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

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description Board member of the European Laminopathies Network
Geographic Reach Europe 
Policy Influence Type Membership of a guideline committee
 
Description Continuation of ENMC workshop neuromuscular laminopathies
Geographic Reach Multiple continents/international 
Policy Influence Type Contribution to new or Improved professional practice
 
Description Continuing ENMC Workshop on muscular laminopathies
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
 
Description ENMC Workshop on Skeletal Muscle Laminopathies
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Funding for EMBO Workshop primary organiser and grant writer
Amount € 34,000 (EUR)
Organisation European Molecular Biology Organisation 
Sector Charity/Non Profit
Country Germany
Start 01/2019 
End 04/2019
 
Description Identification of convergent gene regulatory pathways as novel targets in Emery-Dreifuss muscular dystrophy
Amount £118,654 (GBP)
Funding ID 18GRO-PG24-0248 
Organisation Muscular Dystrophy UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2019 
End 12/2021
 
Title NET39/PLPP7 R252P mutant mouse model for Emery-Dreifuss muscular dystrophy 
Description This is a mouse carrying a point mutation we identified in human patients with Emery-Dreifuss muscular dystrophy. As the grant only started 7 months ago, this mouse model has been ordered and is in the process of being generated now. Based on our in vitro myogenesis data we expect this mouse to be a model for particularly Emery-Dreifuss muscular dystrophy, but likely also other nuclear envelope-linked muscular dystrophies such as Limb-Girdle muscular dystrophy as we think that both muscular dystrophies are caused by disruption of complexes containing lamin A, emerin and other nuclear envelope transmembrane proteins of which NET39/PLPP7 is a muscle-specific component that can cause the pathology to be focused in muscle. This model will be used heavily in our work on the grant and will be deposited in Central Bio-research Services (CBS) Transgenic Core at the University of Edinburgh (for local researchers access once it arrives) and once published it will be also donated to JAX Repository (Jackson Laboratories) where it will be made available to other researchers. If they are interested, this model will also be donated to 'Shared Ageing Research Models" www.ShARMUK.org which is biobank supported by Wellcome Trust in partnership with MRC Harwell and CIMA (Centre for Integrated research into Musculoskeletal Ageing) 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2019 
Provided To Others? No  
Impact As the mouse is in the process of being made for the grant that just started there are no outputs as yet. 
 
Title NET39/PLPP7 conditional knockout model for nuclear envelope-linked muscular dystrophies 
Description As the grant only started 7 months ago, this mouse model has been ordered and is in the process of being generated now. Based on our in vitro myogenesis data we expect this mouse to be a model for particularly Emery-Dreifuss muscular dystrophy, but likely also other nuclear envelope-linked muscular dystrophies such as Limb-Girdle muscular dystrophy as we think that both muscular dystrophies are caused by disruption of complexes containing lamin A, emerin and other nuclear envelope transmembrane proteins of which NET39/PLPP7 is a muscle-specific component that can cause the pathology to be focused in muscle. This model will be used heavily in our work on the grant and will be deposited in Central Bio-research Services (CBS) Transgenic Core at the University of Edinburgh (for local researchers access once it arrives) and once published it will be also donated to JAX Repository (Jackson Laboratories) where it will be made available to other researchers. If they are interested, this model will also be donated to 'Shared Ageing Research Models" www.ShARMUK.org which is biobank supported by Wellcome Trust in partnership with MRC Harwell and CIMA (Centre for Integrated research into Musculoskeletal Ageing) 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2019 
Provided To Others? No  
Impact As the model is in the process of being made for the grant no impacts have been generated as yet. 
 
Title Tmem38a D260N mouse 
Description This was generation of a CRISPR mouse carrying the D260N mutation we found in Emery-Dreifuss muscular dystrophy patients. We are currently characterising this mouse as part of this award. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2021 
Provided To Others? No  
Impact We are currently characterising this mouse as a model for Emery-Dreifuss muscular dystrophy. Thus, at this point there has not been an impact, but we anticipate that there will be once the experiments are finished and it is published and deposited for general use. 
 
