Regulation of genome organization and gene expression by nuclear mechanotransduction.

It is well established that cells sense chemical signals from their local microenvironment and transduce them to the nucleus to regulate gene expression programmes. Although a number of experiments have shown that mechanical cues can also modulate gene expression, the underlying mechanisms are far from clear. Nevertheless, we are now beginning to understand how mechanical cues are transduced to the nucleus and how they influence nuclear mechanics, genome organization and transcription. In particular, recent progress in super-resolution imaging, in genome-wide application of RNA sequencing, chromatin immunoprecipitation and chromosome conformation capture and in theoretical modelling of 3D genome organization enables the exploration of the relationship between cell mechanics, 3D chromatin configurations and transcription, thereby shedding new light on how mechanical forces regulate gene expression.

ATR mediates a checkpoint at the nuclear envelope in response to mechanical stress.

ATR controls chromosome integrity and chromatin dynamics. We have previously shown that yeast Mec1/ATR promotes chromatin detachment from the nuclear envelope to counteract aberrant topological transitions during DNA replication. Here, we provide evidence that ATR activity at the nuclear envelope responds to mechanical stress. Human ATR associates with the nuclear envelope during S phase and prophase, and both osmotic stress and mechanical stretching relocalize ATR to nuclear membranes throughout the cell cycle. The ATR-mediated mechanical response occurs within the range of physiological forces, is reversible, and is independent of DNA damage signaling. ATR-defective cells exhibit aberrant chromatin condensation and nuclear envelope breakdown. We propose that mechanical forces derived from chromosome dynamics and torsional stress on nuclear membranes activate ATR to modulate nuclear envelope plasticity and chromatin association to the nuclear envelope, thus enabling cells to cope with the mechanical strain imposed by these molecular processes.

Chromatin Remodeling Due to Transient-Link-and-Pass Activity Enhances Subnuclear Dynamics.

Spatiotemporal coordination of chromatin and subnuclear compartments is crucial for cells. Numerous enzymes act inside nucleus-some of those transiently link and pass two chromatin segments. Here, we study how such an active perturbation affects fluctuating dynamics of an inclusion in the chromatic medium. Using numerical simulations and a versatile effective model, we categorize inclusion dynamics into three distinct modes. The transient-link-and-pass activity speeds up inclusion dynamics by affecting a slow mode related to chromatin remodeling, viz., size and shape of the chromatin meshes.

Fibroblast rejuvenation by mechanical reprogramming and redifferentiation.

Over the course of the aging process, fibroblasts lose contractility, leading to reduced connective-tissue stiffness. A promising therapeutic avenue for functional rejuvenation of connective tissue is reprogrammed fibroblast replacement, although major hurdles still remain. Toward this, we recently demonstrated that the laterally confined growth of fibroblasts on micropatterned substrates induces stem-cell-like spheroids. In this study, we embedded these partially reprogrammed spheroids in collagen-I matrices of varying densities, mimicking different three-dimensional (3D) tissue constraints. In response to such matrix constraints, these spheroids regained their fibroblastic properties and sprouted to form 3D connective-tissue networks. Interestingly, we found that these differentiated fibroblasts exhibit reduced DNA damage, enhanced cytoskeletal gene expression, and actomyosin contractility. In addition, the rejuvenated fibroblasts show increased matrix protein (fibronectin and laminin) deposition and collagen remodeling compared to the parental fibroblast tissue network. Furthermore, we show that the partially reprogrammed cells have comparatively open chromatin compaction states and may be more poised to redifferentiate into contractile fibroblasts in 3D-collagen matrix. Collectively, our results highlight efficient fibroblast rejuvenation through laterally confined reprogramming, which has important implications in regenerative medicine.

Guiding Irregular Nuclear Morphology on Nanopillar Arrays for Malignancy Differentiation in Tumor Cells.

For more than a century, abnormal nuclei in tumor cells, presenting subnuclear invaginations and folds on the nuclear envelope, have been known to be associated with high malignancy and poor prognosis. However, current nuclear morphology analysis focuses on the features of the entire nucleus, overlooking the malignancy-related subnuclear features in nanometer scale. The main technical challenge is to probe such tiny and randomly distributed features inside cells. We here employ nanopillar arrays to guide subnuclear features into ordered patterns, enabling their quantification as a strong indicator of cell malignancy. Both breast and liver cancer cells were validated as well as the quantification of nuclear abnormality heterogeneity. The alterations of subnuclear patterns were also explored as effective readouts for drug treatment. We envision that this nanopillar-enabled quantification of subnuclear abnormal features in tumor cells opens a new angle in characterizing malignant cells and studying the unique nuclear biology in cancer.

