Pask integrates hormonal signaling with histone modification via Wdr5 phosphorylation to drive myogenesis
- University of Utah School of Medicine, United States
- Howard Hughes Medical Institute, University of Utah School of Medicine, United States
Figures
Figure 1 with 4 supplements
Pask is required for skeletal muscle regeneration after acute muscle injury.
(A) Differentiation of C2C12 myoblasts was assessed after Pask was either knocked down using pooled siRNA or inhibited using 25 µM BioE-1197. Control cells were transfected with pooled non-targeting …
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Figure 1—source data 1
Numerical values from graphs represennted in Figure 1.
- https://doi.org/10.7554/eLife.17985.003
Figure 1—figure supplement 1
Pask is required for adipogenesis of mesenchymal stem cell.
(A) C3H10T1/2 cells were treated with either DMSO or 25µM BioE-1197 48 hr prior to induction of adipocyte differentiation. Adipocyte differentiation was induced as described in Experimental …
Figure 1—figure supplement 2
Pask is enriched in stem cells and is required for terminal differentiation of neuronal, adipogenic and myogenic cell types.
(A) Comparison of Pask mRNA expression profile from GeneAtlas MOE430 dataset obtained from BioGPS (B) Induction of Pask mRNA expression during iPSC formation from indicated differentiated cell types …
Figure 1—figure supplement 3
Pask is required for differentiation of ES cells but not for iPS reprograming.
(A) Mouse embryonic fibroblasts (MEFs) containing an IRES-GFP cassette expressed from the Oct4 locus were induced for reprogramming by mSTEMCCA factors as described in Materials and methods in the …
Figure 1—figure supplement 4
Genetic and pharmacological inhibition of Pask suppresses myogenesis of mouse and human myoblasts.
(A) MHC staining of C2C12 cells with Cas9 guided deletion of Pask during differentiation. (B) Fusion index calculation from Figure (A). (B) Western blot analysis MHC expression during …
Figure 2 with 3 supplements
Pask is required for transcriptional activation of MyoG in response to differentiation cues.
(A) Schematic of myogenesis from satellite cells that depicts the progression of transcription factors during myogenesis. (B) qRT-PCR analysis of WT and Pask-/- satellite cells prior to (day 0) or …
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Figure 2—source data 1
Numerical data from qPCR analysis off WT vs Pask-/- satellite cells from the graph represented in Figure 2B and quantification of Pax7+ and MyoG+ cell numbers.
- https://doi.org/10.7554/eLife.17985.009
Figure 2—figure supplement 1
Pask is required for transcriptional activation of MyoG in response to differentiation cues.
(A) Western blot analysis of abundance of the indicated proteins during myoblast differentiation in control or Pask-silenced C2C12 cells using 2% Horse Serum media. (B) qRT-PCR analysis of DMSO or …
Figure 2—figure supplement 2
Pask does not regulate MyoD+ cell population.
(A) Immunofluorescence microscopy showing MyoD expression and localization in control, Pask-siRNA or 25 µM BioE-1197 treated cells on Day 0 of differentiation. (B) Quantification of MyoD+ cell …
Figure 2—figure supplement 3
Pask promoter is occupied by MyoG and MyoD during differentiation.
(A) ChIP-Seq data from the ENCODE dataset for H3K4me3 levels, MyoG, MyoD and POL II binding at the indicated time-points at the Pask promoter during C2C12 differentiation. The vertical turquois line …
Figure 3 with 1 supplement
Pask is required for myogenic conversion of C3H10T1/2 cells by MyoD.
(A) Schematic depiction of the mechanism by which MyoD-induces the myogenic conversion of adipogenic C3H10T1/2 cells. (B) qRT-PCR analysis of the indicated mRNAs in C3H10T1/2 cells expressing MyoD …
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Figure 3—source data 1
Numerical values from qPCR analysis in Figure 3A and quantification of MyoD+ and MyoG+ cell numbers from Figure 3D.
- https://doi.org/10.7554/eLife.17985.014
Figure 3—figure supplement 1
Pask is required for myogenic conversion of mesenchymal stem cells by MyoD.
