Unravelling the microRNA-chromatin remodelling circuitry that drives myogenesis

THE 'BIG' PICTURE: Multi-cellular organisms contain many distinct cell types with very specialized functions. For example, we need skeletal muscle to be able to move while our skin prevents dehydration and protects us from injury and infections. Amazingly all these different cells arise from a single cell, the fertilized egg. The development of an embryo begins when the egg starts dividing to give rise to many cells. The cells are initially multi-potent, but they gradually become specialized and restricted to perform specific tasks. This cell-lineage restriction is regulated by factors that influence the activity of genes and regulate the accessibility of the genome in the nucleus of the cell. Some genes are 'off' and DNA is packaged tightly, other regions are more loosely packaged and therefore 'open' and accessible to factors that can switch a gene 'on'. If a gene is 'on', it means the gene will be expressed and actively transcribed. The regulation of gene activity is crucial for cells to become specialised in their functions.

THE QUESTION: We are interested in the molecules that control the development of muscle in an embryo. Why do progenitor cells develop into muscle rather than other related cell types, such as fat, cartilage, bone or connective tissue? Our studies focus on a class of RNA molecules, which we found regulate the factors that control the accessibility of the genome. These non-coding RNAs were discovered recently and because they are very small, they were called 'microRNAs' (miRs), they control the translation of genes into protein - a fundamental job required in all cells.

WHY IS THIS IMPORTANT? Regulating the accessibility of the genome is very important during cellular differentiation in embryo development. Understanding this genetic programme is also important for muscle regeneration and repair after injury or long-term bed rest. The process of cell lineage determination is also crucial for the differentiation of stem cells, or for the reprogramming of already specialized cells towards a different fate. We know many of the components involved, but we still do not understand in detail how they function, or how they are put together. We recently discovered that microRNAs influence the composition of these 'reprogramming' complexes and we have a well-defined system in which to study this more systematically. This offers the unique opportunity to identify all of the important players and will provide a deeper mechanistic insight at the molecular level. This is needed, not only to fully understand how specialized cells form in developing embryos, but also to be able to use stem cells in regenerative medicine and tissue engineering, emerging fields of increasing importance and with significant future potential for medicine and health. We will learn how an embryo makes normal, healthy, working muscle and this will in the long-term benefit people who suffer from various conditions that affect muscle health or help to alleviate age related muscle-loss.

EXPERIMENTAL MODEL SYSTEM: We use very early chick embryos, which are very similar in morphology to early human embryos, to investigate the roles of important microRNAs present in muscle. We will use protocols to identify how they affect the the factors that control the accessibility of the genome in naïve and differentiating muscle cells. We have experience with these state-of-the-art molecular methods in the chick embryo, an accessible experimental system and we have assembled a highly skilled team of researchers to execute this programme of research.

Recruitment of chromatin regulators to specific genomic loci is crucial for cell fate decisions during development. We have documented a dramatic effect of myomiRs, miR-1/206 and miR-133, on the composition of BAF/Brg1 chromatin remodeling complexes, which contain one of three alternate BAF60 variants bound to the core ATPase, Brg1. We showed that myomiRs inhibit expression of two variants, BAF60a and BAF60b, thus enabling incorporation of the BAF60c variant and effective activation of the myogenic programme.
The project will address a number of exciting new questions resulting from this work.
(1) Additional miRNAs are predicted or confirmed to target additional subunits of the chromatin remodeling complex. What is the functional significance of these interactions for myogenic lineage specification? This project will examine the intriguing possibility that composition of the BAF/Brg1 chromatin remodeling complexes is fine-tuned by miRNAs. A combination of in vitro and in vivo approaches will investigate this hypothesis in developing somites of chick embryos (Objectives 1, 2a, 2b).
(2) All three BAF60 variants (a, b and c) are present in early epithelial somites, but in committed myoblasts BAF60c is predominantly complexed with Brg1. What is the function of different BAF60-containing complexes in myogenic progenitors? We will use chromatin immunoprecipitation followed by next-generation sequencing (ChIPseq), to identify genome-wide the loci associated with alternative complexes (Objective 3).
Specific tasks:
-validate specific miRNA target gene interactions and examine their functional significance for muscle development
-determine expression and function of BAF subunits during embryo myogenesis (PCR, in situ, immune staining)
-characterize somite specific regulatory elements for physiological level expression (existing ATACseq profiles)
-perform ChIPseq with endogenous Brg1, tagged Brg1, and tagged BAF60a/b and c proteins in developing somite

INTRODUCTION: The role of miRNAs in vertebrate development and in particular in skeletal muscle development and differentiation is currently not understood in detail. In particular, nothing is known about how miRs orchestrate embryonic myogenesis through targeting components of the BAF/Brg1 chromatinremodeling complex. We recently obtained intriguing results for a role of myomiRs in regulating BAF60 subunits "a" and "b", and thus enabling selective incorporation of BAF60c. This project will build and expand on this exciting data to investigate the role of myomiRs and other miRNAs in regulating BAF-subunit switching in developing somites. miRNAs represent a novel mechanism by which gene expression can be tightly controlled and this project will provide a mechanistic link between microRNAs and chromatin remodeling complexes during cell lineage determination.

BASIC SCIENCE: This is a basic science project; it addresses fundamental questions about epigenetic mechanisms that control embryonic development, specifically the role of chromatin remodeling factors and their regulation by microRNAs in skeletal myogenesis. Similar mechanisms will be important for stem cells and therefore findings will be relevant to human and animal health. The project is most likely to have longer-term impacts in the biomedical and health science areas.

IMPACT FOR HUMAN (AND ANIMAL) HEALTH AND APPLIED TRANSLATIONAL RESEARCH: Chromatin remodeling is of fundamental importance for cellular differentiation programmes in humans and animals. Deregulation of this process contributes to developmental abnormalities and leads to diseases in the adult. Understanding the molecular mechanisms that regulate the specificity of the transcriptional and cellular response in different cells will underpin the development of strategies aimed at the use of stem cell-based therapies in regenerative medicine. This includes for example skeletal muscle regeneration.

IMPACT ON GENERATION OF A SCIENTIFICALLY LITERATE WORKFORCE: This project will help train the next generation of biomedical researchers by supporting the research career of a postdoctoral researcher, by training a technical assistant and by involving a bioinformatician in cutting edge genomics techniques. Indirect benefits will come from their contributions to a research-led environment for teaching of postgraduate and undergraduate students. The PIs laboratory has an excellent training record and researchers have successfully obtained independent positions in academia or been appointed in industry (e.g. University of Nottingham, University of Liverpool, Pfizer, Genentech/Roche).

IMPACT ON WIDER PUBLIC: Members of the public are interested in scientific progress, particularly when relevant to health. Increased understanding of gene regulatory mechanisms during skeletal myogenesis will be important for the healthy maintenance of this tissue. Our discoveries are likely to be relevant for regenerative medicine and stem cell science. New information gathered in this project will be disseminated to a general audience in an accessible form using web based tools.

IMPACT ON PHARMA AND BIOTECH INDUSTRY: RNA based therapeutics are of potential importance for drug development and longer-term beneficiaries will be biotech and pharmaceutical industry. The project will increase our knowledge base, a prerequisite to design more sophisticated drugs targeting specific pathways in specific contexts.

CONCLUSION: This study will directly and indirectly contribute to both improved health and economic wealth.