Multiple repeat regions within mouse DUX recruit chromatin regulators to facilitate an embryonic gene expression program

Reviewer #1 (Public Review):

In this manuscript, the authors identified and characterized the five C-terminus repeats and a 14aa acidic tail of the mouse Dux protein. They found that repeat 3&5, but not other repeats, contribute to transcriptional activation when combined with the 14aa tail. Importantly, they were able to narrow done to a 6 aa region that can distinguish "active" repeats from "inactive" repeats. Using proximal labeling proteomics, the authors identified candidate proteins that are implicated in Dux-mediated gene activation. They were able to showcase that the C-terminal repeat 3 binds to some proteins, including Smarcc1, a component of SWI/SNF (BAF) complex. In addition, by overexpressing different Dux variants, the authors characterized how repeats in different combinations, with or without the 14aa tail, contribute to Dux binding, H3K9ac, chromatin accessibility, and transcription. In general, the data is of high quality and convincing. The identification of the functionally important two C-terminal repeats and the 6 aa tail is enlightening. The work shined light on the mechanism of Dux function.

Reviewer #2 (Public Review):

In this manuscript, Smith et al. delineated novel mechanistic insights into the structure-function relationships of the C-terminal repeat domains within the mouse DUX protein. Specifically, they identified and characterised the transcriptionally active repeat domains, and narrowed down to a critical 6aa region that is required for interacting with key transcription and chromatin regulators. The authors further showed how the DUX active repeats collaborate with the C-terminal acidic tail to facilitate chromatin opening and transcriptional activation at DUX genomic targets.

Reviewer #3 (Public Review):

Dux (or DUX4 in human) is a master transcription factor regulating early embryonic gene activation and has garnered much attention also for its involvement in reprogramming pluripotent embryonic stem cells to totipotent "2C-like" cells. The presented work starts with the recognition that DUX contains five conserved c. 100-amino acid carboxy-terminal repeats (called C1-C5) in the murine protein but not in that of other mammals (e.g. human DUX4). Using state-of-the-art techniques and cell models (BioID, Cut&Tag; rescue experiments and functional reporter assays in ESCs), the authors dissect the activity of each repeat, concluding that repeats C3 and C5 possess the strongest transactivation potential in synergy with a short C-terminal 14 AA acidic motif. In agreement with these findings, the authors find that full-length and active (C3) repeat containing Dux leads to increased chromatin accessibility and active histone mark (H3K9Ac) signals at genomic Dux binding sites. A further significant conclusion of this mutational analysis is the proposal that the weakly activating repeats C2 and C4 may function as attenuators of C3+C5-driven activity.

By next pulling down and identifying proteins bound to Dux (or its repeat-deleted derivatives) using BioID-LC/MS/MS, the authors find a significant number of interactors, notably chromatin remodellers (SMARCC1), a histone chaperone (CHAF1A/p150) and transcription factors previously (ZSCAN4D) implicated in embryonic gene activation.

The experiments are of high quality, with appropriate controls, and thus provide a rich compendium of Dux interactors for future study. Indeed, a number of these (SMARCC1, SMCHD1, ZSCAN4) make biological sense, both for embryonic genome activation and for FSHD (SMCHD1).

The central question raised by this study, however, concerns the function of the Dux repeats, apparently unique to mice. While it is possible, as the authors propose, that the weak activating C1, C2 C4 repeats may exert an attenuating function ("sub-functionalization") on activation mediated by C3 and/or C5, it could similarly be argued that the different repeats are indeed expected to display different activation potentials, chromatin opening, cofactor recruitment, due to, simply, the differences in their sequences. The argument for an active attenuating function would have been strengthened, for example, by the finding of repressor recruitment by C1/C2/C4 (and not just less of everything). The possible biological relevance of these repeats thus remains to be established.

Author Response

The following is the authors’ response to the original reviews.

First, the authors would like to thank the reviewers and editors for their thoughtful comments. The comments were used to guide our revision, which is substantially improved over our initial submission. We have addressed all comments in our responses below, through a combination of clarification, new analyses and new experimental data.