Title mouse model for EDMD with Tmem38a D260? mutation 
Description CRISPR mouse made in parallel with the D260N point mutation. This mouse has the amino acid deleted while maintaining reading frame as opposed to changed. Both were separate mutations found in different families with Emery-Dreifuss muscular dystrophy. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2020 
Provided To Others? No  
Impact This model has not yet been published or had an impact because it is currently being amplified to be characterised. Due to Covid both these new mouse models were not able to be amplified or tested yet. However, once published the mice will be made available in a mouse repository. 
 
Title Adipogenesis Superseries matched DamID and microarray data 
Description In characterising changes in spatial genome organisation and its consequences for genome function during tissue differentiation or activation we have generated several matched DamID, microarray, and Hi-C datasets, all of which have been/ are being made publicly available upon publication. This specific dataset is for adipogenesis using the 3T3-L1 model system and a TMEM120A knockout mouse we generated. 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? Yes  
Impact This study has significant impact both because of the lipodystrophic phenotype of the mouse and as there are many ways in development that the spatial genome and expression changes during myogenesis and adipogenesis mirror one another. This study was the subject of press reports with an altimetric score of 25 when it was published as: Czapiewski, R., Batrakou, D. G., de las Heras, J. I., Carter, R. N., Sivakumar, A., Sliwinska, M., Dixon, C. R., Webb, S., Lattanzi, G., Morton, N. M., and Schirmer, E. C. (2022) Genomic loci mispositioning in Tmem120a knockout mice yields latent lipodystrophy. (note original version Gene/miRNA locus mispositioning in Tmem120a-/- mice yields a lipodystrophic pathology. (Preprint version: https://www.biorxiv.org/content/10.1101/2021.04.12.439495v1) Nat Commun 13(1), 321.doi:10.1038/s41467-021-27869-2. PMID:35027552 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE150507
 
Title Hi-C datasets from NET39/PLPP7 mouse Emery-Dreifuss muscular dystrophy models 
Description We generated Hi-C datasets from skeletal muscle using 3 wild-type, 3 PLPP7 knockout, and 2 PLPP7 disease point mutation mice. As for the RNA-Seq datasets, these data will be made publicly available upon publication of the study; however, this has been delayed by not having a post to finish the project since the grant was not renewed. Note that this was generated after the end of the grant because we pre-paid for the Hi-C, but the ex-PDRA was not able to get time to come in to the lab to process the samples from the freezer until 2023. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? No  
Impact The likely impact is currently under assessment as the ex-PDRA can only come in occasionally to try to finish his paper; however, we can say at this point that the quality of the data is good and that based on the fact that we are finding many interesting facets of genome organization during memory T-cell activation from the Hi-C data generated in our current collaborative MRC grant on HIV integration sites, it is likely that same will apply for these datasets. 
 
Title LaminB1-DamID analysis of differentiating 3T3L1 mouse pre-adipocytes with knock-down of Tmem120a and Tmem120b. 8 samples 
Description This is the DamID datasets for nuclear envelope associated chromatin changes during adipogenesis +/- the presence of fat-specific genome organizing protein Tmem120A 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? Yes  
Impact This mouse model resulted in a lipodystrophy pathology, consistent with our hypothesis that Tmem120A mediates Lamin A-linked lipodystrophies - especially as we separately found that wild-type lamin A binds to Tmem120A while lamin A carrying lipodystrophy point mutations exhibits greatly reduced binding. This study was the subject of press reports with an altimetric score of 25 when it was published as: Czapiewski, R., Batrakou, D. G., de las Heras, J. I., Carter, R. N., Sivakumar, A., Sliwinska, M., Dixon, C. R., Webb, S., Lattanzi, G., Morton, N. M., and Schirmer, E. C. (2022) Genomic loci mispositioning in Tmem120a knockout mice yields latent lipodystrophy. (note original version Gene/miRNA locus mispositioning in Tmem120a-/- mice yields a lipodystrophic pathology. (Preprint version: https://www.biorxiv.org/content/10.1101/2021.04.12.439495v1) Nat Commun 13(1), 321.doi:10.1038/s41467-021-27869-2. PMID:35027552 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE150053
 
