    (A) Western blot analyses for H3K18 and H3K27 acetylation in LHCN-imDux and iC2C12-imDux cells continually induced with dox (200 ng/ml) and treated with 0.25 μM iP300w for 12 hours. (B) RT-qPCR for mDux target genes in LHCN-mDux after 12-hour induction with dox (200 ng/ml) and 0.25 μM iP300w. Data are presented as means ± SEM; ****P < 0.001 by one-way ANOVA with Tukey’s post hoc test. Results are presented as relative expression to GAPDH (n = 3). (C) RT-qPCR for mDux target genes in iC2C12-mDux cells after 12-hour induction with dox (200 ng/ml) and 0.25 μM iP300w (n = 3).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaaw7781/DC1

    Fig. S1. iP300w protects 293T cells from DUX4-induced toxicity.

    Fig. S2. Doxycycline effect on morphology and viability of parent cell lines.

    Fig. S3. iP300w inactivates induction of DUX4 target genes.

    Fig. S4. iP300w inactivates induction of DUX4 target genes in different cell types.

    Fig. S5. Dose- and time-dependent DUX4 inactivation by iP300w in FSHD myoblasts.

    Fig. S6. Effect of doxycycline on profibrotic gene expression in wild-type mice.

    Fig. S7. Low levels of DUX4 induce H3 acetylation through p300 in human myoblasts.

    Fig. S8. DUX4 induces H3 acetylation in nonmyogenic cells.

    Fig. S9. Low levels of DUX4 induce H3 acetylation through p300 in mouse myoblasts.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. iP300w protects 293T cells from DUX4-induced toxicity.
    • Fig. S2. Doxycycline effect on morphology and viability of parent cell lines.
    • Fig. S3. iP300w inactivates induction of DUX4 target genes.
    • Fig. S4. iP300w inactivates induction of DUX4 target genes in different cell types.
    • Fig. S5. Dose- and time-dependent DUX4 inactivation by iP300w in FSHD myoblasts.
    • Fig. S6. Effect of doxycycline on profibrotic gene expression in wild-type mice.
    • Fig. S7. Low levels of DUX4 induce H3 acetylation through p300 in human myoblasts.
    • Fig. S8. DUX4 induces H3 acetylation in nonmyogenic cells.
    • Fig. S9. Low levels of DUX4 induce H3 acetylation through p300 in mouse myoblasts.

    Download PDF

    Files in this Data Supplement:

Navigate This Article