Reviewer #1 (Public Review):

In this manuscript, the authors identified and characterized the five C-terminus repeats and a 14aa acidic tail of the mouse Dux protein. They found that repeat 3&5, but not other repeats, contribute to transcriptional activation when combined with the 14aa tail. Importantly, they were able to narrow done to a 6 aa region that can distinguish "active" repeats from "inactive" repeats. Using proximal labeling proteomics, the authors identified candidate proteins that are implicated in Dux-mediated gene activation. They were able to showcase that the C-terminal repeat 3 binds to some proteins, including Smarcc1, a component of SWI/SNF (BAF) complex. In addition, by overexpressing different Dux variants, the authors characterized how repeats in different combinations, with or without the 14aa tail, contribute to Dux binding, H3K9ac, chromatin accessibility, and transcription. In general, the data is of high quality and convincing. The identification of the functionally important two C-terminal repeats and the 6 aa tail is enlightening. The work shined light on the mechanism of DUX function.

A few major comments that the authors may want to address to further improve the work:

We thank the reviewer for their efforts and constructive comments, which have guided our revisions.

1. The summary table for the Dux domain construct characteristics in Fig. 6a could be more accurate. For example, C3+14 clearly showed moderate weaker Dux binding and H3K9ac enrichment in Fig 3c and 3e. However, this is not illustrated in Fig. 6a. The authors may consider applying statistical tests to more precisely determine how the different Dux constructs contribute to DNA binding (Fig. 3c), H3K9ac enrichment (Fig. 3e), Smarcc1 binding (Fig. 5e), and ATAC-seq signal (Fig. 5f).

We thank the reviewer for this comment, and agree that there were some modest differences in construct characteristics that were not captured in the Summary Table (6a). To better reflect the differences between constructs, we added additional dynamic range to our depiction/scoring, and believe that the new scoring system provides sufficient qualitative range to capture the difference without imposing a statistical approach.

1. Another concern is that exogenous overexpressed Dux was used throughout the experiments. The authors may consider validating some of the protein-protein interactions using spontaneous or induced 2CLCs (where Dux is expressed).

We agree that it would be helpful to determine endogenous DUX interaction with our BioID candidates. Here, we attempted co-IPs for endogenous DUX protein with the DUX antibody and were unsuccessful, which indicated that the DUX antibody is useful for detection but not efficient in the primary IP. This is why we utilized the mCherry tag for DUX IP experiments, which worked exceptionally well.

1. It could be technically challenging, but the authors may consider to validate Dux and Smarcc1 interaction in a biologically more relevant context such as mouse 2-cell embryos where both proteins are expressed. Whether Smarcc1 binding will be dramatically reduced at 4-cell embryos due to loss of Dux expression?

While we agree that it would be interesting to validate the in vivo interaction of DUX and SMARCC1 in the early embryo, it is not technically feasible for us to conduct the experiment, as the IP would require thousands of two-cell embryos, and we have the issue of poor co-IP quality with the DUX antibody.

Reviewer #2 (Public Review):

In this manuscript, Smith et al. delineated novel mechanistic insights into the structure-function relationships of the C-terminal repeat domains within the mouse DUX protein. Specifically, they identified and characterised the transcriptionally active repeat domains, and narrowed down to a critical 6aa region that is required for interacting with key transcription and chromatin regulators. The authors further showed how the DUX active repeats collaborate with the C-terminal acidic tail to facilitate chromatin opening and transcriptional activation at DUX genomic targets.

Although this study attempts to provide mechanistic insights into how DUX4 works, the authors will need to perform a number of additional experiments and controls to bolster their claims, as well as provide detailed analyses and clarifications.

We thank this reviewer for their constructive comments, and have conducted several new analyses, additional experiments and clarifications – which have strengthened the manuscript in several locations. Highlights include a statistical approach to the similarity of mouse repeats to themselves and to orthologs (Figure S1d) and clarified interpretations, a wider dynamic range to better reflect changes in DUX construct behaviors (Figure 6a), and additional data on construct behavior, including ‘inactive’ constructs (e.g C1+14aa in Figure 1a,d, new ATAC-seq in Figure S1g), and active constructs such as C3+C5+14aa and C3+C514aa (in Figure S1b).