Title RNA-Seq dataset of Emery-Dreifuss muscular dystrophy patients 
Description We analysed gene expression levels of a wide range of Emery-Dreifuss muscular dystrophy patients ranging from lamin and emerin mutations to nesprin, SUN, FHL1, and newly described Emery-Dreifuss muscular dystrophy mutations in muscle-specific nuclear envelope transmembrane proteins. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact Interestingly, the analysis of this data is consistent with some of the biomarkers being investigated with the genes changed in most patients relating principally to cytokine production and fibrosis, however with more specific genes identified. This analysis also revealed several previously uninvestigated defects in oxidative and glucose metabolism and splicing. This study is currently being written up for publication and the datasets will be publicly deposited upon publication. The datasets have already been shared freely for analysis with other researchers in the field and the results shared in the ENMC supported effort to establish diagnostic biomarkers and standards of care for laminopathies. The datasets were made fully publicly available through the NCBI GEO database when the paper was published: de las Heras, J. I., Todorow, V., Krecinic-Balic, L., Hintze, S., Czapiewski, R., Webb, S., Schoser, B., Meinke, P., and Schirmer, E. C. (2023) Metabolic, fibrotic, and splicing pathways are all altered in Emery-Dreifuss muscular dystrophy spectrum patients to differing degrees. (Preprint version https://biorxiv.org/cgi/content/short/2022.05.20.492778v1) Hum Mol Genet. 25:ddac264. PMID:36282542 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE204804
 
Title RNA-Seq datasets from EDMD mouse models 
Description We have performed RNA-Seq on skeletal muscle and heart from our mouse models for EDMD. This data has been analysed and has many parallels with our previous RNA-Seq analysis of human EDMD patients as well as reflecting genome organisation changes in the model and will be included in the paper; however, since the grant was not renewed we do not have anyone currently employed to finish the paper and the PDRA can only come in typically for only a couple days every couple months to try to finish the paper, so it has not been published yet. Once it is published the data will be made publicly available through the NCBI GEO repository. 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? No  
Impact Analysis of these RNA-Seq datasets from our mouse models of EDMD against the RNA-Seq datasets we have performed on EDMD patients is revealing for understanding the disorder and the critical pathways to focus on for treatments to ameliorate pathologies. 
 
Title RNA-Seq datasets from fat from Tmem120A mouse lipodystrophy model 
Description A conditional fat-specific knockout of Tmem120A was generated in mice that has a lipodystrophy phenotype. RNA-Seq analysis was performed on fat deposits from control and knockout mice. 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? Yes  
Impact The Tmem120A knockout mouse has a lipodystrophy phenotype that phenocopies the human pathology of Lamin-linked lipodystrophy mutations better than the lamin A mouse models. This, together with our findings that: 1) Tmem120A is a nuclear membrane protein that interacts with lamin A, 2) Tmem120A regulates genome organization changes during adipogenesis with many of the genes under its positional/expression regulation being ones that could explain the lipodystrophy phenotype of lamin A-linked lipodystrophies, 3) many of these normal Tmem120A regulated gene repositionings/expression changes fail to occur in human lamin A-linked lipodystrophy patients, and 4) lipodystrophy point mutations in lamin A exhibit greatly weakened binding to Tmem120A, all suggest that Tmem120A mediates the pathology in Lamin-linked lipodystrophy. These findings were published in: Czapiewski, R., Batrakou, D. G., de las Heras, J. I., Carter, R. N., Sivakumar, A., Sliwinska, M., Dixon, C. R., Webb, S., Lattanzi, G., Morton, N. M., and Schirmer, E. C. (2022) Genomic loci mispositioning in Tmem120a knockout mice yields latent lipodystrophy. (note original version Gene/miRNA locus mispositioning in Tmem120a-/- mice yields a lipodystrophic pathology. (Preprint version: https://www.biorxiv.org/content/10.1101/2021.04.12.439495v1) Nat Commun 13(1), 321.doi:10.1038/s41467-021-27869-2. PMID:35027552 which was also the subject of press reports. 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE147016
 
