Nuclear Envelope Directed Genome Organization in Myogenesis and Emery-Dreifuss Muscular Dystrophy

Each individual's genome is identical in all the cells of their bodies. Yet these cells are able to achieve incredibly different forms from those making the heart to the liver or brain. There are many aspects of genome regulation that enable different subsets of the genes in the genome to be expressed to help each tissue develop. One of the least understood of these is how the position of a gene in the nucleus contributes to its regulation. Many genes important for tissue development move from the edge of the nucleus to the interior concomitant with their being expressed. Other genes that are antagonistic to tissue development move to the edge of the nucleus and get shut down. In general the genes located at nuclear periphery tend to be shut down and there is evidence that disruption of nuclear spatial genome organisation underlies some developmental defects and diseases.
We have previously identified proteins located at the edge of the nucleus that are important for establishing spatial genome organisation. Some of these proteins were found only in liver and affected genome organisation in liver, while others were found only in fat and affected genome organisation in fat and yet others were found only in muscle and affected genome organisation in muscle. We found five such proteins in muscle and different ones affected distinct subsets of genes important for muscle development. In separate work we were studying the muscle-wasting disease Emery-Dreifuss muscular dystrophy. Mutations in seven different proteins have been found to cause this disease, but a little over half of clinically diagnosed Emery-Dreifuss patients do not have mutations in these seven proteins and so must have other causes. We analysed 62 such Emery-Dreifuss patients and found that 11 of them have mutations in four of the five genome-organising muscle proteins.
The goal of the proposed project is to test whether the mutations found can explain the pathology of the disease by engineering the mutations into a cell system where muscle development can be followed in a dish and also into mice. The former can give information about when and where specific defects occur in individual muscle cells while the latter can give information about systemic muscle function, metabolism and pathology. Levels of muscle metabolic markers will be assayed as well as markers of other tissues in case the mutant genome-organising proteins fail to shut down genes from other tissues as this could help explain Emery-Dreifuss muscle defects. Muscle size, muscle regeneration and muscle stem cell function will also be assayed. To ascertain whether genome organisation defects can explain the cell and tissue pathology, the expression of different genes and other genome regulatory elements will be determined by genome-wide sequencing approaches and the position of genes in normal versus mutant cells and tissues observed using microscopy. The above studies will be done both using mutants in the genome-organising proteins and in other proteins that cause Emery-Dreifuss muscular dystrophy to determine if the underlying defects match. Finally, we will investigate the mechanism behind the genome organisation changes by making specific modifications to the genome around the affected genes and determining whether these modifications block the normal gene movements to or from the nuclear edge. This study should both provide insights into the fundamental mechanisms underlying genome organisation and also identify the gene and metabolic pathways altered in Emery-Dreifuss muscular dystrophy. This combined understanding could lead to therapeutic approaches focused on restoring the proper metabolic balance by supplying missing metabolites and enzymes.

One of the least understood remaining aspects of genome regulation to decipher is how tissue-specific patterns of spatial genome organisation are established and the mechanism by which they alter gene expression. Though general genome organisation derives from heterochromatin interactions with lamins and nuclear envelope transmembrane proteins (NETs), the genome changes orchestrated by tissue-specific NETs do not target heterochromatin. Several recent studies found that defective spatial genome organisation can lead to developmental defects or disease and we recently found that Emery-Dreifuss muscular dystrophy (EDMD) patients with no mutations in previously linked proteins have mutations in muscle specific NETs (mNETs) that direct muscle-specific genome organisation patterns. Thus we propose both to study these mutations in context of the disease and use EDMD as a model to study mechanisms of spatial genome organisation.
Specifically, we will engineer candidate mNET mutations into the C2C12 myogenesis system to study their effect on genome organisation, gene expression and differentiation in a readily manipulable tissue culture experimental system. The strongest mutations will be engineered into mice to study systemic and metabolic effects and the penetrance of genome organisation effects in situ. Of note, our preliminary studies found both myogenic genes and several loci for regulatory RNAs repositioning during myogenesis in a manner dependent on mNET function. Regulatory RNAs have not been investigated with respect to EDMD and we will analyse miRNAs and lncRNAs for positioning, expression and effects on myogenesis and pathology in our experimental system and in patient biopsies if available. Finally, we will investigate how nuclear envelope tethering of genome regions can influence internal nuclear organisation by specifically modifying adjacent sequences to shorten or extend the length between a tether site and activated gene in the nuclear interior.

Who will benefit from this research? How would they benefit from this research?
(1) Scientific community: This is a basic biomedical research project and as such the primary beneficiaries of the outputs arising from this research will be from the international scientific community. How they will benefit from this research is outlined above.
(2) Non-academic beneficiaries: In both the short and long term, the main beneficiaries of this research will be patients with Emery-Dreifuss muscular dystrophy (EDMD) along with those overseeing their care. Moreover, as there are likely overlapping principles underlying EDMD and other nuclear envelope disorders - a principle underscored by our independent studies showing genome misorganisation and misregulation with fat-, liver-, and blood-specific NETs - the findings obtained from this work will potentially help resolve other nuclear envelopathies/ laminopathies that include Limb-girdle muscular dystrophy, cardiomyopathy, lipodystrophies, neuropathy, dermopathy, osteopoikilosis, mandibuloacral dysplasia, Pelger-Huet anomaly, and several premature ageing progeroid syndromes. The outputs of the research from this project will benefit these groups of patients by:
(i) increasing the level of understanding of the molecular mechanisms leading to phenotypic manifestation of EDMD.
(ii) availability of model systems that can be used to test for reversibility of the phenotype/ disease state thus offering possibilities for translational research and therapeutic approaches.
(iii) the first investigation of regulatory RNAs in EDMD that could lead directly to therapeutics involving administration of miRNAs.
(3) Benefits to society and the UK economy: This research will provide outstanding training opportunities and acquisition of new multidisciplinary professional research skills by staff employed on the project. Of note, for those previously working on this project, student Mike Robson is now a Henry Wellcome Fellow in the laboratory of Stefan Mundlos (Max Planck-Berlin), student Nikolaj Zuleger has his own grant working in the laboratory of Marino Zerial (Max Planck-Dresden), and post-doctoral research associate Peter Meinke is now a group leader at Ludwig Maximilians University in Munich. The project provides potential for staff to develop skills using in vivo and tissue culture differentiation approaches, molecular biology methods in genome engineering, computational and bioinformatic analyses of the high-throughput sequencing data (RNA-Seq, DamID, Hi-C), quantitative and statistical analyses, and personal skills required in the modern work environment. These training opportunities will broaden their horizon and improve their employment potential in diverse sectors. This will ensure the international competitiveness of UK biomedical research and will benefit the society and the UK economy.
(4) Beneficiaries in the area of intellectual property: Although this is largely a basic biomedical research project, tools for data analysis, plasmids, mouse strains, cell lines and assays may be
useful in a broader context and could be potentially commercialised.
(5) General public: The dissemination of the results of the project via outreach events and press-releases will benefit the general public as they will enhance the public understanding of the importance of cutting-edge MRC-funded biomedical research and the benefits that such research brings in longer-term to society and patients suffering from EDMD and other nuclear envelopathies/ laminopathies.