Reviewer #3 (Public Review):

Dux (or DUX4 in human) is a master transcription factor regulating early embryonic gene activation and has garnered much attention also for its involvement in reprogramming pluripotent embryonic stem cells to totipotent "2C-like" cells. The presented work starts with the recognition that DUX contains five conserved c. 100-amino acid carboxy-terminal repeats (called C1-C5) in the murine protein but not in that of other mammals (e.g. human DUX4). Using state-of-the-art techniques and cell models (BioID, Cut&Tag; rescue experiments and functional reporter assays in ESCs), the authors dissect the activity of each repeat, concluding that repeats C3 and C5 possess the strongest transactivation potential in synergy with a short C-terminal 14 AA acidic motif. In agreement with these findings, the authors find that full-length and active (C3) repeat containing Dux leads to increased chromatin accessibility and active histone mark (H3K9Ac) signals at genomic Dux binding sites. A further significant conclusion of this mutational analysis is the proposal that the weakly activating repeats C2 and C4 may function as attenuators of C3+C5-driven activity.

By next pulling down and identifying proteins bound to Dux (or its repeat-deleted derivatives) using BioID-LC/MS/MS, the authors find a significant number of interactors, notably chromatin remodellers (SMARCC1), a histone chaperone (CHAF1A/p150) and transcription factors previously (ZSCAN4D) implicated in embryonic gene activation.

The experiments are of high quality, with appropriate controls, thus providing a rich compendium of Dux interactors for future study. Indeed, a number of these (SMARCC1, SMCHD1, ZSCAN4) make biological sense, both for embryonic genome activation and for FSHD (SMCHD1).

A critical question raised by this study, however, concerns the function of the Dux repeats, apparently unique to mice. While it is possible, as the authors propose, that the weak activating C1, C2 C4 repeats may exert an attenuating function on activation (and thus may have been selected for under an "adaptationist" paradigm), it is also possible that they are simply the result of Jacobian evolutionary bricolage (tinkering) that happens to work in mice. The finding that Dux itself is not essential, in fact appears to be redundant (or cooperates with) the OBOX4 factor, in addition to the absence of these repeats in the DUX protein of all other mammals (as pointed out by the authors), might indeed argue for the second, perhaps less attractive possibility.

In summary, while the present work provides a valuable resource for future study of Dux and its interactors, it fails, however, to tell a compelling story that could link the obtained data together.

We appreciated the reviewer’s views regarding the high quality of the work and our generation of an important dataset of DUX interactors. We also appreciate the comments provided to improve the work, and have performed and included in the revised version a set of clarifications, additional analyses and additional experiments that have served to reinforce our main points and provide additional mechanistic links. We also agree that more remains to be done to understand the function and evolution of repeats C1, C2 and C4.

Reviewer #1 (Recommendations For The Authors):

1. For immuno-blots, authors may indicate the expected bands to help readers better understand the results.

Agreed, and we have included the predicted molecular weight of proteins in the Figure Legends. We note that our work shows that the C-terminal domains confer anomalous migration in SDS-PAGE.

1. Fig. 5b, a blot missing for the mCherry group?

Figure 5b is a volcano blot, so we believe the reviewer is referring to Figure 5d, which is a coimmunoprecipitation experiment between SMARCC1 and mCherry-tagged DUX constructs. However, we are unsure of the comment as an anti mCherry sample is present in that panel.

1. Line 99-100, Fig. S1d, it seems that repeat2, but not repeat3, is more similar to human DUX4 C-terminal region.

This comment and one by another reviewer have prompted us to re-examine the similarities of the DUX repeats, and we have new analyses (Figure S1d) and an alternative framing in the manuscript as a result. We have expanded on this in our response to Reviewer #2, point #1 – and direct the reviewer there for our expanded treatment.

1. There are a few references are misplaced. For example, line 48, the studies that reported the role of Dux in inducing 2CLCs should be from Hendrickson et al., 2017, De Iaco et al., 2017, and Whiddon et al., 2017. The authors may want to double check all references.

Thanks for pointing these out. These issues have been corrected in the manuscript.

1. In the materials & methods section, a few potential errors are noticed. For example, concentrations of PD0325901 and CHIR99021 in mESC medium appear ~1000-fold higher than standards.

Thanks – corrected.

Reviewer #2 (Recommendations For The Authors):

Major Points

1. Line 99 - The authors claimed that the "human DUX4 C-terminal region is most similar to the 3rd repeat of mouse DUX", but based on Supp. Fig. 1d, the human DUX4 C-term should be most similar to the 2nd repeat of mouse DUX. If this is indeed the case, it will undermine the rest of this study, since the authors claim that the 3rd repeat is transcriptionally active, whereas the 2nd repeat is transcriptionally inactive, and the bulk of this study largely focused on how the active repeats, not the inactive repeats, are critical in recruiting key transcriptional and chromatin regulators to induce the embryonic gene expression program.