Title Transcriptome analysis of differentiating 3T3L1 mouse pre-adipocytes with knock-down of Tmem120a and Tmem120b. 11 samples 
Description Gene expression analysis of adipocyte differentiation +/- the presence of fat-specific genome-organising nuclear membrane proteins Tmem120A and Tmem120B. 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? Yes  
Impact These datasets link with genome organization datasets to show that many genes that move between the nuclear envelope and the nuclear interior during adipocyte differentiation depend on the nuclear membrane Tmem120 proteins for that movement and change expression associated with that movement. This is relevant then to the mouse model we generated deleting Tmem120A which has a lipodystrophy phenotype and argues that lamin A-linked lipodystrophy pathologies are mediated through Tmem120A. This study was the subject of press reports with an altimetric score of 25 when it was published as: Czapiewski, R., Batrakou, D. G., de las Heras, J. I., Carter, R. N., Sivakumar, A., Sliwinska, M., Dixon, C. R., Webb, S., Lattanzi, G., Morton, N. M., and Schirmer, E. C. (2022) Genomic loci mispositioning in Tmem120a knockout mice yields latent lipodystrophy. (note original version Gene/miRNA locus mispositioning in Tmem120a-/- mice yields a lipodystrophic pathology. (Preprint version: https://www.biorxiv.org/content/10.1101/2021.04.12.439495v1) Nat Commun 13(1), 321.doi:10.1038/s41467-021-27869-2. PMID:35027552 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE150022
 
Title miRNA-Seq of EDMD patients 
Description The same set of EDMD patients for which RNA-Seq was performed in the first entry was used for miRNA-Seq as well. Interestingly, the miRNA-Seq revealed that many of the miRNAs that were altered in levels have roles in regulating the gene pathways that were changing in the RNA-Seq data. Thus, this also raises levels of certain miRNAs as another potential biomarker for use in EDMD diagnostics and prognostics. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact As for the RNA-Seq datasets these data have been shared with researchers at the ongoing ENMC Workshop of neuromuscular laminopathies and are being used in developing better clinical and diagnostic approaches. This data has now been published together with the RNA-Seq data in the manuscript: de las Heras, J. I., Todorow, V., Krecinic-Balic, L., Hintze, S., Czapiewski, R., Webb, S., Schoser, B., Meinke, P., and Schirmer, E. C. (2023) Metabolic, fibrotic, and splicing pathways are all altered in Emery-Dreifuss muscular dystrophy spectrum patients to differing degrees. (Preprint version https://biorxiv.org/cgi/content/short/2022.05.20.492778v1) Hum Mol Genet. 25:ddac264. PMID:36282542 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE204826
 
Title nuclear envelope partner protein datasets of STING 
Description This dataset is the first identification of nuclear envelope-specific partners of the protein originally identified in the nuclear envelope as NET23 that has multiple sub-cellular localisations and now mostly goes by the name STING. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact This was the first identification of nuclear-specific partners for STING. While the field initially ignored the original identification of this protein in a nuclear envelope fraction, the recent demonstration of functions in DNA-damage repair and cancer along with cGAS in the nucleus have made this a very important finding. It was published in: Dixon, C. R., Malik, P., de las Heras, J. I., Saiz-Ros, N., de Lima Alves, F., Tingey, M., Gaunt, E., Richardson, A. C., Kelly, D. A., Goldberg, M. W., Towers, G. J., Yang, W., Rappsilber, J., Digard, P., and Schirmer, E. C. (2021) STING nuclear partners contribute to innate immune signaling responses. iScience 24(9), 103055. PMID:34541469 
URL https://doi.org/10.17632/xgwb62nznh.1
 
Description A. Mark Evans on calcium flux in nuclear envelope invaginations of pulmonary smooth muscle aorta cells 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided expertise on the nuclear envelope, reagents, and performed staining assays, FISH, and RNA-Seq and bioinformatic analysis.
Collaborator Contribution Professor Evans team did the rest of the work, particularly all the calcium flux and most of the eventual stainings used in the paper.
Impact Duan, J., Navarro-Dorado, J., Clark, J. H., Kinnear, N. P., Meinke, P., Schirmer, E. C., and Evans, A. M. Ca2+ signals by tunneling across a cell-wide circuit of cytoplasmic nanocourses demarcated by SR. Submitted.
Start Year 2014
 