Regulation of nuclear architecture, mechanics, and nucleocytoplasmic shuttling of epigenetic factors by cell geometric constraints.

Cells sense mechanical signals from their microenvironment and transduce them to the nucleus to regulate gene expression programs. To elucidate the physical mechanisms involved in this regulation, we developed an active 3D chemomechanical model to describe the three-way feedback between the adhesions, the cytoskeleton, and the nucleus. The model shows local tensile stresses generated at the interface of the cell and the extracellular matrix regulate the properties of the nucleus, including nuclear morphology, levels of lamin A,C, and histone deacetylation, as these tensile stresses 1) are transmitted to the nucleus through cytoskeletal physical links and 2) trigger an actomyosin-dependent shuttling of epigenetic factors. We then show how cell geometric constraints affect the local tensile stresses and subsequently the three-way feedback and induce cytoskeleton-mediated alterations in the properties of the nucleus such as nuclear lamina softening, chromatin stiffening, nuclear lamina invaginations, increase in nuclear height, and shrinkage of nuclear volume. We predict a phase diagram that describes how the disruption of cytoskeletal components impacts the feedback and subsequently induce contractility-dependent alterations in the properties of the nucleus. Our simulations show that these changes in contractility levels can be also used as predictors of nucleocytoplasmic shuttling of transcription factors and the level of chromatin condensation. The predictions are experimentally validated by studying the properties of nuclei of fibroblasts on micropatterned substrates with different shapes and areas.

Predicting cell lineages using autoencoders and optimal transport.

Lineage tracing involves the identification of all ancestors and descendants of a given cell, and is an important tool for studying biological processes such as development and disease progression. However, in many settings, controlled time-course experiments are not feasible, for example when working with tissue samples from patients. Here we present ImageAEOT, a computational pipeline based on autoencoders and optimal transport for predicting the lineages of cells using time-labeled datasets from different stages of a cellular process. Given a single-cell image from one of the stages, ImageAEOT generates an artificial lineage of this cell based on the population characteristics of the other stages. These lineages can be used to connect subpopulations of cells through the different stages and identify image-based features and biomarkers underlying the biological process. To validate our method, we apply ImageAEOT to a benchmark task based on nuclear and chromatin images during the activation of fibroblasts by tumor cells in engineered 3D tissues. We further validate ImageAEOT on chromatin images of various breast cancer cell lines and human tissue samples, thereby linking alterations in chromatin condensation patterns to different stages of tumor progression. Our results demonstrate the promise of computational methods based on autoencoding and optimal transport principles for lineage tracing in settings where existing experimental strategies cannot be used.

Mechanogenomic coupling of lung tissue stiffness, EMT and coronavirus pathogenicity.

In this Current Opinion, we highlight the importance of the material properties of tissues and how alterations therein, which influence epithelial-to-mesenchymal transitions, represent an important layer of regulation in a number of diseases and potentially also play a critical role in host-pathogen interactions. In light of the current SARS-CoV-2 pandemic, we here highlight the possible role of lung tissue stiffening with ageing and how this might facilitate increased SARS-CoV-2 replication through matrix-stiffness dependent epithelial-to-mesenchymal transitions of the lung epithelium. This emphasizes the need for integrating material properties of tissues in drug discovery programs.

Laterally confined growth of cells induces nuclear reprogramming in the absence of exogenous biochemical factors.