We thank the reviewer for their comments here. Since submission,and as mentioned above for reviewer #1 we have revisited the issue of similarity of the DUX4 C-terminal region to the mouse C-terminal repeats, with a BLAST-based approach that is more rigorous and informed by statistics – which is in Author response table 1 and now in the manuscript as Figure S1d, and has affected our interpretation. Our prior work involved a simple % identity comparison table and we now appreciate that some of the similarity analyses did not meet statistical significance, and therefore we are unable to draw certain conclusions. We make the appropriate modifications in the text. For example, we no longer state that the DUX4 C-terminus appears to be most similar to mouse repeats 3 and 5. This does not affect the main conclusions of the paper regarding interactions of the C-terminus with chromatin-related proteins, only our speculation on which repeat might have represented the original single repeat in the mouse – an issue we think of some interest, but did not rise to the level of mentioning in the original or current abstract.

Author response table 1.

Parameters: PAM250 matrix. Gap costs of existence: 15 and extension: 3. Numbers represent e-value of each pairwise comparison

*No significant similarities found (>0.05).

1. In Supp Fig 1d, it seems that the rat DUX4 C-terminal region is most similar to the 4th repeat of mouse DUX, which according to the author is supposedly transcriptionally inactive. This weakens the authors justification that the 3rd or 5th repeat is likely the "parental repeat for the other four", and further echoes my concern in point 1 where the human DUX4 C-term is most similar to the 2nd (inactive) repeat of mouse DUX.

The reviewer’s point is well taken and is addressed in point #1 above.

1. In Fig. 1d, the authors showed that DUX4-containing C3 and C5, but lacking acidic tail, can promote MERVL::GFP expression, albeit to a slightly lower extent compared to FL. However, in Fig. 2b, C3 or C5 alone (lacking acidic tail) completely failed to promote MERVL::GFP expression. However, in the presence of the acidic tail, both versions were able to promote MERVL::GFP expression, similar to that of FL. The latter would suggest that it is the acidic tail that is crucial for MERVL::GFP expression, and this does not quite agree with Fig 1b, where C12345 (lacking acidic tail) was able to promote MERVL::GFP expression. Although C12345 did not activate MERVL to a similar level as FL, it is clearly proficient, compared to C3 or C5 alone (lacking acidic tail) where there is no increase in MERVL at all. Additional constructs will be helpful to clarify these points. For example, 'C3+C5 minus acidic tail' and 'HD1+HD2+acidic tail only' constructs.

We agree that constructs such as those mentioned would add to the work. First, we have done the additional construct HD1+HD2+14aa tail, which is presented as ΔC12345+14aa in Figure 2a and in S2a. Additionally, we performed experiments on the requested C3+C5+14aa and C3+C5Δ14aa (see samples 6 and 7 in Author response image 1, which are now included in Supplemental Figure 2b). The results reinforce our hypothesis of an additive effect toward DUX target gene activation by increasing C-terminal repeats and including the 14aa tail.

Author response image1.

1. Related to the above, the flow cytometry data for the MERVL::GFP reporter as presented in Figures 1 and 2, as well as in Supp. Fig. 2, show a considerably large difference in the %GFP|mCherry for the FL construct, ranging from ~6-26%. This makes it difficult to convince the reader which of the different DUX domain constructs cannot or can partially induce GFP|mCherry signal when compared to FL, and hence it is tough to definitively ascertain the exact contribution of each of the 5 C-terminal repeats with high confidence, as it appears that there exists a significant amount of variability in this MERVL::GFP reporter system. The authors need to address this issue since this is their primary method to elucidate the transcriptional activity of each of the mouse DUX repeat domains.

We note that with the Dux-/- cell lines we used throughout the timeline of the study, the percent of %GFP|mCherry expression progressively and slowly decreased – possibly due to slow/modest epigenetic silencing of the reporter. However, we always used the full-length DUX construct to establish the dynamic range. We emphasize that the relative differences between constructs over multiple cell line replicates remained relatively consistent. However, we elected to show absolute values in each experiment, rather than simply normalizing the full-length to 100% and showing relative.

1. Lines 140-142 - The authors claimed that the functional difference between the transcriptionally active and inactive repeats could be narrowed down to a "6aa region which is conserved between repeats C3 and C5, but not conserved in C1, C2 and C4". Assuming the 6aa sequence is DPLELF, why does C1C3a elicit almost twice the intensity of GFP|mCherry signal compared to C3C1c, despite both constructs having the exact same 6aa sequence?

Indeed, C1C3a and C3C1c both containing the ‘active’ DPL sequence but having different relative levels of %GFP|mCherry. This is consistent with these sequences having a positive role in DUX target gene regulation – but likely in combination with other other regions which potentiate its affect, possibly through interacting proteins or post-translational modifications.

Why does DPLEPL (the intermediate C3C1b construct) induce a similar extent of GFP|mCherry signal as the FL construct, even though the former includes 3aa from a transcriptionally inactive repeat? In contrast, GSLELF (the other intermediate C1C3b construct) that also includes 3aa from a transcriptionally inactive repeat is almost completely deficient in inducing any GFP|mCherry signal. Why is that so? Is DPL the most crucial sequence? It will be important to mutate these 3 (or the above 6) residues on FL DUX4 to examine if its transcriptional activity is abolished.

These are interesting points. DPL does appear to be the most important region in the mouse DUX repeats. However, DPL is not shared in the C-terminus of human DUX4. Notably, the DUX4 C-terminus is sufficient to activate the mouse MERVL::GFP reporter when cloned to mouse homeodomains (see Author response image 2, second sample) and other DUX target genes (initially published in Whiddon et al. 2017). One clear possibility is that the DPL region is helping to coordinate the additive effects of multiple DUX repeats, which only exist in the mouse protein.

Author response image 2.

1. Line 154 - The intermediate DUX domain construct C1C3b occupied a different position on the PCA plot from the C1C3c construct that does not contain any of the critical 6aa sequence, as shown in Fig. 2e. However, both these constructs appear to be similarly deficient in inducing any GFP|mCherry signal, as seen in Fig. 2c. Why is that so?

The PCA plot assesses the impact on the whole transcriptome and not just the MERVL::GFP reporter, suggesting the 3aa region has transcriptional effects on the genome beyond what is detected in the MERVL::GFP reporter.

1. To strengthen the claim that "Chromatin alterations at DUX bindings sites require a transcriptionally active DUX repeat", the authors should also perform CUT&Tag for constructs containing transcriptionally inactive DUX repeats (e.g. C1+14aa), and show that such constructs fail to occupy DUX binding sites, as well as are deficient in H3K9ac accumulation.

This is a good comment. We elected to control this with constructs containing or lacking an active repeat. Although we have not pursued this by CUT&TAG, we have examined the impact of DUX constructs with inactive repeats (including the requested C1+14aa, new Figure S1g) by ATAC-seq (see #12, ATAC-seq section, below), and observe no chromatin opening, suggesting that the lack of transcriptional activity is rooted in the inability to open chromatin.

1. It would be good if the authors could also include CUT&Tag data for some of the C1C3 chimeric constructs that were used in Fig. 2, since the authors argued that the minimal 6aa region is sufficient to activate many of the DUX target genes. This would also strengthen the authors’ case that the transcriptionally active, not inactive, repeats are critical for binding at DUX binding sites and ensuring H3K9ac occupancy.

We agree that these would be helpful, and have examined the inactive repeats in transcription and ATAC-seq formats during revision (new data in Figures 1d and S1g), but not yet the CUT&TAG format.

1. Line 213 - "SMARCA4" should have been "SMARCA5"? Based on Fig. 4d, SMARCA5 is picked up in the BirA*-DUX interactome, not SMARCA4.

Thanks – corrected.

1. Lines 250-252 - The authors compared the active BirA-C3 against the inactive BirA-C1 to elucidate the interactome of the transcriptionally active C3 repeat, as illustrated in Fig. 5c. They found 12 proteins more enriched in C1 and 154 proteins in C3. This information should be presented clearly as a separate tab in Supp Table 2. What are the proteins common to both constructs, i.e. enriched to a similar extent? Do they include chromatin remodellers too? Although the authors sought to identify differential interactors between the 2 constructs, it is also meaningful to perform 2 separate comparisons - active BirA-C3 against BirA alone control, and inactive BirA-C1 against BirA alone control - like in Fig. 4d, so as to more accurately define whether the active C3 repeat, and not the inactive C1 repeat, interacts with proteins involved in chromatin remodeling.