Description Benedikt Schoser for patient samples 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Country Germany 
Sector Academic/University 
PI Contribution Sequencing patient samples and performing experiments with patient cells for protein and gene localisations.
Collaborator Contribution Providing patient samples and maintaining contact with patients.
Impact Meinke, P., Kerr, A. R. W., Czapiewski, R., de las Hera, J. I., Dixon, C. R., Harris, E., Kolbel, H., Muntoni, F., Schara, U., Straub, V., Schoser, B., Wehnert, M., and Schirmer, E. C. (2019) A multistage sequencing strategy pinpoints novel candidate alleles for Emery-Dreifuss muscular dystrophy and supports gene misregulation as its pathomechanism. EbioMedicine 102587.doi:10.1016/j.ebiom.2019.11.048. Note the Lancet journals also published a commentary highlighting this study by Hui Xiong: https://www.thelancet.com/action/showPdf?pii=S2352-3964%2819%2930835-7 This study collected unlinked Emery-Dreifuss muscular dystrophy patients, sequenced exomes on those for which enough family members were represented to do partial linkage analysis, then generated a primer library from this plus other candidate genes based on our muscle nuclear envelope proteomics and genome organization studies and sequenced additional unlinked patients. We identified many new clear and strong candidate Emery-Dreifuss alleles, finding also that most were in genome organizing muscle-specific nuclear membrane proteins and the mutations destroy this function. The novel approach used in this study can be applied to other genetically heterogeneous orphan diseases. Note that this study presents a new iterative approach for disease allele identification in genetically diverse orphan diseases and could have a direct impact on approaches used in future and the collaboration that started with the ending Wellcome SRF is being continued in the MRC and MDUK grants.
Start Year 2011
 
Description Glenn E. Morris for making antibodies 
Organisation Oswestry Hospital
Country United Kingdom 
Sector Hospitals 
PI Contribution We prepare constructs and/or protein for antibody generation and do other experimental work.
Collaborator Contribution Professor Morris's team generates monoclonal antibodies for the NETs we identified.
Impact Zuleger, N., Boyle, S., Kelly, D. A., de las Heras, J., Lazou, V., Korfali, N., Batrakou, D. G., Randles, K. N., Morris, G. E., Harrison, D. J., Bickmore, W. A., and Schirmer, E. C. (2013) Specific nuclear envelope transmembrane proteins can promote the location of chromosomes to and from the nuclear periphery. Genome Biol. 14(2), R14. PMID: 23414781 Professor Morris made antibodies to NET45 for the above paper supporting my Wellcome SRF and is currently trying to make additional antibodies to muscle NETs with links to Emery-Dreifuss muscular dystrophy in support of the MRC and MDUK grants.
Start Year 2011
 
Description Jan Lammerding for cellular mechanics 
Organisation Cornell University
Country United States 
Sector Academic/University 
PI Contribution We generate cell lines with point mutations or knockdowns/knockouts of muscle NETs for Professor Lammerding to test for cell and nuclear mechanical stability
Collaborator Contribution Professor Lammerding's lab tests cell lines we make for their mechanical stability and their speed/ability to move through constricted spaces.
Impact In process
Start Year 2019
 
Description Laurence Florens mass spectrometry 
Organisation Stowers Institute for Medical Research
Country United States 
Sector Academic/University 
PI Contribution Preparing samples and other cell biology and biochemistry experiments.
Collaborator Contribution Mass spectrometry analysis of samples.
Impact Laurence Florens is one of the best technical mass spectrometrists in the world and has supported several papers for my Wellcome SRF and we continue to collaborate where she will analyse samples for both the MRC and MDUK grants. Korfali, N., Wilkie, G. S., Swanson, S. K., Srsen, V., de las Heras, J., Batrakou, D. G., Malik, P., Zuleger, N., Kerr, A. R. W., Florens, L., and Schirmer, E. C. (2012) The nuclear envelope proteome differs notably between tissues. Nucleus 3(6), 552-564. PMID: 22990521 Korfali, N., Florens, L., and Schirmer, E. C. (2016) Isolation, proteomic analysis and microscopy confirmation of the liver nuclear envelope proteome. Methods Mol Biol. 1411, 3-44. PMID: 27147032 Capitanchik, C., Dixon, C., Swanson, S. K., Florens, L., Kerr, A. R. W., and Schirmer, E. C. (2018) Analysis of RNA-Seq datasets reveals enrichment of tissue-specific splice variants for nuclear envelope proteins. Nucleus 9(1), 410-430. PMID:29912636 Saiz-Ros, N., Czapiewski, R., Epifano, I., Stevenson, A., Swanson, S. K., Dixon, C. R., Zamora, D. B., McElwee, M., Vijayakrishnan, S., Richardson, C. A., Dong, L., Kelly, D. A., Pytowski, L., Goldberg, M. W., Florens, L., Graham, S. V., and Schirmer, E. C. (2019) Host vesicle fusion protein VAPB contributes to the nuclear egress stage of herpes simplex virus type-1 (HSV-1) replication. Cells 8(2), pii: E120 doi: 10.3390/cells8020120. PMID:30717447 Salpingidou, G., de las Heras, J. I., Swanson, S. K., Florens, L., Schirmer, E. C., Hutchison, C. J., and Goldberg, M. W. An integrated model for nuclear envelope assembly based on proteomic analysis of distinct nuclear membrane precursor vesicles. In preparation.
Start Year 2011
 
