Role of mechanical signalling at the nuclear envelope in pluripotent stem cell differentiation

Mammalian development and subsequent organ maintenance relies on the ability to deliver the right cells to the right place at the right time. This highly coordinated process relies on the proper regulation of cell fate transitions in stem cells. However, at this time, how cell fate transitions are regulated is poorly understood.

One aspect to how transitions are regulated is how the instructive genes are positioned and expressed in the nucleus. Indeed, the spatial organisation of the genome, and how it changes in a cell, helps to control when and how genes are activated. Proteins found at the outside of the nucleus, referred to as nuclear envelope proteins, tether specific genes to the periphery of the nucleus and play an important role in regulating those genes.

As evidence of their importance, mutations in nuclear envelope proteins often result in developmental disorders. Mutant nuclear envelope proteins have been implicated in a range of human pathologies such as premature ageing (e.g. Hutchinson-Gilford progeria syndrome) and neuromuscular diseases (e.g.

Emery-Dreifuss muscular dystrophy). It is therefore important to understand how these proteins influence mammalian development to shed light not only on how these diseases emerge, but also to exploit the therapeutic potential of differentiating stem cells towards specific lineages of choice. Nevertheless, how nuclear envelope proteins influence gene expression in stem cells during development remains unclear.

At the same time, the nuclear envelope proteins in question have also been shown to be sensitive to forces, which are ubiquitous during development and organ maintenance. Furthermore, stem cells are highly responsive to these forces, altering gene positioning and expression in response to them. In this proposal, we hypothesize that forces on the nucleus alter the function of nuclear envelope proteins and thus gene positioning and expression.

We further hypothesize that this force-mediated genetic regulation is an important aspect of fate transitions. To test our hypotheses, we will focus on two questions. First, how do nuclear envelope proteins regulate gene expression in stem cells?

Second, how do forces affect nuclear envelope proteins and subsequent gene expression during stem cell differentiation? In the proposed research, we will investigate these questions to bring greater understanding not only to spatial regulation of gene expression in stem cells, but also to how stem cells respond to, and exploit, forces in their environment.

Here, we address how nuclear envelope proteins influence early mammalian development by taking advantage of recent advances in next-generation sequencing that allow us to study how the genome folds at the level of a single cell and 'state-of-the art' microscopes capable of tracking individual proteins and genes in living cells.

We will first identify genes that are controlled by nuclear envelope proteins during stem cell differentiation (Aim 1). We will then use high-resolution microscopes to track individual genes 'live', allowing us to determine how nuclear envelope proteins control expression of these genes: is it by positioning or tethering them at the nuclear periphery where they are silenced, or by recruiting specific proteins to these genes that influence their expression (Aim 2)?

Finally, to understand which nuclear envelope proteins influence the ability of a cell to sense forces in the cell, we will use a bespoke cell stretcher to apply mechanical stress to cells and use approaches described in Aim 1 and 2 to determine which specific nuclear envelope proteins are required for genes to respond to these external forces (Aim 3).

These results will shed light on the role of nuclear envelope proteins in human development and disease, enhance understanding of the role of forces in development, and also improve strategies for differentiating pluripotent stem cells towards specific lineages for regenerative medicine.