We thank the reviewer for this comment, and we have modified the manuscript by adding a second sheet in Supplementary Table 2 including the results for enriched proteins in BirA-C1 vs. C3. Additionally, due to limitations of annotation between BirA alone and BirA*-C3 being sequenced in different mass spectrometry experiments, it is difficult to quantitatively compare the two datasets with pairwise comparisons.

1. Fig 5d: The authors mentioned in the legend that endogenous IP was performed for SMARCC1. However, in line 266, they stated Flag-tagged SMARCC1. Is SMARCC1 overexpressed? The reciprocal IP should also be presented. More importantly, C1 constructs (e.g. C1+14aa and C1Δ14aa) should also be included.

To clarify, Figure 4e used exogenously overexpressed FLAG-SMARCC1 in HEK-293T cells to confirm the results of the full-length DUX BioID experiment. Figure 5d was performed with overexpressed DUX construct, but involved endogenous SMARCC1 in mESCs. This has now been made clearer in the revised manuscript.

1. For both the SMARCC1 CUT&Tag and ATAC-seq experiments shown in Figures 5e and 5f respectively, the authors need to include DUX derivatives that contain transcriptionally inactive repeats with and without the 14aa acidic tail, i.e. C1+14aa and C1Δ14aa, and show that these constructs prevent the binding/recruitment of SMARCC1 to DUX genomic targets, and correspondingly display a decrease in chromatin accessibility. Only then can they assert the requirement of the transcriptionally active repeat domains for proper DUX protein interaction, occupancy and target activation.

We agree that examination of an inactive repeat in certain approaches would improve the manuscript. Importantly, we have now included C1+14 in our ATAC-seq experiments, and in Author response image 3 two individual replicates, which constitute a new Figure S1g. Compared to the transcriptionally active DUX constructs, which see opening at DUX binding sites, we do not see chromatin opening at DUX binding sites with transcriptionally inactive C1+14.

Author response image 3.

1. To prove that DUX-interactors are important for embryonic gene expression, it will be important to perform loss of function studies. For instance, will the knockdown/knockout of SMARCC1 in cells expressing the active DUX repeat(s) lead to a loss of DUX target gene occupancy and activation?

We agree that it would be interesting to better understand SMARCC1 cooperation with DUX function in the embryo, but we believe this is beyond the scope of this paper.

Minor Points

1. Lines 124-126 - What is the reason/rationale for why the authors used one linker (GGGGS2) for constructs with a single internal deletion, but 2 different linkers (GGGGS2 and GAGAS2) for constructs with 2 internal deletions?

With Gibson cloning, there are homology overhang arms for each PCR amplicon that are required to be specific for each overlap. Additionally, each PCR amplicon needs to be specific enough from one another so that all inserts (up to 5 in this manuscript) are included and oriented in the right order. The linker sequences were included in the homology arm overlaps, so the nucleotide sequences for each linker needed to be specific enough to include all inserts. This is a general rule to Gibson cloning. Additionally, both GGGGS2 and GAGAS2 are common linker sequences used in molecular biology and the amino acids structures are similar to one another, suggesting there is no functional difference between linkers.

1. Line 704 - 705: In the figure legend, the authors stated that 'Constructs with a single black line have the linker GGGGS2 and constructs with two black lines have linkers with GGGGS2 and GAGAS2, respectively.'. This was not obvious in the figures.

Constructs used for flow and genomics experiments that are depicted in Figure 2, Supplementary Figure 2, Figure 3, Figure 4, and Figure 5 have depicted black lines where deletions are present. Where these deletions are present, there are linkers in order to preserve spacing and mobility for the protein.

1. Line 160 - Clusters #1 and #2 are likely written in the wrong order. It should have been "activating the majority of DUX targets in cluster #2, not cluster #1" and "failed to activate those in cluster #1, not cluster #2", based on the RNA-seq heatmap in Fig. 2f.

We thank the reviewer for this comment, and the error has been corrected in the manuscript.

1. Line 188 - Delete the word "of" in the following sentence fragment: "DUX binding sites correlating with the of transcriptional".

Thanks – corrected.

1. Line 191 - Delete the word "aids" in the following sentence fragment: "important for conferring H3K9ac aids at bound".

Thanks – corrected.

1. Line 711 - "C1-C3 a,b,d" should be "C1-C3 a,b,c".

Thanks – corrected.