Description Nik Morton for metabolism studies 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution We did pretty much all the actual work on the study, generating animals and doing experiments.
Collaborator Contribution Professor Morton's lab provided intellectual input, training on a number of metabolism assays, and use of their metabolic cages and Seahorse machine.
Impact Our adipocyte-specific Tmem120A knockout mouse fails to accumulate white adipose tissue while accumulating brown adipose tissue on a high fat diet and has insulin resistance and glucose intolerance and other characteristics of a cryptic lipodystrophy that only presents on high-fat diet. We have finished all mouse work and bioinformatics analysis and are currently in the process of writing up the paper while wrapping up the last FISH experiments. For our muscle work we continue collaborating with Professor Morton for metabolism experiments and we are currently growing the animal models we have made in our MRC award for the first experiments.
Start Year 2012
 
Description Peter Meinke collaborator for investigating metabolic and mitochondrial functions in EDMD patient cells 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Country Germany 
Sector Academic/University 
PI Contribution We performed RNA-Seq and miRNA-Seq on a pool of EDMD patients and performed pathway analysis that highlighted several pathways uniformly altered in all patients. One of those pathways was metabolic and mitochondria function.
Collaborator Contribution Based on our RNA-Seq and miRNA-Seq analysis of EDMD patients, we are collaborating with former PDRA Peter Meinke who now has own lab at the LMU to do metabolic and mitochondria analysis in patient samples to confirm the functional deficits are uniform across a wider range of EDMD patients.
Impact We have now concluded the experimental analysis and are currently writing up a paper that describes the pathways that are uniformly altered in all EDMD patients as well as distinguishing several subgroups of patients specific gene expression defect clustering that may reflect poor clinical diagnoses or might reflect different severity outcomes in which case altered expression of these particular genes could serve prognostically and recommend particular variations for treatment regimes.
Start Year 2021
 
Description Volker Straub on Emery-Dreifuss muscular dystrophy patient samples 
Organisation Newcastle University
Country United Kingdom 
Sector Academic/University 
PI Contribution NGS sequencing on patient samples and cell biology analysis of said samples.
Collaborator Contribution Providing patient biopsy samples.
Impact Meinke, P., Kerr, A. R. W., Czapiewski, R., Trippe, H., Sewry, C. A., Muntoni, F., Lochmuller, H., Straub, V., Schoser, B., Wehnert, M., and Schirmer, E. C. An iterative multi-pronged sequencing strategy identifies many novel and candidate disease alleles for orphan disease Emery-Dreifuss muscular dystrophy. Submitted. Note that this study presents a new iterative approach for disease allele identification in genetically diverse orphan diseases and could have a direct impact on approaches used in future and the collaboration that started with the ending Wellcome SRF is being continued in the MRC and MDUK grants.
Start Year 2011
 
Description 10 media outlets carried articles on Duan et al 2019 Nature Communications The cell-wide web coordinates cellular processes by directing site-specific Ca2+ flux across cytoplasmic nanocourses. 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Duan, J., Navarro-Dorado, J., Clark, J. H., Kinnear, N. P., Meinke, P., Schirmer, E. C., and Evans, A. M. (2019) The cell-wide web coordinates cellular processes by directing site-specific Ca2+ flux across cytoplasmic nanocourses. Nat. Commun. 10(1), 2299 doi 10.1038/s41467-019-19955-w. PMID:31127110
This study found that invaginations of the nuclear envelope in smooth muscle cells contain calcium stores that have both specific RyR-SERCA subtype combinations and that yield specific responses to signaling cascades from other calcium stores in the cell. These invaginations moreover segregate epigenetic marks and the data suggest that the combination of segregated calcium signaling and segregated epigenetic genome regions provides for genome regulation in this cell type. The study received considerable press with at least 10 press articles that included outlets as far as Australia and already has an Altmetric score of 124 and a YouTube video created about it.
Year(s) Of Engagement Activity 2019
 