Cells in tissues undergo transdifferentiation programs when stimulated by specific mechanical and biochemical signals. While seminal studies have demonstrated that exogenous biochemical factors can reprogram somatic cells into pluripotent stem cells, the critical roles played by mechanical signals in such reprogramming process have not been well documented. In this paper, we show that laterally confined growth of fibroblasts on micropatterned substrates induces nuclear reprogramming with high efficiency in the absence of any exogenous reprogramming factors. We provide compelling evidence on the induction of stem cell-like properties using alkaline phosphatase assays and expression of pluripotent markers. Early onset of reprogramming was accompanied with enhanced nuclear dynamics and changes in chromosome intermingling degrees, potentially facilitating rewiring of the genome. Time-lapse analysis of promoter occupancy by immunoprecipitation of H3K9Ac chromatin fragments revealed that epithelial, proliferative, and reprogramming gene promoters were progressively acetylated, while mesenchymal promoters were deacetylated by 10 days. Consistently, RNA sequencing analysis showed a systematic progression from mesenchymal to stem cell transcriptome, highlighting pathways involving mechanisms underlying nuclear reprogramming. We then demonstrated that these mechanically reprogrammed cells could be maintained as stem cells and can be redifferentiated into multiple lineages with high efficiency. Importantly, we also demonstrate the induction of cancer stemness properties in MCF7 cells grown in such laterally confined conditions. Collectively, our results highlight an important generic property of somatic cells that, when grown in laterally confined conditions, acquire stemness. Such mechanical reprogramming of somatic cells demonstrated here has important implications in tissue regeneration and disease models.

Network analysis identifies chromosome intermingling regions as regulatory hotspots for transcription.

The 3D structure of the genome plays a key role in regulatory control of the cell. Experimental methods such as high-throughput chromosome conformation capture (Hi-C) have been developed to probe the 3D structure of the genome. However, it remains a challenge to deduce from these data chromosome regions that are colocalized and coregulated. Here, we present an integrative approach that leverages 1D functional genomic features (e.g., epigenetic marks) with 3D interactions from Hi-C data to identify functional interchromosomal interactions. We construct a weighted network with 250-kb genomic regions as nodes and Hi-C interactions as edges, where the edge weights are given by the correlation between 1D genomic features. Individual interacting clusters are determined using weighted correlation clustering on the network. We show that intermingling regions generally fall into either active or inactive clusters based on the enrichment for RNA polymerase II (RNAPII) and H3K9me3, respectively. We show that active clusters are hotspots for transcription factor binding sites. We also validate our predictions experimentally by 3D fluorescence in situ hybridization (FISH) experiments and show that active RNAPII is enriched in predicted active clusters. Our method provides a general quantitative framework that couples 1D genomic features with 3D interactions from Hi-C to probe the guiding principles that link the spatial organization of the genome with regulatory control.

Cell geometry dictates TNFα-induced genome response.

Cells in physiology integrate local soluble and mechanical signals to regulate genomic programs. Whereas the individual roles of these signals are well studied, the cellular responses to the combined chemical and physical signals are less explored. Here, we investigated the cross-talk between cellular geometry and TNFα signaling. We stabilized NIH 3T3 fibroblasts into rectangular anisotropic or circular isotropic geometries and stimulated them with TNFα and analyzed nuclear translocation of transcription regulators -NFκB (p65) and MKL and downstream gene-expression patterns. We found that TNFα induces geometry-dependent actin depolymerization, which enhances IκB degradation, p65 nuclear translocation, nuclear exit of MKL, and sequestration of p65 at the RNA-polymerase-II foci. Further, global transcription profile of cells under matrix-TNFα interplay reveals a geometry-dependent gene-expression pattern. At a functional level, we find cell geometry affects TNFα-induced cell proliferation. Our results provide compelling evidence that fibroblasts, depending on their geometries, elicit distinct cellular responses for the same cytokine.

How cells respond to environmental cues - insights from bio-functionalized substrates.

Biomimetic materials have long been the (he)art of bioengineering. They usually aim at mimicking in vivo conditions to allow in vitro culture, differentiation and expansion of cells. The past decade has witnessed a considerable amount of progress in soft lithography, bio-inspired micro-fabrication and biochemistry, allowing the design of sophisticated and physiologically relevant micro- and nano-environments. These systems now provide an exquisite toolbox with which we can control a large set of physicochemical environmental parameters that determine cell behavior. Bio-functionalized surfaces have evolved from simple protein-coated solid surfaces or cellular extracts into nano-textured 3D surfaces with controlled rheological and topographical properties. The mechanobiological molecular processes by which cells interact and sense their environment can now be unambiguously understood down to the single-molecule level. This Commentary highlights recent successful examples where bio-functionalized substrates have contributed in raising and answering new questions in the area of extracellular matrix sensing by cells, cell-cell adhesion and cell migration. The use, the availability, the impact and the challenges of such approaches in the field of biology are discussed.

