WDR5 is required for DUX4 expression and its pathological effects in FSHD muscular dystrophy

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.

Figure 1.

A reporter system to study DBE-T activity in mammalian cells. (A) Schematic representation (not in scale) of the Gal4-λN-BoxB targeting system to study DBE-T ability to regulate transcription in which the firefly Luciferase reporter gene is under the control of five copies of the sequence recognized by the yeast transcription factor GAL4 (5X GAL4-UAS). The GAL4 DNA binding domain fused to the λN RNA-binding domain that specifically recognizes the BoxB sequence fused with DBE-T is shown. Figure created with BioRender.com. The RNA structure is based only on BioRender templates. (B) Luciferase activity measured in CHO cells transfected with Gal4-λN and 5X GAL4-UAS-Luciferase in combination with empty vector (EV), BoxB-LacZ or BoxB-DBE-T. Luciferase activity relative to EV control is shown. N = 3. Student's t-test. ***P< 0.001. (C) Top. Schematic representation (not in scale) of the FSHD locus (see Supplementary Figure S1A for details). Bottom. Enlargement showing the DBE-T portion and its non-redundant fragments used for the luciferase assays (see Materials and Methods for details). (D) Luciferase activity in CHO cells transfected with the Gal4-λN-BoxB targeting system in combination with BoxB-fused to LacZ, the indicated DBE-T fragments or DBE-T. Luciferase activity relative to BoxB-LacZ control is shown. N = 3 Student's t-test. ***P< 0.001. (E) Luciferase activity in CHO cells transfected with the Gal4-λN-BoxB targeting system in combination with empty vector (EV) or BoxB-fused to LacZ, DBE-T, DBE-T deleted of Frag.3 (Δ3) or DBE-T deleted of Frag.5 (Δ5). Luciferase activity relative to EV control is shown. N = 3. Student's t-test. **P< 0.01, ***P< 0.001. (F) Luciferase activity in CHO cells transfected with the Gal4-λN-BoxB targeting system in combination with empty vector (EV) or BoxB-fused to DBE-T, DBE-T Frag.3 or the antisense of DBE-T Frag.3 (AS). Luciferase activity relative to EV control is shown. N = 3. Student's t-test. **P< 0.01.

Figure 2.

Identification of DBE-T Frag.3 interactors. (A) Schematic representation (not in scale) of the RNA-targeting systems coupled with proximity-dependent biotinylation. The fusion protein BirA*-Csy4* biotinylates proteins associated to the RNA fused to the Csy4 RNA-tag. Then, biotinylated proteins are isolated and identified by mass spectrometry. (B) Schematic representation of the Csy4-DBE-T Frag.3 specifically associated proteins. (C) RNA immunoprecipitation following UV crosslinking (UV-RIP) of CHO cells transfected with BoxB-LacZ or BoxB-DBE-T using anti-WDR5 antibodies or IgG (control). The extracted RNA was processed for RT-qPCR showing the enrichment of DBE-T Frag.3 over the control LacZ RNA. N = 3. (D) In vitro pull-down experiments using purified, recombinant GST-WDR5 and in vitro transcribed DBE-T Frag.3 showing the direct interaction between GST-WDR5 and DBE-T Frag.3 and the significant preference for interacting with DBE-T Frag.3 compared to its antisense control. N = 4. Student's t-test. *P< 0.05. (E) WDR5 depletion caused a significant decrease in reporter gene activation by DBE-T. N = 4. Student's t-test. ***P< 0.001. (F) Gal4-λN-BoxB reporter assay showing WDR5 overexpression significantly boosted reporter gene activation by DBE-T and a significant reduction using a DBE-T version deleted of Frag.3, while a DBE-T mutant lacking Frag.5 is equally stimulated by WDR5 overexpression. N = 6. Student's t-test. **P< 0.01, ***P< 0.0001. (G) Gal4-λN-BoxB reporter assay showing WDR5 can specifically stimulate gene activation by DBE-T Frag.3. N = 5. Student's t-test. *P< 0.05, **P< 0.01, ***P< 0.001. (H) ChIP-qPCR showing WDR5 enrichment at the FSHD locus that is reduced upon DBE-T knockdown while WDR5 enrichment at the control locus GAPDH was unaffected by DBE-T knockdown. N = 3.

