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The Transcription Factor DUX4 Orchestrates Translational Reprogramming by Broadly Suppressing Translation Efficiency and Promoting Expression of DUX4-induced mRNAs

Danielle C. Hamm, Ellen M. Paatela, Sean R. Bennett, Chao-Jen Wong,Amy E. Campbell, Cynthia L. Wladyka, Andrew A. Smith, Sujatha Jagannathan, Andrew C. Hsieh, Stephen J. Tapscott 

Abstract

Translational control is critical for cell fate transitions during development, lineage specification, and tumorigenesis. Here, we show that the transcription factor double homeobox protein 4 (DUX4), and its previously characterized transcriptional program, broadly regulates translation to change the cellular proteome. DUX4 is a key regulator of zygotic genome activation in human embryos, whereas misexpression of DUX4 causes facioscapulohumeral muscular dystrophy (FSHD) and is associated with MHC-I suppression and immune evasion in cancer. We report that translation initiation and elongation factors are disrupted downstream of DUX4 expression in human myoblasts. 

Introduction

The double homeobox protein 4 (DUX4) gene encodes a transcription factor that is expressed in immune-privileged niches such as the preimplantation embryo [1,2], testis [3], and, possibly, thymus [4]. DUX4 is briefly expressed in the 4-cell human embryo and serves as a key transcriptional activator of the zygotic genome, driving expression of hundreds of coding genes and repetitive retroelements [1,2]. In addition to zygotic genome activation (ZGA), regulation of mRNA degradation and translation is essential to rapidly diversify the proteome during early development [5] and has been associated with increased developmental potential of human preimplantation embryos [6]. It is becoming abundantly clear that translational control, both globally and at the level of individual transcripts, helps mediate cell fate transitions. This includes the shift from the maternal to the embryonic developmental program, the balance of stem cell self-renewal and differentiation, and the plasticity of cancer [7,8].

Materials and methods

MB135 myoblasts were grown in Ham’s F-10 supplemented with 10% FBS, 1% penicillin/streptomycin, 10 ng/mL rhFGF, 1 μM dexamethasone, and 3 μg/mL puromycin as appropriate to maintain lines carrying the DUX4 transgene. SuSa cells were grown in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin. Differentiation of FSHD myoblasts into myotubes was achieved by switching myoblast grown to confluence into DMEM, 1% penicillin/streptomycin, 10 μg/ml insulin, and 10 μg/ml transferrin for 48 hours. Pulsed MB135iDUX4 myoblasts were treated with 1 to 2 μg/mL DOX for 4 hours, rinsed with PBS, and fresh growth media added. MB135iDUX4 myoblasts with continuous DUX4 induction were treated with 1 μg/mL DOX for 20 hours. All cell types were stimulated with 50 ng/mL IFNγ where specified. MB135iDUX4 myoblasts were pulsed with DOX for 4 hours, incubated for 48 hours, supplemented with MG132 (0.5 μM), Bafilomycin A1 (0.5 μM), or ONX-0914 (200 nM) with the addition of 50 ng/mL IFNγ for 16 hours. 

Results

We recently reported that DUX4 blocks IFNγ-stimulated induction of MHC-I and surface antigen presentation [31]. To determine the mechanism of DUX4-induced MHC-I regulation, we used a well-characterized cellular model system of human myoblasts with a doxycycline (DOX)-inducible DUX4 transgene (MB135iDUX4) [32]. DUX4 expression occurs in transient bursts in rare populations of ESCs [2,16] and is sporadically misexpressed in FSHD muscle cells [3,23], making it difficult to characterize downstream mechanisms endogenously. We have previously demonstrated that a short “pulse” of DUX4 in MB135iDUX4 myoblasts induced a transcriptional program representative of FSHD muscle cells and the early cleavage-stage embryo [33]. Pulsed DUX4 expression in this cell culture system enabled reproducible and synchronized DUX4 induction, permitting the investigation of mechanisms downstream of DUX4 that may have otherwise been masked by heterogeneous populations of DUX4-expressing cells.

