DUX4 vs. PAX7–Who’s the fairest one of all?

by Amanda Hill

For several years, DUX4 has enjoyed center stage in the yet-unfolding story of facioscapulohumeral muscular dystrophy (FSHD). DUX4 was first identified in 1999¹, but only in the past five to 10 years have scientists reached consensus about the full underlying genetics of FSHD. Since then, researchers have worked tirelessly to understand how DUX4 causes muscle toxicity and cell death², and how it prevents muscle regeneration.

Recently, however, a Snow White-like story is unfolding, and scientists are starting to pay attention. While no one questions the foundational role of DUX4 in the pathogenesis of FSHD, three separate groups of researchers are now also calling attention to a protein called PAX7 and its close cousin, PAX3. It seems that DUX4 is like the evil, jealous stepmother who banished and put to sleep the “fairest one of all”—the kindhearted princess, PAX7.

Both DUX4 and PAX7/3 are members of a family of proteins known as “transcription factors.” Transcription factors bind DNA to regulate expression of genes that are important in embryonic development and in differentiation of cells into specific tissues. PAX7 and PAX3 are key regulators of the formation of skeletal muscle, though PAX3 generally operates during embryonic development while PAX7 generally operates later in development and in adult muscle regeneration³. In our Snow White story, PAX7 is the princess that everyone wants to see prevail.

About 10 years ago, Darko Bosnakovski, DVM PhD, and Michael Kyba, PhD, then at the University of Texas Southwestern Medical Center, discovered that in a cell culture model of FSHD, the presence of DUX4, the evil queen, causes the function of PAX7 to be suppressed. When researchers manipulated the cells to artificially increase expression of PAX7, the FSHD cells survived more robustly. This indicated that the function of PAX7 may somehow counter the deleterious effects of DUX4⁴.

Last year, these studies were resurrected and greatly expanded upon. A team at the University of Washington, led by Daniel Miller, MD PhD, found that in early development, FSHD stem cells have normal expression of both PAX7 and PAX3 as they differentiate into muscle cells, consistent with the observation that most children with FSHD have normal muscle development and growth. Interestingly though, DUX4 expression was not observed at the predicted rates in individual cells also expressing PAX7 or PAX3. This indicates that in early development, conditions promoting PAX7/3 expression may counter DUX4 expression, and that expression of DUX4 and PAX7/3 may be mutually exclusive⁵.

In parallel, Christopher Banerji, PhD, and Peter Zammit, PhD, at King’s College London reanalyzed several gene expression datasets previously generated from muscle biopsies from patients affected by FSHD. They found that the suppression of PAX7-regulated genes is, in many instances, actually a stronger and more reliable biomarker of FSHD than the expression of DUX4-regulated genes.

The network of genes regulated by PAX7 and suppressed in FSHD could also explain many FSHD phenotypes, including less effective antioxidant properties, cell death, inability to regenerate muscle, and the expression of DUX4 itself. These researchers also manipulated healthy cells to co-express both DUX4 and PAX7, and found that they mutually inhibited each other’s ability to activate their target genes⁶.

However, exactly how DUX4 and PAX7 interact remains to be deciphered. It seems that the relationship between the two is more complex than a simple, direct competition model would suggest⁶. Some non-mutually exclusive hypotheses that have been put forth include 1) DUX4 and PAX7 compete to bind DNA at a few key toxicity-related sites, and/or 2) DUX4 and PAX7 compete to interact with a third, yet-unknown protein necessary for their activity, and/or 3) DUX4 may interfere with the DNA-binding capabilities of PAX7.

I hope that at this point you can appreciate how DUX4 and PAX7 seem to have an antagonistic, seesaw-like relationship. As Drs. Banerji and Zammit explain, it appears that DUX4 operates in two ways to cause FSHD: 1) by direct activation of certain genes that results in muscle cell death (the evil queen is simply toxic), and 2) by suppressing the work of PAX7, such as in muscle regeneration (the evil queen puts Snow White to sleep). In theory, if Snow White were awake, she may help counter the toxic effects of the evil queen.

It is clear that scientists are just starting to get to know PAX7, our Snow White, and what role she plays in the FSHD story. And understanding PAX7 biology is valuable because it may shed light on additional therapeutic opportunities. We’ll continue to watch this Snow White and the evil queen storyline unfold with great interest, as it will inevitably lead to an even greater understanding of FSHD pathogenesis, and may help contribute to a more “happily ever after” for FSHD sufferers.

Footnotes

1. Gabriels J et al. Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element. Gene.1999;236(1): 25-32. PubMed.
2. Lemmers, RJLF et al. A unifying genetic model for facioscapulohumeral muscular dystrophy. Science.2010: 1189044. PubMed.
3. Buckingham, Margaret, and Frederic Relaix. PAX3 and PAX7 as upstream regulators of myogenesis. Seminars in Cell & Developmental Biology. Vol. 44. Academic Press, 2015. PubMed.
4. Bosnakovski, D et al. An isogenetic myoblast expression screen identifies DUX4‐mediated FSHD‐associated molecular pathologies. The EMBO Journal. 2008;27(20):2766-2779. PubMed.
5. Haynes, P et al. Expression patterns of FSHD-causing DUX4 and myogenic transcription factors PAX3 and PAX7 are spatially distinct in differentiating human stem cell cultures. Skeletal Muscle. 2017;7(1): 13. PubMed.
6. Banerji, CRS et al. PAX7 target genes are globally repressed in facioscapulohumeral muscular dystrophy skeletal muscle. Nature Communications. 2017;8(1):2152. PubMed.
7. Bosnakovski, D et al. The DUX4 homeodomains mediate inhibition of myogenesis and are functionally exchangeable with the PAX7 homeodomain. J Cell Sci.2017: jcs-205427. PubMed.