If you ask me, one of the most beautiful aspects of science is its iterative, accretionary nature. Even the most extensive publication or exciting new finding doesn’t stand on its own—rather, individual studies contribute bits and pieces of information which eventually coalesce into a model explaining some biological process. At Fred Hutch, a striking example of this process can be found in work of the Tapscott Lab, which devotes itself to studying an enigmatic protein called DUX4 whose complexity is nothing short of stunning. A recent publication from the lab, led by postdoctoral scholar-turned staff scientist Dr. Danielle Hamm, builds beautifully on previous work and furthers the lab’s mission by identifying a new facet of DUX4 function with potentially broad implications for developmental biology and cancer.
Fans of Science Spotlight may already be familiar with DUX4 (which shows its face around here quite often), but for those who aren’t, here’s the quick version: DUX4 is a transcription factor which is in part responsible for initializing embryonic gene expression very early in development (for this reason, it is often known as a pioneer transcription factor). As you may imagine, turning on development genes is only desirable during development, so DUX4 is normally silenced after the embryo doesn’t need it anymore. Unfortunately for some, however, DUX4 doesn’t always stay quiet; DUX4 re-expression in muscle cells causes a progressive disease known as facioscapulohumeral dystrophy (FSHD), and DUX4 is also re-expressed in some cancer cells, where it drives an early embryonic gene program thought to be important for tumor progression. Independently of its role as a transcription factor, DUX4 also acts to suppress immune responses—it is here that our latest story starts, with a curious observation.
“We had previously reported that DUX4 suppresses innate immune signaling in cancer,” begins Dr. Hamm. “In order to better understand how it does this, we used an experimental system where we could essentially give cells a quick ‘pulse’ of DUX4 expression and monitor the downstream effects on innate immune responses.” In particular, Hamm and colleagues were checking DUX4-pulsed cells for their ability to induce major histocompatibility complex class I (MHC-I), a cell surface complex which the majority of cells use to alert the immune system of threats. Hamm pulsed cells with DUX4, applied the inflammatory cytokine IFN to trigger an immune response, and—lo and behold—the cells were silent, showing no increase in protein levels of MHC-I subunits. Except that this wasn’t surprising at all to Dr. Hamm, because the Tapscott Lab had recently published a separate study describing a mechanism by which DUX4 may accomplish this very task. That study (which I covered here), found that DUX4 physically interacts with the immune modulator STAT1 and prevents it from turning on immune response genes at the mRNA level.
However, there appeared to be more to this story. As Dr. Hamm puts it, “what surprised us most wasn’t the fact that DUX4 expression suppressed MHC-I induction, but how durable the immune suppression effect was following a relatively short pulse of DUX4 expression.” Remarkably, the cells weren’t just silent when DUX4 was expressed—they remained silent for days afterward, long after the DUX4 protein itself was gone. Still not convinced that this was a distinct mechanism of immune suppression, Hamm and colleagues checked mRNA levels of MHC-I subunits and found that they were robustly induced in response to IFN, even after a DUX4 pulse. Finally, the team made one small adjustment to their approach; instead of pulsing cells with wild-type DUX4, they pulsed them with a mutant DUX4 unable to bind DNA (but which could still interact with STAT1, per the previous study). While this transcriptionally-inactive DUX4 still suppressed short-term immune responses, it was unable to keep cells silent in the long-term. “Altogether, this convinced us that brief DUX4 expression suppresses MHC-I induction long-term by a distinct mechanism from the STAT1 interaction,” Hamm notes. Even better, the team now had several clues: since MHC-I subunit mRNAs were still reliably induced, DUX4 had to be suppressing MHC-I on a post-transcriptional level. Moreover, this suppression was probably carried out not directly by DUX4, but by some of its targets, since the mutant DUX4 lost this suppressive capability.
Armed with these clues, Hamm and colleagues embarked on an extensive series of experiments to find out how DUX4 suppressed MHC-I protein induction. After ruling out several candidate mechanisms, the team discovered that DUX4 expression leads to a cascade of signaling events which includes differential phosphorylation of several proteins including eIF2-alpha, eIF4E, and eEF2. Besides having complicated names, these proteins are responsible for regulating translational initiation and elongation—in other words, protein production from mRNAs! “Here, we were surprised again, because these factors regulate translation of many more mRNAs than just those encoding MHC-I,” Hamm remarks. Using metabolic labeling and high-throughput strategies (in collaboration with the Hsieh Lab) to measure protein production in cells following a pulse of DUX4, the team’s suspicions were confirmed—brief DUX4 expression led to a broad reduction in protein translation. This effect corresponded with the inhibition of multiple key translation factors to rapidly control protein expression at both the initiation and elongation stages downstream of DUX4 activity.
Now, the more astute readers may begin to notice an interesting contradiction revolving around DUX4’s canonical role as a transcriptional activator in light of these new results. A transcription factor which induces mRNAs but simultaneously prevents their translation into functional proteins? Gimme a break… but don’t stop reading now! This contradiction didn’t escape the attention of Dr. Hamm and the team; they confirmed that—despite a broad suppression of translation following DUX4 expression—DUX4 target genes appeared to escape this suppression. Using several predictive computational tools to see what made these genes distinct from those that were suppressed, Hamm and colleagues hypothesize that these mRNAs possess secondary structures which are less susceptible to translation inhibition, although this remains to be tested experimentally. Because DUX4 regulates gene expression in the early embryo, this dual inductive-suppressive function may reflect its normal function in zygotic genome activation—the developmental event whereby an embryo switches from translating maternally-sourced mRNAs to transcribing and translating mRNAs from its own genome.
“What started as a relatively specific mechanistic question quickly blossomed into a much more complicated and interesting finding,” admits Hamm, “and while we struggled a bit to make sense of this complexity, we’re excited to use these findings as a foundation to better understand DUX4-mediated translation suppression, which may end up being a crucial aspect of its function in both developmental cell-state conversions and disease contexts.” All in all, it appears that DUX4 has something to say, and it’ll do anything to be heard—whether that means belting out the lyrics to its set of target genes or quieting down the rest of the proteome and basking in the limelight.
The spotlighted research was funded by the National Institutes of Health, the Friends of FSH Research, and the Chris Carrino Foundation for FSHD.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium members Drs. Stephen J. Tapscott and Andrew Hsieh contributed to this study.
Hamm, D. C., Paatela, E. M., Bennett, S. R., Wong, C.-J., Campbell, A. E., Wladyka, C. L., Smith, A. A., Jagannathan, S., Hsieh, A. C., & Tapscott, S. J. 2023. The transcription factor DUX4 orchestrates translational reprogramming by broadly suppressing translation efficiency and promoting expression of DUX4-induced mRNAs. PLOS Biology. 21(9), e3002317.