Human DUX4 and mouse Dux interact with STAT1 and broadly inhibit interferon-stimulated gene induction
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The transcription factor DUX4 is emerging as a key molecule in early mammalian development and in diverse pathologies including muscular dystrophy and solid tumors. While DUX4 has been linked to immune evasion, the mechanisms have not been delineated. In this study, the authors demonstrate that DUX4 functions as a negative regulator of interferon signaling by inhibiting STAT1, thereby suppressing interferon-stimulated gene induction. These studies provide a critical mechanistic link between DUX4 expression and the modulation of immune signaling pathways.
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Abstract
DUX4 activates the first wave of zygotic gene expression in the early embryo. Mis-expression of DUX4 in skeletal muscle causes facioscapulohumeral dystrophy (FSHD), whereas expression in cancers suppresses IFNγ induction of major histocompatibility complex class I (MHC class I) and contributes to immune evasion. We show that the DUX4 protein interacts with STAT1 and broadly suppresses expression of IFNγ-stimulated genes by decreasing bound STAT1 and Pol-II recruitment. Transcriptional suppression of interferon-stimulated genes (ISGs) requires conserved (L)LxxL(L) motifs in the carboxyterminal region of DUX4 and phosphorylation of STAT1 Y701 enhances interaction with DUX4. Consistent with these findings, expression of endogenous DUX4 in FSHD muscle cells and the CIC-DUX4 fusion containing the DUX4 CTD in a sarcoma cell line inhibit IFNγ induction of ISGs. Mouse Dux similarly interacted with STAT1 and suppressed IFNγ induction of ISGs. These findings identify an evolved role of the DUXC family in modulating immune signaling pathways with implications for development, cancers, and FSHD.
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eLife assessment
The transcription factor DUX4 is emerging as a key molecule in early mammalian development and in diverse pathologies including muscular dystrophy and solid tumors. While DUX4 has been linked to immune evasion, the mechanisms have not been delineated. In this study, the authors demonstrate that DUX4 functions as a negative regulator of interferon signaling by inhibiting STAT1, thereby suppressing interferon-stimulated gene induction. These studies provide a critical mechanistic link between DUX4 expression and the modulation of immune signaling pathways.
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Reviewer #1 (Public Review):
The authors of this manuscript report that human DUX4 and mouse Dux4 interact with STAT1 and inhibit interferon-stimulated gene transcription (ISG). The different functional domains of DUX4 were investigated to evaluate which ones are necessary for ISG. DUX4 transcriptional activity was found not to be necessary for ISG, rather the DUX4 C-terminal domain (CTD) was necessary and sufficient to suppress ISG. Employing liquid chromatography-mass spectroscopy (LC-MS), the DUX4 CTD was found to interact with several polypeptides present in human myoblasts. Two key regulators of innate immune signaling, STAT1, and DDX3X, ranked at the top of the list of candidate DUX4-CTD interactors. Immunoprecipitation confirmed DUX4-CTD interaction with STAT1, DDX3X, and several other polypeptides identified by LC-MS. Two …
Reviewer #1 (Public Review):
The authors of this manuscript report that human DUX4 and mouse Dux4 interact with STAT1 and inhibit interferon-stimulated gene transcription (ISG). The different functional domains of DUX4 were investigated to evaluate which ones are necessary for ISG. DUX4 transcriptional activity was found not to be necessary for ISG, rather the DUX4 C-terminal domain (CTD) was necessary and sufficient to suppress ISG. Employing liquid chromatography-mass spectroscopy (LC-MS), the DUX4 CTD was found to interact with several polypeptides present in human myoblasts. Two key regulators of innate immune signaling, STAT1, and DDX3X, ranked at the top of the list of candidate DUX4-CTD interactors. Immunoprecipitation confirmed DUX4-CTD interaction with STAT1, DDX3X, and several other polypeptides identified by LC-MS. Two regions of DUX4 were found to mediate interaction with STAT1. Amino acids 271-372 were necessary for co-IP of STAT1, and amino acids 372-424, containing (L)LxxL(L) motifs, could enhance binding to phosphorylated STAT1. IFN-gamma treatment enhanced DUX4-CTD binding to wild-type STAT1 and of the STAT1-S727A mutant. In contrast, IFN-gamma did not enhance the binding of DUX4 to the STAT1-Y701A mutant, indicating that DUX4-CTD and STAT1 interaction is promoted by STAT1 -Y701 phosphorylation. A mechanistic investigation of DUX4-STAT1 interaction was conducted by chromatin immunoprecipitation which revealed reduced IFN-gamma-induced STAT1 binding and Pol-II recruitment at promoters of several ISGs. Treatment with IFN-gamma of myoblasts derived from patients affected by facioscapulohumeral dystrophy (FSHD) showed that myoblasts expressing endogenous DUX4 failed to express the IDO1 gene which was, on the other hand, expressed in FSHD myoblasts not expressing DUX4. The majority of Ewing fusion-negative small blue round cell sarcomas have a genetic rearrangement between the CIC and DUX4 genes creating a fusion protein containing the C-terminal (L)LxxL(L) motif of DUX4. The Kitra-SRS sarcoma cell line expresses CIC-DUX4. IFN-gamma treatment of the Kitra-SRS cells showed very low induction of ISGs. Knock-down of the CIC-DUX4 fusion RNA resulted in a substantially increased IFN-gamma induction of ISGs whereas a corresponding knock-down in human myoblasts, which do not express CIC-DUX4, did not alter ISG induction.
