DUX in a row: Exploring genetic repression across the D4Z4 repeat

In the late 19^th century, two French neurologists described a new form of muscular dystrophy which they named for the specific muscles it affects—the facial (facio-), shoulder (-scapulo-), and upper arm muscles (-humeral). Facioscapulohumeral dystrophy, or FSHD, causes progressive muscle weakness and wasting in these areas, affecting about 1 in every 10,000 individuals. Unfortunately, the best treatment options currently available for this debilitating condition remain limited to physical therapy and pain relief aimed at preserving mobility and comfort.

FSHD is caused by the abnormal expression of the DUX4 transcription factor in muscle cells. Normally, DUX4 is turned on only briefly during early embryonic development and is kept off in most other tissues. When genetic or epigenetic disruptions relax the normally repressive chromatin around a macrosatellite repeat region called D4Z4 on the long arms of chromosomes 4 and 10, it allows access to a normally “off-limits” region of the genome and enables DUX4 misexpression. A majority of FSHD cases (called FSHD1) happen because of mutations that result in fewer of those repeated DNA segments containing the DUX4 gene, while a smaller group (FSHD2) is due to defects in proteins that regulate gene repression (such as SMCHD1, DNMT3B, or LRIF1, which help maintain DNA methylation and repressive chromatin at D4Z4).

In an ode to our beloved Seattle summers, let’s think of these D4Z4 repeats and DNA modifications as a blackberry bush. As anyone who’s recently been on the Burke Gilman Trail probably knows, once a blackberry bush grows in, it creates a highly effective barrier that represses growth of any other plant species under it. In healthy muscle cells, the D4Z4 repeats act like these blackberry bushes, creating a physical barrier to DUX4 gene expression. While some blackberry bushes can be a nuisance, having an intact D4Z4 is a good thing—these repeats serve an important role by keeping the DUX4 gene tightly repressed during the post-developmental period where expression becomes pathogenic.

While it’s well established that the D4Z4 represses DUX4 expression, we don’t yet fully understand whether specific regions within these repeats are essential for epigenetic repression. To answer this question, researchers from the Tapscott lab led by graduate student Ellen Paatela and Dr. Stephen Tapscott published a study in Human Molecular Genetics describing a novel silencing GFP reporter system which tests whether specific DNA sequences within the D4Z4 region can turn off (or reduce) the expression of DUX4. When a tested DNA sequence has silencing activity, there’s less GFP expressed, indicating that it can suppress DUX4 expression.

After validating their system using the LRIF1 promoter, a region known to recruit D4Z4-relevant repressors, they divided the D4Z4 region into 14 overlapping segments and found that the fifth segment (S5) was the only region that caused a significant suppression of GFP. Breaking this fragment into even smaller pieces revealed that a single 146 nucleotide fragment, which they named S5.4, was responsible for a vast majority of the reduction in GFP signal.

"We were surprised and excited to find only one specific sequence within the D4Z4 repeat that robustly recruits repressors, identifying D4Z4-S5 as a potential key regulatory element for DUX4 expression," comments Paatela.

When cells were treated with a DNA demethylating drug (5-aza-dC) or a histone deacetylase inhibitor (entinostat), GFP levels rose, indicating a loss of silencing. This effect was temporary—silencing reappeared within a week—suggesting the repressive state can be reestablished.

After pinpointing S5.4 as a key silencing region, the team explored the underlying epigenetic mechanism driving their repression. Using bisulfite nanopore sequencing to investigate individual methylation events, the authors revealed that GFP^Low cells (DUX4 repressed) had higher DNA methylation across most CpG sites in D4Z4-S5 compared to GFP^High cells. However, because some silenced cells still had unmethylated DNA, this points to a multi-layered silencing mechanism where DNA methylation works in concert with other epigenetic marks, such as repressive histone modifications.

The authors next investigated whether known histone modifiers involved in D4Z4 repression also help silence DUX4 at these key DNA regions. While knockdown of NuRD complex components CHD4 and MBD1 had minimal effect, knockdown of the H3K9 methyltransferase SETDB1 caused robust de-repression of GFP, as did loss of its partner ATF7IP, indicating their key role in silencing. Interestingly, knockdown of TRIM28, a known SETDB1 recruiter, did not affect silencing, suggesting alternative recruitment mechanisms. Additional histone modifiers, SIN3A and SIN3B (histone deacetylases), also contributed to repression, whereas KDM1A (an H3K4 demethylase) did not.

The team also explored how certain drugs known to reduce DUX4 levels affect these key DNA regions. They found that inhibitors targeting the p38 signaling pathway, such as losmapimod and SB203580, were particularly effective at silencing reporter expression through the D4Z4-S5 sequence. Other drug classes, like beta-2-adrenergic receptor agonists and BET inhibitors, showed little impact in this system. Interestingly, a compound called berberine, known to stabilize special DNA structures called G-quadruplexes, strongly suppressed DUX4 activity via D4Z4-S5, even though this sequence isn’t known to form such structures—an unexpected finding that calls for further study.

To test if their reporter system could help screen potential treatments, the researchers tracked GFP expression over several days using live-cell imaging. They confirmed that p38 inhibitors could maintain suppression of DUX4-linked reporters over time, highlighting this system’s promise as a tool to find new therapies for FSHD.

With the D4Z4-S5 reporter now proven in action, the researchers see it as more than just a laboratory tool—it’s a new way to scout out the next generation of FSHD therapies. As Paatela puts it, “We hope this reporter system with D4Z4-S5 will be a useful tool for future screens to discover novel DUX4 regulators and FSHD therapeutics.”

Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Members Drs. Stephen Tapscott and Taran Gujral contributed to this research.

The spotlighted research was funded by the National Institutes of Health, the Friends of F.S.H. Research, and the Chris Carrino Foundation for FSHD.

Paatela EM, St. Amant FG, Hamm DC, Bennett SR, Gujral TS, van der Maarel SM, Tapscott SJ. 2025. A discrete region of the D4Z4 is sufficient to initiate epigenetic silencing. Human Molecular Genetics. https://doi.org/10.1093/hmg/ddaf114.