Discussion

In this study, we have shown that brief expression of the developmental transcriptional factor DUX4 results in prolonged translational reprogramming of the cell. DUX4 expression drives an early embryonic gene program that facilitates a totipotent-like state [1,2,16]. Reprogramming cells to totipotency requires both the activation of a new gene expression program and suppression of existing gene products to erase the previous cellular identity. Broad suppression of protein synthesis is often accompanied by selective translation of mRNA networks during instances of cell stress and cellular reprogramming [60–63]. Critically, we found that DUX4 induced relatively broad translational regulation, where translation of many classes of mRNAs—including factors involved in antigen presentation, translation, and somatic cell lineage specification—were suppressed while DUX4-induced transcripts were translated. Thus, DUX4-induced transcription and downstream translational regulatory mechanisms ultimately result in reprogramming of protein synthesis, underscoring the importance of understanding DUX4 biology beyond its role as a transcriptional activator.

Acknowledgments

We thank the Fred Hutchinson Cancer Center Flow Cytometry Core and Genomics Core for providing technical assistance. We thank Dr. Bradley Cairns, Dr. Bradley Weaver, Dr. Adam Geballe, and Dr. Lucas Sullivan for their perspectives regarding this project.

Citation: Hamm DC, Paatela EM, Bennett SR, Wong C-J, Campbell AE, Wladyka CL, et al. (2023) The transcription factor DUX4 orchestrates translational reprogramming by broadly suppressing translation efficiency and promoting expression of DUX4-induced mRNAs. PLoS Biol 21(9): e3002317. https://doi.org/10.1371/journal.pbio.3002317

Academic Editor: Jeff Coller, Johns Hopkins University, UNITED STATES

Received: July 6, 2023; Accepted: August 31, 2023; Published: September 25, 2023

Copyright: © 2023 Hamm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All data needed to evaluate the conclusions in the paper are present in the paper, supporting information, or the Gene Expression Omnibus repository. The Ribo-seq, Poly-seq, and RNA-seq data generated in support of this publication have been deposited in the Gene Expression Omnibus (GSE206439). The processed datasets, including gene expression, ribosome footprint p-sites, metadata, shell scripts and R code for preprocessing and downstream analysis, are available on Zenodo (https://zenodo.org/record/7822959; DOI: 10.5281/zenodo.7822959). A GitBook with detailed description of our analysis is also available (https://fredhutch.github.io/DUX4-IFNg-ribosome-footprints). Flow cytometry data generated in this study are available in the FlowRepository.org (FR-FCM-Z6XR, FR-FCM-Z6XT, FR-FCM-Z6XS).

Funding: This research was supported by the Flow Cytometry Shared Re-source, RRID:SCR_022613, and the Genomics & Bioinformatics Shared Resource, RRID:SCR_022606, of the Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium (P30CA015704). This work was supported by grants from the National Institutes of Health P50AR065139 (SJT), R01AR045203 (SJT), F32CA254805 (DCH), R37CA230617 (ACH), R01GM135362 (ACH), and the Friends of FSH Research https://www.fshfriends.org/ and the Chris Carrino Foundation for FSHD https://chriscarrinofoundation.org/ (SJT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: SJT and DCH are co-inventors on a patent application submitted by the Fred Hutchinson Cancer Center that covers research presented here. Other authors declare that they have no competing interests."

Abbreviations: 2CLC, 2-cell-like cell; 4EBP1, 4E-binding protein 1; DOX, doxycycline; dsRNA, double-stranded RNA; DUX4, double homeobox protein 4; eEF2, eukaryotic elongation factor 2; eEF2K, eukaryotic elongation factor 2 kinase; eIF4E, eukaryotic initiation factor 4E; ESC, embryonic stem cell; FACS, fluorescence-activated cell sorting; FSHD, facioscapulohumeral muscular dystrophy; GO, Gene Ontology; HPG, L-homopropargylglycine; IFNγ, interferon gamma; iPSC, induced pluripotent stem cell; ISG, interferon-stimulated gene; KO, knockout; m7GTP, 7-methylguaniosine 5′-triphosphate; MFE, minimum free energy; MHC-I, major histocompatibility complex class I; mTORC1, mechanistic target of rapamycin complex 1; nt, nucleotide; RNA-seq, RNA sequencing; RPF, ribosome-protected fragment; TE, translational efficiency; TIS, translation initiation site; TOP, terminal oligopyrimidine; TSS, transcription start site; WCL, whole-cell lysate; WT, wild-type; ZGA, zygotic genome activation.

 

https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002317#abstract0

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