Pask integrates hormonal signaling with histone modification via Wdr5 phosphorylation to drive myogenesis

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1) The authors claim that PASK is dispensable for iPSC reprograming but necessary for differentiation. Several prior studies have suggested that WDR5 is important for self-renewal, proliferation and reprogramming through promotion of H3K4me3, and that loss of WDR5 function leads to activation of genes that promote differentiation (Ang et al. Cell 2011, Yang et al. eLife 2014, Wang et al. Nature 2011). This issue should at least be addressed in the Discussion.

This is an interesting and very important question that we also probed in a limited way during our studies. In C2C12 myoblasts, Wdr5 silencing resulted in stunted cell proliferation (Author response image 1). When myoblasts were induced to differentiate, control myoblasts efficiently differentiated, however, Wdr5 silenced myoblasts failed to form multi-nucleated myofibers (Author response image 2)and induce myogenin and MHC- two markers of differentiation we have used in this manuscript (Author response image 3). More important for this question, we did not see any myogenin or MHC expression at Day 0 (in proliferative condition) when Wdr5 was silent. Thus, at least in myoblasts in our hands, Wdr5 knockdown is not sufficient to cause exit from self-renewal and differentiation. Our data show that Wdr5 is required for both the proliferative phase and differentiation process in myoblasts. This is not surprising since Wdr5 is an essential intermediate in many transcriptional events during both proliferation and differentiation states. For example, in myoblasts, Wdr5 has been shown to associate with Pax7 and regulate myoblast proliferation. During differentiation, MyoD and other transcription factors are known to recruit MLL, KAT2A-B and Set complexes of which Wdr5 is a core member to the Myog promoter to promote differentiation. We have included the data here, but have not included it in the manuscript per se, because it seemed a bit tangential to the logic of the already complex story. At the discretion of the editor and reviewers, we could include it in the paper.

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2) Are there any effects on proliferation rate due to loss of WDR5 phosphorlyation under proliferative culture conditions?

We do not see any effect on proliferation rate upon either PASK inhibition (Manuscript Figure 1—figure supplement 4F) or Wdr5 S49A expression in myoblasts (Author response image 4).

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3) PASK knockdown had no effect on H3K4me3 signature at the MyoD promoter (or expression of MyoD) although it did influence H3K4me3 and MyoD occupancy at MyoG promoter to drive the expression of MyoG in response to differentiation cues. While MyoD is required for the driving myogenesis, is it specifically necessary for PASK dependent changes in trimethylation of H3K4 at the MyoG promoter? In other word.s given the multiple positive reinforcing feedback loops in the proposed model, could it be that MyoD acts cooperatively with WDR5 to change chromatin accessibility? The hierarchical relationship between these factors can be clarified. For example, are there differences in the observed changes in chromatin signatures with pask inhibition or deletion upon MyoD knockdown?

This is a very interesting mechanistic question. While the interplay between MyoD and pWdr5 is an important and complex area, we have tried to address this question partially here. MyoD is extensively involved in remodeling of the Myog promoter at the onset of differentiation by recruiting MLL complexes, HATs and SWI/SNF complexes. MyoD serves a pioneering function at the Myog promoter to activate its transcription in response to differentiation signaling (Cao et al., 2006). While MyoD occupies Myog promoter during the proliferative phase, signaling inputs are required to stimulate chromatin remodeling and activation of MyoG transcription (Cao et al., 2006). On the other hand, inhibition of MyoD aborts the differentiation process. Thus, we hypothesize that MyoD acts synergistically with signaling inputs such as those that activate the PASK-Wdr5 pathway to remodel Myog promoter. We tested this by examining levels of H3K4me3 on the Myog promoter in control or Myod1- silenced C2C12 before and after the onset of differentiation. This was done in cells expressing WT, S49A and S49E Wdr5 as a surrogate for activation or inactivation of PASK signaling (Author response image 5). We observed that knockdown of Myod1 partially prevented H3K4me3 induction on the Myog promoter in all samples, including Wdr5_S49E. We also examined the effect of Myod1 knockdown on differentiation in Wdr5 WT, S49A or S49E cells. Again, we noticed that in non-targeting siRNA (Control) samples, Wdr5_S49E expressing cells made robust myofibers and Wdr5_S49A expressing cells were defective in myotube formation (Author response image 6). Myod1 knockdown, however, significantly suppressed myotube formation in Wdr5_S49E expressing cells. Hence, we think that Wdr5 phosphorylation is not sufficient to overcome the loss of MyoD function and that MyoD acts synergistically with phosphorylated WDR5 to induce epigenetic changes at the Myog promoter.

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