Description A press release carried by 2 news outlets for Czapiewski et al paper in Nature Communications 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Press release carried in two media outlets for Czapiewski et al paper to which PDRA on this award Jose de las Heras contributed. This paper was about the same principle we are working on in the MDUK award of a different disease paradigm for nuclear envelope disorders where minor changes in multiple genes from genome organisation disruption can yield the same phenotypic outcomes as one gene being completely shut down as in the historical view of one gene-one disease. It further revealed a gradient between the nuclear envelope muscular dystrophies and nuclear envelope lipodystrophies.
Year(s) Of Engagement Activity 2022
 
Description Cell Block Science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact This is an outreach activity organised by Centre Outreach Coordinator Sarah Keer-Keer where Centre scientists teach science to criminals in the prison system. PDRA Jose de las Heras and PhD student Aishwarya Sivakumar have participated in several individual visits to the prisons for this.
Year(s) Of Engagement Activity 2018
 
Description Cell Block Science - 23 Apr 2019 (~50 inmates) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact Cell Block Science is a unique programme run by University of Edinburgh and supported by the Wellcome Trust, where a series of workshops discussing basic concepts and recent advances in research on cell and molecular biology, genetics, bioethics, etc are run once a week for 8 consecutive weeks for inmates at HMP Edinburgh.
Both PhD student Aishwarya Sivakumar and post-doctoral research associate Jose de las Heras participated in this workshop.
Year(s) Of Engagement Activity 2019
 
Description EdinCell Workshop 29 January 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact PhD student Aishwarya Sivakumar conducted a workshop for P4 students on Inheritance at Royal High Primary School, Edinburgh
Year(s) Of Engagement Activity 2019
 
Description European Laminopathies Network 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact The initial activity described here was a meeting in Bologna, Italy in November 2018 to establish the framework of a European Laminopathies Network using COST funding and to establish those to invite to its board of directors. The Network would have on the board one clinician and one basic researcher from each European country with active engagement of laminopathy patients and research and I am on the board representing the UK from a basic research standpoint and invited Volker Straub (Newcastle) to represent the UK clinicians, who accepted. The Network will have functions engaging with patients and clinicians, establishing standard practices for tissue collection and centralising resources, organising conferences, organising attempts at European Network funding, and engaging with policy makers. The Network has already fused a patient-clinician-researcher conference onto the UK Nuclear Envelope and Chromatin Organisation bi-annual meeting that I established back in 2007 and I am one of the organisers for this meeting that will happen at Bush House facility of KCL on Sept 2-5 this year, which will also be coupled with a board meeting for the European Laminopathies Network.
Year(s) Of Engagement Activity 2018,2019
URL https://www.laminopathiesmeeting2017.com/
 
Description Podcast by Alex Makarov on our lamin structural mechanics paper in Nature Communications from 2019 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact In February of 2020 a podcast was produced about our important 2019 study on nuclear mechanics: Makarov, A. A., Zou, J., Houston, D. R., Spanos, C., Solovyova, A. S., Cardinale-Peralta, C., Rappsilber, J., and Schirmer, E. C. (2019) Lamin A molecular compression and sliding as mechanisms behind nucleoskeleton elasticity. Nat Communs 10(1), 3056 doi 10.1038/s41467-019-11063-6. PMID:31296869 This study used cross-linking mass spectrometry to investigate interactions in both in vitro and ex vivo assembled lamins. This demonstrated that the linker regions use electrostatic interactions to pull the start of the following coiled coil over the end of the preceding coiled coil and thus place lamins in the assembled polymer into a starting shortened state. This provides a new spring-like mechanism that can explain better than any study preceding it why intermediate filaments are the most elastic of filament systems, as it provides a simple mechanism for the molecules to contract again after a stretch force.
In the podcast Dr Makarov is interviewed about the study and general aspects of science and healthy lab environments. The podcast was paired with an ending segment on Vegan diets that may have increased the number of people listening to it.
Year(s) Of Engagement Activity 2020
URL https://media.ed.ac.uk/media/BioPOD+February+2020A+Lamin-A+Proteins+/0_tsjcexer/116435451
 
Description Press stories on Tollis et al 2022 ACS Chem Biol carried in 7 media outlets 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Press about paper from the lab carried in 7 media outlets. Intended to disseminate information about research. No known impacts at this time.
Year(s) Of Engagement Activity 2022
 
