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

  1. Howard Hughes Medical Institute, Department of Oncological Sciences and Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA
  2. Green Center for Reproductive Biological Sciences, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, USA


  • Reviewing Editor
    Jerry Workman
    Stowers Institute for Medical Research, Kansas City, United States of America
  • Senior Editor
    Kevin Struhl
    Harvard Medical School, Boston, United States of America

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:

  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).

  2. 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).

  3. 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?

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.

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.

  1. Howard Hughes Medical Institute
  2. Wellcome Trust
  3. Max-Planck-Gesellschaft
  4. Knut and Alice Wallenberg Foundation