Study uncovers new proteins involved in regulating muscular dystrophy-linked gene

CRISPR-based proteomics technique reveals potential therapeutic targets for FSHD, a rare but devastating disease
Dr. Stephen Tapscott
Dr. Stephen Tapscott Fred Hutch file photo

A new study has revealed more players in the pathway of facioscapulohumeral muscular dystrophy, or FSHD, the most common form of muscular dystrophy.

Led by Fred Hutchinson Cancer Research Center biologist Dr. Stephen Tapscott and staff scientist Dr. Amy Campbell, the study, published today in the journal eLife, is the first to systematically identify proteins involved in repressing the FSHD-triggering gene, DUX4.

Normally, the DUX4 gene is only turned on in very early embryonic development, shutting off before the embryo even implants in the uterus. But in people with FSHD, the DUX4 gene comes back on, progressively destroying muscle cells. The researchers have been trying to figure out how to shut it off again with the hopes of pointing to new therapeutic targets that could halt that progression.

In their latest study, they used a CRISPR-based proteomics technique to find proteins that attach to the DUX4 gene and its neighboring DNA. They then asked whether those proteins are involved in shutting off the gene in muscle cells and in embryonic stem cells. Those experiments identified two large groups of proteins involved in shutting off, or repressing, DUX4, called NuRD and CAF-1. Tapscott and his colleagues then went on to identify a protein that represses those repressors, known as MBD3L2, which they believe could be a potential new therapeutic target for FSHD.

The disease, which afflicts nearly 900,000 people around the world, is caused by an uncommon quirk of DNA. A genetic disease, FSHD is not triggered by a mutation in a gene in the way we might normally think about an inherited condition. Rather, it’s caused by having too few copies of the DUX4 gene — healthy people have 11 or more copies of DUX4 on chromosome 4; having 10 or fewer copies triggers FSHD. Paradoxically, having fewer repeats of the gene causes DUX4 to occasionally switch on when it shouldn’t, wreaking havoc in skeletal muscles.

It’s not clear why DUX4 turning on is particularly damaging for muscle cells over other cell types, the researchers said. But their study points to a possible model for what could be going on.

Dr. Amy Campbell
Dr. Amy Campbell

In skeletal muscles, a single muscle fiber is made up of many muscle cells, but they’ve all fused together and lost the barriers between them. All their DNA-storage compartments, known as nuclei, are bundled together. Campbell and Tapscott think that in people with FSHD, the DUX4 gene may spontaneously turn back on only in a few cells at a time, but because muscle-cell nuclei don’t have walls between them, DUX4 could “spread” its way from nucleus to nucleus along the muscle fiber, wreaking havoc along the way. In other cell types, if DUX4 turns itself back on, that cell would just die without harming nearby cells.

“The environment is able to compensate for the loss of a single cell,” Campbell said. “Whereas in a muscle, you’re affecting these large muscle fibers that maybe can’t catch up.”

The MBD3L2 protein that their study identified seems to be at least partly responsible for turning DUX4 back on, and they wonder if that protein could be responsible for the spreading — and the progressive nature of FSHD. Those are still big unknowns, Tapscott said.

“If the spreading from nucleus to nucleus is the basis of progression, then blocking the spreading by blocking MBD3L2 could slow down progression,” he said.

Campbell also found that the NuRD and CAF-1 protein clusters shut DUX4 off in embryonic cells; next they want to ask whether MBD3L2 is also involved in normal embryonic development in the same way it seems to be acting in FSHD muscle cells.

Additionally, DUX4 was recently found to be involved in some rare types of leukemia and sarcoma, although it’s not clear if the gene is behaving in the same way in these cancers as it does in muscular dystrophy. Campbell and Tapscott are planning to address that question next.

This study was their first foray into using the specific CRISPR technique, known as enChIP, and they’re excited to see what else they and their colleagues might discover with further similar studies.

“This approach worked, and we can go further with it,” Tapscott said. “We can now go deeper into this data and deeper into this approach, perhaps for some of these [mutations] in cancer as well.”

The National Institutes of Health, the FSH Society, Friends of FSH Research and Fred Hutch Reservoir Fund funded this study.

Read more about Fred Hutch achievements and accolades.

Rachel Tompa is a former staff writer at Fred Hutchinson Cancer Center. She has a Ph.D. in molecular biology from the University of California, San Francisco and a certificate in science writing from the University of California, Santa Cruz. Follow her on Twitter @Rachel_Tompa.

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