In much of today’s biology research, the humble lab mouse is thought of as the ‘gold standard’ of physiological relevance—after all, we can’t experiment on humans, and mice are the next best thing, right? And while experiments in mice have provided innumerable advances to our knowledge of how our bodies function (and malfunction!), it’s important to remember that the assumed biological equivalence between mouse and man can only stretch so far.
Such is the case for DUX4, an unassuming gene with a fascinating backstory, and a core subject of research in the Tapscott Lab in the Human Biology Division at Fred Hutch. DUX4 is a transcription factor normally involved in turning on some of the earliest genes expressed by developing embryos whose misexpression in muscle tissue later in life causes the severe disease fascioscapulohumeral muscular dystrophy (FHSD). A better understanding of DUX4 in FHSD pathology requires a model system—mice would’ve been the natural choice, but there was one important problem: previous research from the Tapscott Lab and others had revealed that the mouse ortholog of this gene, mDux, has diverged significantly from human DUX4 (hDUX4), to the point that hDUX4 expressed in mouse cells does not robustly induce its expected collection of target genes. Since scientists’ goal is to study pre-clinical interventions which target hDUX4 in a non-human animal model, this fact limits the utility of mouse models of DUX4-linked FHSD.
Faced with this challenge, Yee Nip and Sean Bennett, two technicians in the Tapscott Lab, turned to a slightly unusual but emerging model system: pigs. The team was encouraged by previous findings from the lab showing greater similarities between canine DUXC and human DUX4 (fun fact: dogs are more closely related to pigs than they are to mice); in their recent publication in Human Molecular Genetics, they make a case for pigs as a viable model system for studying DUX4-linked FHSD.
Before they could determine how closely pig DUXC resembled its human counterpart, Nip and Bennett faced a crucial roadblock—nobody had yet identified the DUXC gene in the pig genome! The team painstakingly combed through a previously-published RNA-sequencing dataset from early pig embryos and was able to annotate a porcine (pig) DUXC gene (pDUXC) whose sequence matched hDUX4 more closely than mDux. Expressing pDUXC in pig skeletal muscle cells led to activation of a collection of early embryonic genes, confirming that pDUXC was indeed the likely pig ortholog of hDUX4. With the pDUXC gene in hand, the duo turned their attention to the next important question: how does hDUX4 behave when expressed in pig muscle cells?
Reassuringly, hDUX4 expressed in pig muscle cells was found to turn on pig orthologs to many of the same gene targets as it does in human muscle cells. However, since hDUX4 and pDUXC didn’t induce the exact same set of genes, the team wondered what features of the proteins accounted for the differences in their gene targets. Suspecting the DNA-binding homeodomain regions as the culprit, Nip and Bennett used an interesting approach: they generated a ‘Frankenstein’ gene with the homeodomains of pDUXC fused to the rest of the hDUX4 protein. When they expressed this protein in pig muscle cells, they were surprised to find that many of the pDUXC-specific genes not induced by hDUX4 were induced by their pDUXC:hDUX4 chimera, supporting the hypothesis that functional differences between these two proteins arose from divergence in this single domain.
In all, this study reminds us that—while mice often represent the top of the animal model hierarchy—they don’t have it all. At least in the case of studying FHSD, the Tapscott team provides valuable evidence that pigs may be the ticket to future treatments and even a cure. “It was super difficult doing bioinformatics in pigs, whose genomes are much more poorly annotated than mice,” Bennett comments, “but we were surprised at how far publicly-available resources could get us, which we used to discover a new gene homolog in pigs with potential clinical relevance.” And with more compact, research-optimized breeds of pigs finishing development, lab mice may soon find that they have a porky adversary.
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 member Dr. Stephen Tapscott contributed to this study.
Nip, Y., Bennett, S. R., Smith, A. A., Jones, T. I., Jones, P. L., & Tapscott, S. J. (2023). Human DUX4 and porcine DUXC activate similar early embryonic programs in pig muscle cells: Implications for preclinical models of FSHD. Human Molecular Genetics.