Polycomb repressor complex protein EZH2 regulation of muscle stem cell migration and differentiation

Muscle is constantly damaged throughout life, due to exercise, age, sickness or poor lifestyle. But unlike many tissues in the adult body, muscle is able to repair itself through the action of specialised muscle stem cells which lie at the edges of muscle fibres. When they detect damage muscle stem cells move to the site of damage and then integrate into the damaged fibres, in a process called differentiation. Muscle weakness, for instance as we age or during disease such as cancer, is thought to occur due to an inability of muscle stem cells to repair damaged muscle, and instead connective and fat cells take the place of muscle fibres. A better understanding of the ways muscle stem cells move to the site of damage and differentiate will ultimately allow the development of therapies to prevent or treat muscle weakness.

In response to damage, muscle stem cells also turn specific genes on or off, using control regions in the DNA called 'epigenetic' switches. This study aims to investigate an enzyme, EZH2, that makes changes to these epigenetic switches in the DNA. EZH2 can therefore potentially turn the correct genes on or off in response to muscle damage and so lead to repair of the muscle. Indeed we know that EZH2 can influence muscle repair, because when this enzyme does not work in the muscle of mice, the muscle is not repaired properly. But we do not know exactly which switches, and therefore genes, are controlled by EZH2 when muscle stem cells repair damaged muscle. One aim of this study is to identify those genes.

We now have exciting preliminary results that reveal EZH2 not only influences differentiation, but also influences muscle stem cell movement towards sites of damage. We now wish to understand how EZH2 can control both migration of muscle stem cells towards damage and their differentiation at the site of damage.

To do this we will use zebrafish larvae, because the muscle of zebrafish regenerates quickly, and the larvae are transparent so that we can easily see the movement of muscle stem cells using a microscope. We will measure how muscle stem cells move towards injury by using zebrafish larvae that have fluorescently labelled muscle stem cells. Fluorescent cells in the fish will be observed by powerful microscopes to make 4D movies and an especially developed computer software will be used to track cells. This is not something that can easily be done in other, non-transparent, animals such as mammals. Muscle repair in zebrafish also occurs in much the same way as in humans and similar genes are turned on and off in response to damage, making this a good system to study muscle repair processes.

We will measure how muscle stem cells behave after injury when EZH2 is prevented from working. We will then ask at which point in the repair process EZH2 is important, as we can visualise which genes are turned on or off in muscle stem cells before, during and after injury. To show if EZH2 controls genes by epigenetic switches we will extract migrating muscle stem cells and measure the switches on the DNA.
Studies of cancer cells suggest that EZH2 can also control the movement of cells separately from regulating how genes are turned on or off. We will also investigate whether EZH2 has this activity in muscle stem cells as well, by using mutated versions of EZH2 found in cancer cells and asking if they change the way that the muscle stem cells move.

Our results will be important for understanding how the 'epigenetic' switches are coordinated with the movement of muscle stem cells to sites of damage to ensure that muscle repair is successful. We will also be able to identify genes that are controlled by EZH2 as muscle is repaired, and so may be able target these genes as therapies for improving muscle strength.

Muscle constantly undergoes repair by muscle stem cells (muSCs) in response to exercise or injury, whilst a decline in the ability of muscle to repair itself is associated with ageing and disease. Thus understanding muscle repair has wide implications for healthcare.

Central to the regeneration process is the migration of muSCs to the site of injury followed by differentiation. To dissect the mechanisms controlling muSC responses we have developed a protocol for observing GFP-expressing muSCs in injured, transparent larval zebrafish. Using confocal imaging and pharmacological manipulations we find the Polycomb protein EZH2 is required for muSC migration and ultimately for effective regeneration.

Despite the importance of muSC migration to muscle regeneration, almost nothing is known about how this migration is controlled in vivo. In this project we aim to identify the mechanisms by which EZH2 directs muSC migration and determine how this is related to differentiation.

Firstly, muSC migration after injury will be captured by time-lapse microscopy in zebrafish larvae in which EZH2 function has been abrogated. Cell shape and migration in 3D will be quantified using customised software, and will be correlated with proliferation, differentiation, and localisation of cell adhesion proteins. EZH2 may control muSC migration by regulating the cytoskeleton directly and/or by tri-methylation of histone H3K27 (H3K27me3) and subsequent repression of genes. To test the relative importance of EZH2 for these processes we will over-express EZH2 variants in muSCs with different sub-cellular localisations and activities. We will also collect RNA-seq and ChIP-seq data from isolated muSCs to identify genes showing EZH2-dependent expression and H3K27me3 modifications. This will allow us to ask if EZH2 controls cell migration by H3K27me3-dependent gene regulation, and by including later time points, how EZH2-dependent control of migration relates to onset of muSC differentiation.

Important outputs from our work are anticipated to be:
1. the identification of genes that are important for muscle stem cell function.
2. an understanding of the importance of EZH2 in controlling muscle stem cell migration and regeneration.

Immediate beneficiaries of the project will include:
- researchers or companies aiming to manipulate muscle stem cell function in vivo who wish to know which genes are associated with migratory behaviours
- developers of drugs that target EZH2 activity who wish to know which genes may be affected in muscle stem cells
- the postdocs employed on the grant who will gain valuable non-academic skills
Long-term beneficiaries will include:
- clinicians and commercial entities aiming to treat people with muscle diseases or weaknesses who will gain a better understanding of which genes are associated with effective muscle stem cell function
- charities who wish to understand why certain treatments prescribed to elderly or sick people may lead to muscle weakness e.g. Age concern, ARUK, BHF
- academic and commercial entities designing more effective wound healing or regenerative medicine approaches will learn which targets of EZH2 to manipulate to enhance muscle repair
- academic and industrial communities who use/develop image processing approaches for quantitative characterisation of complex multi-dimensional imaging datasets
- educationalists interested in promoting scientific research, who will have access to the stunning images we will generate in the course of this project

Results from this project would be of value to medical researchers and pharmaceutical companies interested in designing therapies to promote stronger muscle or to design treatments for cancers associated with elevated EZH2 activity. As EZH2 is known to be involved in the migration of many cells types (cancer, immune, development, wound healing), it is important to understand how any drugs generated by the pharmaceutical industry that target this pathway may affect the muscle repair process. Conversely, it would be valuable for medical practitioners or pharmaceutical companies interested in designing treatments for invasive cancers to understand the importance of EZH2 in controlling cell migration in vivo.
By understanding the importance of EZH2 in muscle repair, we will have identified a potential for designing interventions that promote more effective regeneration. This would be of immense interest to clinicians, charities and companies interested in promoting better muscle strength in people with a number of diseases (cancer, chronic diseases such as muscle dystrophies) or in ageing people who are infirm. It would also be of interest to companies marketing products to athletes who wish to swiftly repair muscle after exercise-induced injury.

The postdoc and bioinformatician employed in this project will benefit by learning new skill sets and gaining an insight into the topical area of regenerative medicine.
By working in an interdisciplinary team they will also be exposed to new techniques and approaches that will help inform their future career paths. They will also gain valuable transferable skills in project and time management, team work and presentation of results.