This is an important and compelling study that sheds light on a molecular mechanism by which DUX4 inhibits IFN-mediated immune response with potential translational relevance for the treatment of DUX4-expressing cancers. The experiments are rigorously executed and controlled for, and the conclusions are well supported by the presented data.
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Reviewer #2 (Public Review):
The goal of this study was to understand the molecular mechanism of how transcription factor DUX4, which has a role in cancer, inhibits the induction of genes stimulated by interferon-gamma. The authors achieved this goal, and their results mostly support their conclusions. They found that DUX4, in their experimental model, interacts with STAT1, thereby decreasing STAT1 and Pol-II recruitment to sites of gene transcription.
The present study has many strengths: The topic is of broad interest, the findings are novel and intriguing, the experiments are well-designed and controlled, the data, with one exception, is carefully interpreted, and the manuscript is very well-written.
Two major weaknesses were identified. One is that all experiments, except Figure 6, rely on one experimental setup, which is a human …
Reviewer #2 (Public Review):
The goal of this study was to understand the molecular mechanism of how transcription factor DUX4, which has a role in cancer, inhibits the induction of genes stimulated by interferon-gamma. The authors achieved this goal, and their results mostly support their conclusions. They found that DUX4, in their experimental model, interacts with STAT1, thereby decreasing STAT1 and Pol-II recruitment to sites of gene transcription.
The present study has many strengths: The topic is of broad interest, the findings are novel and intriguing, the experiments are well-designed and controlled, the data, with one exception, is carefully interpreted, and the manuscript is very well-written.
Two major weaknesses were identified. One is that all experiments, except Figure 6, rely on one experimental setup, which is a human skeletal muscle cell line with an integrated doxycycline-inducible transgene. The concern is that both the treatment of cells with the drug doxycycline and the fact that signaling pathways could be disrupted in this (immortalized?) cell line could lead to artifacts that skew results. Indeed, results in Figure 4C indicate that total STAT1 is completely localized in the nucleus even prior to interferon stimulation when it should be in the cytoplasm. The other weakness is the use of the DUX4-C-terminal-domain (DUX4-CTD) mutant for the majority of the mechanistic experiments. The concern here is that although the phenotype of ISG repression is observed in this truncated mutant, important regulatory domains could be missing that modulate the interaction with STAT1 or other proteins. Is the NLS added after the flag tag identical to the endogenous NLS? Related, I disagree with the interpretation of Figure 4C that "this interaction happens within the nuclei of DUX4-CTC expressing cells". The interaction could happen prior to STAT1 shuttling to the nucleus.
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Author Response:
We thank the reviewers for their thoughtful comments. We would like to respond to one point made by reviewer 2. We agree with the recommendations of this reviewer for improving the manuscript, including additional studies in non-transformed cells. However, we would also like to clarify one point. Reviewer 2 stated that “results in Figure 4C indicate that total STAT1 is completely localized in the nucleus even prior to interferon stimulation when it should be in the cytoplasm.” Figure 4C uses proximity ligation assays to show that the interaction of STAT1 with DUX4-CTD occurs in the nucleus, at a lower level without interferon and a higher level with interferon, but does not measure the distribution of total STAT1. Supplemental Figure S3A/3B shows a combined cytoplasmic and nuclear distribution of STAT1 without …
Author Response:
We thank the reviewers for their thoughtful comments. We would like to respond to one point made by reviewer 2. We agree with the recommendations of this reviewer for improving the manuscript, including additional studies in non-transformed cells. However, we would also like to clarify one point. Reviewer 2 stated that “results in Figure 4C indicate that total STAT1 is completely localized in the nucleus even prior to interferon stimulation when it should be in the cytoplasm.” Figure 4C uses proximity ligation assays to show that the interaction of STAT1 with DUX4-CTD occurs in the nucleus, at a lower level without interferon and a higher level with interferon, but does not measure the distribution of total STAT1. Supplemental Figure S3A/3B shows a combined cytoplasmic and nuclear distribution of STAT1 without interferon treatment and shows increased nuclear STAT1 with interferon treatment, as would be expected in cells with an intact signaling pathway, although we also agree that the presentation of this finding can be improved with additional images that specifically address this point. Again, we thank both reviewers for their careful reading and helpful comments on our study.
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