Description Science on a Summers evening 4/9/18 at JCMB on Kings Buildings Campus University of Edinburgh 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Chromosome demonstrations
Year(s) Of Engagement Activity 2018
 
Description Workshop on Molecular Biology Techniques titled "A Question of Taste" At National Museum - 24 Oct and 28 Nov 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact This unique workshop is conducted once a year for high school students studying Advanced Biology. The workshop gives them hands on experience in routinely used molecular biology techniques such as DNA isolation, polymerase chain reactions, restriction digestion and gel electrophoresis, which are only covered theoretically in classes at schools. The workshop helps them understand the difference and correlation between phenotypes and genotypes. PhD student Aishwarya Sivakumar participated in this event.
Year(s) Of Engagement Activity 2019
 
Description Workshops on 'Inheritance' for primary school students with EdinCell at South Queensferry Primary School - 23 Oct 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact The workshop aims at discussing the basic principles of inheritance using examples of four genes in fruit flies which encode physical characteristics like eye colour, vestigial wings, etc. Through actual fruit flies containing these mutations and with interactive activities that demonstrate how two copies of each gene is inherited from parents, the workshop elaborates on the concepts of the genetic foundations of life. Both PhD student Aishwarya Sivakumar and Post-doctoral research associated Jose de las Heras participated.
Year(s) Of Engagement Activity 2019
 
Description press article picked up by 6 media outlets on Meinke et al 2019 A multistage sequencing strategy pinpoints novel candidate alleles for Emery-Dreifuss muscular dystrophy and supports gene misregulation as its pathomechanism. 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact News article on paper from lab on new muscular dystrophy alleles we identified using a new sequencing strategy that could be applied generally to genetically variable orphan diseases. Note that we also developed a primer library in this study that could be more effectively used to sequence clinically diagnosed patients than existing NHS strategies. In addition to the press article noted below the Lancet journals also published a commentary.
MEDICAL X PRESS
DECEMBER 18, 2019
Genetic test could aid quest to reveal causes of rare diseases
The causes of rare diseases could be uncovered using an approach created to identify genetic mutations that trigger a muscle-wasting condition, a study suggests.

Researchers have devised a way to pinpoint defective genes that are linked to a rare form of muscular dystrophy, which causes muscle weakening and heart problems.
The approach could help doctors perform a faster and cheaper diagnosis of the condition-called Emery-Dreifuss muscular dystrophy, or EDMD.
It could also be adapted to screen for gene mutations involved in other rare diseases, researchers say.
Discovering the cause of rare conditions can be challenging. Robust genetic screening tests are difficult when only a relatively small number of people are affected.
Previous research has shown that mutations in six different genes cause EDMD, which affects around one in 100,000 people worldwide.
However, these mutations are found in less than half of people diagnosed with the disease, indicating that other genes can also trigger it, researchers say.
Researchers from the University of Edinburgh designed a new multi-stage sequencing approach to discover other genetic mutations that might lead to EDMD.
They initially identified more than 300 genes that could be involved, including ones linked to other forms of muscular dystrophy and those that perform a similar function to the genes known to cause EDMD.
By analysing these alongside the genetic code of 56 people from the UK and Germany diagnosed with EDMD, the team identified more than 20 new mutations that appear to cause the condition. These are likely most of the remaining genes linked to EDMD, researchers say.
The study, published in the journal EBioMedicine, was funded by Wellcome, Muscular Dystrophy UK, the Medical Research Council, and the European Union's Seventh Framework Programme.
Professor Eric Schirmer, of the University of Edinburgh's School of Biological Sciences, who led the study, said: "As well as helping to identify mutations in rare diseases, using our approach in initial NHS screening would also save money and help spot misdiagnoses. This is particularly important as it can take many years to get a clear diagnosis of EDMD."
Dr. Kate Adcock, Director of Research and Innovation at Muscular Dystrophy UK, said: "We know that many people with neuromuscular conditions are living without a genetic diagnosis. This research could pave the way to help people to get a diagnosis earlier. This will help people to manage their condition thereby helping to provide a better quality of life."
Year(s) Of Engagement Activity 2019
URL https://medicalxpress.com/news/2019-12-genetic-aid-quest-reveal-rare.html