(A) Immunofluorescence of MyoD expression in DMSO or BioE-1197 treated samples at Day 1 and Day 2. See main text Figure 4D for quantification. (B) Immunofluorescence of MyoG expression in DMSO or …
Figure 4 with 1 supplement
Pask directly interacts with and phosphorylates Wdr5 at Ser49.
(A) Endogenous Pask was immunoprecipitated from C2C12 cells, either before (day -1, 0) or after induction of differentiation (Day 1). Immunoprecipitates were analyzed by western blot for Pask and …
Figure 4—figure supplement 1
Pask directly interacts with and phosphorylates Wdr5 at Ser49.
(A) Flag-tagged YFP or indicated members of various protein complexes of which Wdr5 is a member were expressed in 293T cells. Flag tagged proteins were immunoprecipitated using anti-Flag antibody. …
Figure 5 with 1 supplement
The phospho-mimetic S49E Wdr5 mutant rescues myogenesis in Pask-silenced cells.
(A) GFP or WT, S49A or S49E Wdr5 were retrovirally expressed in Pask-siRNA C2C12 cells. qRT-PCR analysis was performed for the indicated mRNA on day 3 of differentiation. 18S rRNA was used as …
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Figure 5—source data 1
Quantification of the Pax7+ and MyoG+ cell numbers and the fusion index in Wdr5S49E expressing cells.
- https://doi.org/10.7554/eLife.17985.019
Figure 5—figure supplement 1
Wdr5S49E expression rescues genetic loss of Pask.
C2C12 myoblasts were transduced with retrovirus carrying WT, S49A or S49E mutants of Wdr5 and were selected with 3µg/ml puromycin for 48 hr. Pask (or control) was knocked down at 70% cell density in …
Figure 6
Pask is required for recruitment of Wdr5 and MyoD to the Myog promoter during differentiation.
(A) A depiction of the Myog genomic locus depicting MyoD and RNAPolII occupancy as well as H3K4me2 and H3K4me3 abundance at 60 hr of differentiation from the ENCODE dataset for the C2C12 cell line. T…
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Figure 6—source data 1
Numerical values from the ChIP analysis represented in Figure 6.
- https://doi.org/10.7554/eLife.17985.022
Figure 7 with 1 supplement
Differentiation induced H3K4me1 to H3K4me3 conversion is dependent upon Pask phosphorylation of Wdr5.
(A) H3K4me1 and (B) total H3 ChIP were performed from control or Pask-siRNA C2C12 cells at the indicated day of differentiation and fold enrichment on the Myog promoter was determined by qRT-PCR …
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Figure 7—source data 1
Numerical values from the ChIP analysis represented in Figure 7.
- https://doi.org/10.7554/eLife.17985.024
Figure 7—figure supplement 1
Pask and phosphomimetic Wdr5 promote H3K4me1 to H3K4me3 conversion and MyoD recruitment to the Myog promoter.
(A) Depiction of the Myog locus showing abundance of position of enhancer and promoter region as well as H3K4me3 levels and MyoD occupancy 60 hr after initiation of differentiation from ENCODE …
Author response image 1
Wdr5 silencing affects proliferation rate in C2C12 myoblasts.
https://doi.org/10.7554/eLife.17985.026
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Wdr5 silencing suppresses myotube formation in C2C12 myoblasts.<Author response image 2>
https://doi.org/10.7554/eLife.17985.027
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Wdr5 is required expression of myogenin (MyoG) and myosin (MHC) during differentiation in C2C12 myoblasts.
https://doi.org/10.7554/eLife.17985.028
Author response image 4
C2C12 cells were infected with retrovirus expressing GFP (Control) or WT, S49A or S49E‐Wdr5 cDNAs. 24 hrs after infection, puromycin selection was performed for four days.
1000 cells from each samples were plated into 96 well plates and after two days, total cell number was counted for each cell types.
Author response image 5
H3K4me3 ChIP was performed from C2C12 myoblasts after control or MyoD knockdown expressing WT, S49A or S49E WDR5 mutants at Day 0 and Day 1 of differentiation.
* P<0.05 between Wdr5S49E vs. Wdr5WT at Day 0 in control samples.
Author response image 6
