Physical forces in the form of substrate rigidity or geometrical constraints have been shown to alter gene expression profile and differentiation programs. However, the underlying mechanism of gene regulation by these mechanical cues is largely unknown. In this work, we use micropatterned substrates to alter cellular geometry (shape, aspect ratio, and size) and study the nuclear mechanotransduction to regulate gene expression. Genome-wide transcriptome analysis revealed cell geometry-dependent alterations in actin-related gene expression. Increase in cell size reinforced expression of matrix-related genes, whereas reduced cell-substrate contact resulted in up-regulation of genes involved in cellular homeostasis. We also show that large-scale changes in geneexpression profile mapped onto differential modulation of nuclear morphology, actomyosin contractility and histone acetylation. Interestingly, cytoplasmic-to-nuclear redistribution of histone deacetylase 3 modulated histone acetylation in an actomyosin-dependent manner. In addition, we show that geometric constraints altered the nuclear fraction of myocardin-related transcription factor. These fractions exhibited hindered diffusion time scale within the nucleus, correlated with enhanced serum-response element promoter activity. Furthermore, nuclear accumulation of myocardin-related transcription factor also modulated NF-κB activity. Taken together, our work provides modularity in switching gene-expression patterns by cell geometric constraints via actomyosin contractility.cell matrix interaction | substrate geometry | MRTF-A signaling | chromatin remodelling | transcription control C ells within the local tissue microenvironment acquire nonrandom geometrical organization by cell-matrix and cell-cell interaction. Cellular geometry has been shown to influence nuclear deformation, cytoskeleton reorganization, chromatin compaction, gene expression, growth, apoptosis, and cell division (1-7). Other physical cues such as substrate stretching, fluid flow, substrate rigidity, and cellular topography have also been shown to alter cellular morphology, nuclear architecture, and gene expression (8-11). Regulation of gene expression requires posttranslational modifications of histone tails (12), which alter higher-order chromatin assembly and, hence, the accessibility of gene-regulatory sites by transcriptional machinery (13). In addition, cytoplasmic to nuclear shuttling of transcription factors (TFs) and cofactors are key signaling intermediates rendering specificity. Some of these factors include NF-κB, STAT, and myocardin-related transcription factor (MRTF-A) (14-16). The transcription coactivator yes-associated protein (YAP)/transcription coactivator with PDZ binding domain (TAZ) and MRTF-A have been implicated in nuclear mechanotransduction (17)(18)(19). In a recent study, alterations in cell shape were shown to influence mesenchymal stem cell differentiation (20). However, the mechanisms underlying geometric control of gene expression by the modulation of cytoplasmi...

For cells to adapt to different tissues and changes in tissue mechanics, they must be able to respond to mechanical cues by changing their gene expression patterns. Biochemical signaling pathways for these responses have been elucidated, and recent evidence points to the involvement of force-induced deformation of the nucleus. However, it is still unclear how physical cues received at the plasma membrane (PM) spatiotemporally integrate to the functional chromatin organization of the cell nucleus. To investigate this issue, we applied mechanical forces through magnetic particles adhered to the PM of single cells and mapped the accompanying changes in actin polymerization, nuclear morphology, chromatin remodeling, and nuclear transport of soluble signaling intermediates using high-resolution fluorescence anisotropy imaging. Using this approach, we show the timescales associated with force-induced polymerization of actin and changes in the F/G actin ratio resulting in nuclear translocation of the G-actin-associated transcriptional cofactor, megakaryoblastic acute leukemia factor-1 (MKL). Further, this method of measuring nuclear organization at high spatiotemporal resolution with simultaneous force application revealed the physical propagation of forces to the nucleus, resulting in changes to chromatin organization, followed by nuclear deformation. We also describe a quantitative model that incorporates active stresses and chemical kinetics to evaluate the observed timescales. Our work suggests that mechanical activation of cells is accompanied by distinct timescales involved in the reorganization of actin and chromatin assembly, followed by translocation of transcription cofactors from the cytoplasm to the nucleus.

Graphical AbstractHighlights d Mechanical stress releases p120-catenin from apical junctions d Loss of p120-catenin increases endocytic E-cadherin turnover d Loss of p120 speeds stress-dependent remodeling of the junctional network d Mechanical stress dependence of E-cadherin turnover sets tissue viscoelasticity SUMMARY Studying how epithelia respond to mechanical stresses is key to understanding tissue shape changes during morphogenesis. Here, we study the viscoelastic properties of the Drosophila wing epithelium during pupal morphogenesis by quantifying mechanical stress and cell shape as a function of time. We find a delay of 8 h between maximal tissue stress and maximal cell elongation, indicating a viscoelastic deformation of the tissue. We show that this viscoelastic behavior emerges from the mechanosensitivity of endocytic E-cadherin turnover. The increase in E-cadherin turnover in response to stress is mediated by mechanosensitive relocalization of the E-cadherin binding protein p120-catenin (p120) from cell junctions to cytoplasm. Mechanosensitivity of E-cadherin turnover is lost in p120 mutant wings, where E-cadherin turnover is constitutively high. In this mutant, the relationship between mechanical stress and stress-dependent cell dynamics is altered. Cells in p120 mutant deform and undergo cell rearrangements oriented along the stress axis more rapidly in response to mechanical stress. These changes imply a lower viscosity of wing epithelium. Taken together, our findings reveal that p120-dependent mechanosensitive E-cadherin turnover regulates viscoelastic behavior of epithelial tissues.

Chromosome territories (CTs) in higher eukaryotes occupy tissue-specific non-random three-dimensional positions in the interphase nucleus. To understand the mechanisms underlying CT organization, we mapped CT position and transcriptional changes in undifferentiated embryonic stem (ES) cells, during early onset of mouse ES cell differentiation and in terminally differentiated NIH3T3 cells. We found chromosome intermingling volume to be a reliable CT surface property, which can be used to define CT organization. Our results show a correlation between the transcriptional activity of chromosomes and heterologous chromosome intermingling volumes during differentiation. Furthermore, these regions were enriched in active RNA polymerase and other histone modifications in the differentiated states. These findings suggest a correlation between the evolution of transcription program in modifying CT architecture in undifferentiated stem cells. This leads to the formation of functional CT surfaces, which then interact to define the three-dimensional CT organization during differentiation.