Title: Using Human Disease and Genome Engineering to Dissect Dynamic Genome Organisation at the Nuclear Lamina in vivo during Development
Funder: Wellcome Trust

Abstract: Much of three-dimensional genome organisation derives from DNA-nuclear envelope interactions, creating lamina-associated domains (LADs), and local chromatin-chromatin interactions, creating topologically-associated domains (TADs). At developmentally critical loci, TADs are frequently internally reconfigured and LADs gained/lost during differentiation to influence gene activity. However, it is unclear how changes to one influence or place constraints on the other. Moreover, while large structural variants (SVs) at developmentally critical loci disrupt TADs and cause gene miss-expression and disease, it is unclear how this affects dynamic lamina-association and the gene regulatory activity this may confer. I will address these questions by empirically determining LAD behaviour following introduction of TAD-disrupting SVs causative of human disease at the Epha4 and Sox9 loci, both in a static single cell-type in vitro and dynamically in multiple cell-types during development by in vivo-DamID. To explore if lamina-association controls genes by introducing additional topological constraints, I will discriminate structures of LAD- and non-LAD-associated TADs in cell-populations of multiple tissues, using a hybrid DamID-Hi-C approach, and at single loci in individual cells, using super-resolution FISH-microscopy. Together, this will allow lamina-association’s functional role in development and impact on TAD structure to be determined, potentially improving diagnostic predictions of SV mutations pathogenic effects.

Lay abstract: The specialisation of an embryo’s cells into the many cell types of the body is achieved by precisely activating or inactivating specific genes at the appropriate time and place. To achieve this precision, genes are isolated from the noise of the wider genome by locally folding with nearby DNA to form discrete structures called topologically-associated domains (TADs). Failed isolation, resulting from removal or rearrangement of TAD boundaries by mutation, causes inappropriate gene activity and disease. During development some TADs also physically attach and detach from the nuclear lamina at the nucleus’s outer edge, turning associated genes off. However, it is unknown if disease-causing TAD-rearranging mutations also disrupt lamina-attachment, thus impairing its control of gene activity. To answer this question, I will measure altered lamina-attachment in developing mouse limbs possessing TAD-rearranging mutations and monitor how this pathogenically affects gene activity and limb formation. More fundamentally, it is also unknown exactly how lamina-attachment alters TAD structure to turn off genes. Hence, I will also develop methods to specifically observe lamina-attached TAD structures and measure how disease-causing mutations disrupt them. Together this will unravel lamina-attachment’s purpose in development, allowing the effects of mutations altering the genomes 3D-organisation to be predicted in patients.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE