Gene expression can be regulated through alterations in nuclear architecture, providing control of genome function. Mechanical loading induces both nuclear deformation and alteration in gene expression in a variety of cell types. One putative transduction mechanism for this phenomenon involves alterations to nuclear architecture, resulting from the mechanical perturbation to the cell, to induce altered gene expression. Moreover, remodeling of the nucleus occurs during stem cell differentiation, causing alterations in nuclear stiffness and potentially influencing nuclear architecture-mediated mechano-regulation of transcription. Indeed, a growing body of literature suggests that stem cells respond to mechanical loading differently depending on their differentiation state. Accordingly this project will focus on this aspect of mechanoregulation of genome function, through the following scientific aims and objectives:

Scientific aims of the project:
To test the following hypotheses
1. Mechanically-induced nuclear deformation causes alterations to the nuclear architecture and chromosomal territories sufficient to affect gene transcription
2. Stem cell differentiation modulates the process set out in hypothesis 1, via differentiation-induced nuclear remodeling that affects the mechanical properties of the nucleus.

Specific Objectives:
1. To characterize nuclear deformation induced by the application of static or dynamic mechanical perturbation and to determine the associated rearrangement of chromosomal territories.
2. To assess the mechanical properties of the nucleus in stem cells in their undifferentiated state and during differentiation
3. To assess the mechanism of transfer of strain to the nucleus through the extracellular matrix and cytoskeleton.
4. To conduct genome-wide analysis of mechanosensitive genes and map the relevant loci to chromosomal territories within the nucleus
5. To assess the functional relevance of mechanical-perturbation-induced nuclear deformation on regulation of transcription using 3-D FISH analysis for genes selected from the results of objective 4.
6. To develop an integrated model of mechanosensitive genome regulation that incorporates the mechanical, morphological and transcriptomic data from previous objectives.

In order to fulfill the stated objectives the studies will utilize model systems involving human mesenchymal stem cells and induced pluripotent stem cells in conjunction with state-of-the-art techniques for the mechanical perturbation of cells, functional analysis of nuclear organization and biomechanical analysis. Ultimately a mechanistic model for functional mechanoregulation of nuclear architecture will be developed to incorporate elements of the modeling of the mechanical properties of the nucleus in conjunction with functional genomic analysis. The proposed project clearly meets the aims of the HFSP as it focuses on elucidating fundamental and complex biological mechanisms associated with genome regulation and stem cell fate. The focus is on basic understanding, but the outcomes will clearly have relevance to more application driven fields, such as regenerative medicine. The proposal further meets the aims of the HFSP by bringing together scientists from different fields and in doing so will develop new approaches to understanding complex biological systems.