1. Lines 711-712 - The colors "pink to blue" and "blue to pink" are likely written in the wrong order. Based on Fig. 2c, the blue to pink bar graphs should represent C1-C3 a,b,c in that order, and likewise the pink to blue bar graphs should represent C3-C1 a,b,c in that order.

Thanks – corrected.

1. There is an overload of data presented in Fig. 2c, such that it is difficult to follow which part of the figure represents each data segment as written in the figure legend. It is recommended that the data presented here is split into 2 sub-figures.

Figure 2c has a supporting figure in Supplementary Figure 2b. While there is both a graphical depiction of the constructions and the data both in the main panel of Figure 2C, we have depicted it as so to be as clear as possible for the reader to interpret the complexity and presentence of amino acids in each of the constructs.

1. Line 717 - "following" is misspelt.

Thanks – corrected.

1. Lines 720-721 - "(Top)" and "(Bottom)" should be replaced with "(Left)" and "(Right)", as the 2 bar graphs presented in Fig. 2d are placed side by side to each other, not on the top and bottom.

Thanks – corrected.

1. Lines 725 and 839 - "Principle" is misspelt. It should be "Principal".

Thanks – corrected.

1. In Figures 3d and 3e, the sample labeled "C3+14_1" should be re-labeled to "C3+14", in accordance with the other sub-figures. Additionally, for the sake of consistency, "aa" should be appended to the relevant constructs, e.g. "C3+14aa" and "C3Δ14aa".

Thanks – corrected.

1. Line 773 - Were the DUX domain constructs over-expressed for 12hr (as written in the figure legend) or 18hr (as labeled in Fig. 5d)?

Thanks – corrected.

1. Related to minor point 19 above, is there a reason/rationale for why some of the experiments used 12hr over-expression of DUX domain constructs (e.g. for CUT&TAG in Fig. 3), whereas in other experiments 18hr over-expression was chosen instead (e.g. flow cytometry for MERVL::GFP reporter in Figures 1 and 2, and co-IP validations of BirA*-DUX interactions in Fig. 4)?

Thanks for the opportunity to explain. In this work, experiments that reported on proteins that are translated following DUX gene activation (e.g. MERVL:GFP via flow) were done at 18hr to allow for enough time for transcription and translation of GFP (or other DUX target genes). For experiments that report on the impact of DUX on chromatin and transcription, such as RNA-seq, CUT&Tag, and ATAC-seq, we induced DUX domain constructs for 12 hours.

1. Line 804 - "ΔHDs" is missing between "C2345+14aa" and "ΔHD1".

Thanks – corrected.

1. In Fig. 5c, "Chromatin remodelers" is misspelt.

Thanks – corrected.

1. There is no reference in the manuscript to the proposed model that is presented in Fig. 6b.

Thanks – corrected.

Reviewer #3 (Recommendations For The Authors):

Given the uncertainty of the function of the Dux peptide repeats in mice, could it not also be possible that the underlying repeated nature of the (coding) DNA? That is, could these DNA repeats exert a regulatory function on Dux transcription itself (also given the dire consequences of misregulated DUX4 expression as seen in FSHD, for example).

Yes, it remains possible that the internal coding repeats within Dux are playing a role in locus regulation, and might be interesting to examine. However, we consider this question as being outside the scope of the current paper.

Finally, it would be interesting to know whether these repeats are, in fact, present in all mouse species. Already no longer present in rat, do they exist, or not, in more "distant" mice, e.g. M. caroli?

Determining whether all mouse strains contain C-terminal repeats in DUX is a question we also considered. However, Dux and its orthologs are present in long and very complex repeat arrays that are not present in the sequencing data or annotation in other mouse strains. Therefore, we are not unable to answer this question from existing sequencing data. Answering would require a considerable genome sequencing and bioinformatics effort, or alternatively a considerable effort aimed at cloning ortholog cDNAs from 2-cell embryos.

Minor points:

line 169: here it seems, in fact, that the 'inactive' C2, C4 repeats are more similar to each other (my calculation: 91 and 96% identity at the protein and DNA level, respectively) than the active C3 and C5 repeats (82 and 89% identity, resp.), the outlier being C1.

Thanks for this comment, which was mentioned by other reviewers as well and has been addressed through new statistical analyses and interpretation (see new Figure S1d).

line 191: I'm not sure this sentence parses correctly ("...14AA tail is important for conferring H3K9Ac aids at bound sites...")

We thank the reviewer for this comment, and we have corrected the sentence in the manuscript.