RNA-mediated Epigenetic Silencing of D4Z4 repeats: Implications for targeted therapy for FSHD

Budget

$60,180 (USD) in total, supported in partnership with Friends of FSH Research (50:50)

Lay Abstract

Dr. Jong Won Lim is a Friends of FSH Research Fellow at the Fred Hutchinson Cancer Research Center in Seattle. He is mentored by Dr. Stephen Tapscott and Dr. Galina Filippova, and has recently published the results from his first round of funding from Friends of FSH Research.

Dr. Lim and team have been resilient in developing the experiments needed to help answer one of the most fundamental questions in FSHD biology – and at the same time have identified a potential way to intervene with disease progression. Whereas some projects we fund are easy to explain to our supporters, this one is not, so bear with me and follow along!! As always, we are here to answer any questions you may have.

The fundamental question underlying this work is why does DUX4 turn on when there are less than 10 copies in a row? (if what I just wrote made no sense, take a minute to review the mechanism of FSHD). We’ve suspected for a long time that the reason is that the DUX4-containing array itself emits some kind of signal telling the cell to shut it down. But what on earth could those signals be? It’s not like a piece of DNA has a flare gun it can light – does it?

Before we delve too deep, let’s talk therapeutics. DUX4 is a protein that is not supposed to be turned on. Because of the nature of the protein, and the fact that it is specifically expressed in patients with FSHD, the most logical strategy for therapy is to stop it from working. This may be a traditional pharmaceutical drug, and we are actively investigating whether there are drugs that will do the job (here is one example). However, there are also experimental therapeutics that target specific DNA sequences using DNA or RNA sequences that match the DUX4 genetic sequence. How’s that for futuristic! Recent progress with treatment of spinal muscular atrophy highlights the potential of this technology, and hopefully we will one day be able to write an article like this one.

DNA drugs are a particularly logical approach for FSHD – in fact, we’ve recently funded a study to test the efficacy of such a compound (soon to be announced) , and yet another that seeks a novel way of targeting the DUX4 gene. Both of these compounds work by interfering with the DUX4 mRNA. If you mess up the DUX4 mRNA, DUX4 protein can’t be made. But the mRNA is still made because you haven’t solved the root of the problem: the DNA encoding DUX4 is not silenced. As a creative analogy, it’s like stopping a baby from crying by putting in earplugs.

In a nutshell, Dr. Lim’s study shows that small chemically modified RNA molecules that target the sequences surrounding the DUX4 gene are capable of re-silencing the DUX4 array. He has swaddled the baby and overfed it.

Here’s what we think is happening. DUX4 is a gene that is encoded in a head to tail array – think of it like a conga-line dancing to Hot Hot Hot in your cells. In between each gene is a sequence that governs the gene’s expression. Think of that sequence as the hands in the conga line. Usually, that regulatory sequence pushes gene expression in one direction so only one gene is made (i.e. your hands in the conga line only touch the hips of the person in front of you) – but we already know this is not the case for the DUX4 array.

We know that the regulatory sequence within the array is bidirectional, meaning it can drive expression of gene expression in both directions. One hand is grabbing the hips of the person in front, and the other is grabbing another body part of the person behind. This means you are generating two different RNAs, and because they are opposite from one another, they can bind one another. When two RNA molecules come together that is the equivalent of firing a flare gun off in the cell.

These double stranded molecules feed into a cellular defense mechanism so that the invading RNA is rapidly destroyed. One of the proteins involved in that defense mechanism is called Argonaut (AGO). To make a long story short, Dr. Lim showed that the DUX4 array is shedding off small pieces of RNA that are getting loaded onto an AGO protein, and finding their way back to the array and telling the cell to shut it down. He also showed that proteins required for the generation of siRNA are necessary for the effect.

Because of the superb progress made on this project, Friends of FSH Research has renewed Dr. Lim’s funding for an additional year.