Defects in spatial genome organisation are linked to human disease and it is speculated that most as yet unidentified disease alleles will occur in non-coding genome regions. Genome organisation changes during differentiation generate tissue-specific spatial patterns of gene positioning that contribute to gene expression regulation. We identified several tissue-specific nuclear envelope transmembrane proteins (NETs) that direct tissue-specific gene repositioning. We used genome-wide approaches to map genome regions located at the nuclear periphery during myogenesis, adipogenesis, in liver, and during lymphocyte activation1-3. In each system important differentiation/metabolic genes fail to reposition/regulate expression with loss of the particular tissue-specific NET leading to inhibition of tissue differentiation1. Tethering at the nuclear envelope of regions flanking and slightly distal to genes can influence the ability of these genes to associate with superenhancers to achieve higher levels of expression3. Accordingly, we generated Hi-C datasets for changes in internal genome organisation during myogenesis to elucidate how nuclear envelope tethering influences internal genome interactions and is altered in disease states. We recently sequenced several unlinked cases of Emery-Dreifuss muscular dystrophy (EDMD) and found mutations in the muscle-specific NETs that direct myogenic genome organisation changes and have obtained an MRC grant to investigate the effects of these mutations on muscle function and pathology in mice. We separately generated a fat-specific NET knockout mouse that has a lipodystrophic phenotype, suggesting that NET-directed genome organisation may contribute to many diseases.
At least some of the following Aims will be addressed for each student project.
1. Point mutations found in fat or muscle-specific NETs that function in genome organisation will be tested for how they alter genome organisation and their effects on tissue development, metabolic function, and pathology to clarify their functioning as EDMD or lipodystrophy disease alleles and elucidate mechanism of disease pathology.
2. As several genes regulated by muscle NETs are also needed for muscle regeneration, the student will test their effects in tissue damage-repair mechanisms and also investigate the relationship of the timing of genome organisation changes to the timing of myogenic fusion events.
3. Genome editing will be engaged to study specific loci and the effects of their disruption independent of muscle NET function. Enzymatically dead CRISPR/Cas9 will be fused to a widely expressed NET and cells made to stably express guide RNAs to a particular critical gene locus so that this locus can be maintained at the periphery when it is normally released and needed.
4. Identification of partner proteins followed by testing them for functional contributions to genome organisation and biochemical purification of NET nucleoplasmic fragments for testing loss of binding interactions with disease mutations and post-translational modification.
The student will learn to produce and analyse genome-wide DamID, Hi-C and RNA-Seq data through supervision from the Schirmer lab bioinformatician and bioinformatics core, while confirming specific results through fluorescence in situ hybridisation and 3C. The student will also learn CRISPR/Cas9 genome editing approaches. Biochemistry and biophysical tools can also be learned.
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