Figure 3.

Effects of WDR5 silencing with siRNA. (A) RT-qPCR for the indicated genes performed on RNA extracted from primary FSHD muscle cells treated with control (siNT) or WDR5 (siWDR5) siRNAs. N = 4. Student's t-test. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001. (B) Representative images of Myosin Heavy-chain (MHC, green) and Nuclei (DAPI, blue) staining performed on primary FSHD muscle cells treated with control (siNT), WDR5 (siWDR5) or DUX4 (siDUX4) siRNAs. (C) Quantification of differentiation index, fusion index and nuclei distribution in primary FSHD muscle cells treated with control (siNT), WDR5 (siWDR5) or DUX4 (siDUX4) siRNAs. No statistical difference between siWDR5 and siDUX4 treated cells was observed. N = 3. One-way ANOVA. **P< 0.01, ***P< 0.001. (D) Representative images obtained from the IncuCyte S3 Imager system monitoring live caspase 3/7 apoptosis. Images were acquired at 10× magnification in bright field. The apoptotic cells (green) were acquired with 300 ms exposure. (E) Analysis of the live-cell, real-time, caspase 3/7 apoptosis assays on primary FSHD muscle cells treated with siWDR5, siDUX4 or siNT (control). N = 3. One-way ANOVA. ^####P< 0.0001, ****P< 0.0001. (F) Area under the curve (AUC) quantification. N = 3. Student's t-test. ^###P< 0.001, ****P< 0.0001.

Figure 4.

Effects of WDR5 pharmacological inhibition with OICR-9429. (A) ChIP-qPCR showing WDR5 enrichment at the FSHD locus that is significantly reduced upon WDR5 pharmacological inhibition with OICR9429. (B) RT-qPCR for the indicated genes performed on RNA extracted from primary FSHD muscle cells treated with OICR-9429 or DMSO (as control). N = 4. Student's t-test, **P< 0.01, ****P< 0.0001. (C) Representative images of Myosin Heavy-chain (MHC, green) and Nuclei (DAPI, blue) staining performed on primary FSHD muscle cells treated with DMSO (control) or OICR-9429. (D) Quantification of differentiation index, fusion index and nuclei distribution in primary FSHD muscle cells treated with DMSO (control) or OICR-9429. N = 3. Student's t-test. *P< 0.05. (E) Representative images obtained from the IncuCyte S3 Imager system monitoring live caspase 3/7 apoptosis. Images were acquired at 10× magnification in brighfield. The apoptotic cells (green) were acquired with 300ms exposure. (F) Analysis of the live-cell, real-time, caspase 3/7 apoptosis on primary FSHD muscle cells treated with OICR-9429 or DMSO, as control. The analysis was performed with the IncuCyte S3 software. N = 4. One-way ANOVA. ****P< 0.0001. (G) Area under the curve (AUC) quantification. N = 4. Student's t-test. *P< 0.05.

Figure 5.

Pharmacological WDR5 targeting restores DUX4-mediated gene alterations. (A) GSEA (Gene set enrichment analysis) performed on the DUX4-associated gene signature. Only in primary FSHD muscle cells treated with OICR-94289, there is a significant overall increase of the genes that have been reported to be downregulated upon DUX4 expression and, vice versa, a global and significant downregulation of the genes that have been reported upregulated when DUX4 is expressed. NES: normalized enrichment score. (B) Hallmark gene sets analysis showing the pathways that are significantly up- or down-regulated selectively in primary FSHD muscle cells after treatment with OICR-9429. (C) Gene Ontology analysis performed on genes commonly altered in FSHD and healthy donor primary muscle cells treated with OICR-9429. N = 6. Only the top 5 GO terms (ranked by P-value) are shown. Significance shown in the figures.