Matrix mechanics controls FHL2 movement to the nucleus to activate p21 expression.

Substrate rigidity affects many physiological processes through mechanochemical signals from focal adhesion (FA) complexes that subsequently modulate gene expression. We find that shuttling of the LIM domain (domain discovered in the proteins, Lin11, Isl-1, and Mec-3) protein four-and-a-half LIM domains 2 (FHL2) between FAs and the nucleus depends on matrix mechanics. In particular, on soft surfaces or after the loss of force, FHL2 moves from FAs into the nucleus and concentrates at RNA polymerase (Pol) II sites, where it acts as a transcriptional cofactor, causing an increase in p21 gene expression that will inhibit growth on soft surfaces. At the molecular level, shuttling requires a specific tyrosine in FHL2, as well as phosphorylation by active FA kinase (FAK). Thus, we suggest that FHL2 phosphorylation by FAK is a critical, mechanically dependent step in signaling from soft matrices to the nucleus to inhibit cell proliferation by increasing p21 expression.

Nuclear deformability and telomere dynamics are regulated by cell geometric constraints.

Forces generated by the cytoskeleton can be transmitted to the nucleus and chromatin via physical links on the nuclear envelope and the lamin meshwork. Although the role of these active forces in modulating prestressed nuclear morphology has been well studied, the effect on nuclear and chromatin dynamics remains to be explored. To understand the regulation of nuclear deformability by these active forces, we created different cytoskeletal states in mouse fibroblasts using micropatterned substrates. We observed that constrained and isotropic cells, which lack long actin stress fibers, have more deformable nuclei than elongated and polarized cells. This nuclear deformability altered in response to actin, myosin, formin perturbations, or a transcriptional down-regulation of lamin A/C levels in the constrained and isotropic geometry. Furthermore, to probe the effect of active cytoskeletal forces on chromatin dynamics, we tracked the spatiotemporal dynamics of heterochromatin foci and telomeres. We observed increased dynamics and decreased correlation of the heterochromatin foci and telomere trajectories in constrained and isotropic cell geometry. The observed enhanced dynamics upon treatment with actin depolymerizing reagents in elongated and polarized geometry were regained once the reagent was washed off, suggesting an inherent structural memory in chromatin organization. We conclude that active forces from the cytoskeleton and rigidity from lamin A/C nucleoskeleton can together regulate nuclear and chromatin dynamics. Because chromatin remodeling is a necessary step in transcription control and its memory, genome integrity, and cellular deformability during migration, our results highlight the importance of cell geometric constraints as critical regulators in cell behavior.

Actin Dynamics Couples Extracellular Signals to the Mobility and Molecular Stability of Telomeres.

Genome regulatory programs such as telomere functioning require extracellular signals to be transmitted from the microenvironment to the nucleus and chromatin. Although the cytoskeleton has been shown to directly transmit stresses, we show that the intrinsically dynamic nature of the actin cytoskeleton is important in relaying extracellular signals to telomeres. Interestingly, this mechanical pathway not only transmits physical stimuli but also chemical stimuli. The cytoskeletal network continuously reorganizes and applies dynamic forces on the nucleus and feeds into the regulation of telomere dynamics. We further found that distal telomeres are mechanically coupled in a length- and timescale-dependent manner and identified nesprin 2G as well as lamin A/C as being essential to regulate their translational dynamics. Finally, we demonstrated that such mechanotransduction events impinge on the binding dynamics of critical telomere binding proteins. Our results highlight an overarching physical pathway that regulates positional and molecular stability of telomeres.

Nuclear transport of paxillin depends on focal adhesion dynamics and FAT domains.

The nuclear transport of paxillin appears to be crucial for paxillin function but the mechanism of transport remains unclear. Here, we show that the nuclear transport of paxillin is regulated by focal adhesion turnover and the presence of FAT domains. Focal adhesion turnover was controlled using triangular or circular fibronectin islands. Circular islands caused higher focal adhesion turnover and increased the nuclear transport of paxillin relative to triangular islands. Mutating several residues of paxillin had no effect on its nuclear transport, suggesting that the process is controlled by multiple domains. Knocking out FAK (also known as PTK2) and vinculin caused an increase in nuclear paxillin. This could be reversed by rescue with wild-type FAK but not by FAK with a mutated FAT domain, which inhibits paxillin binding. Expressing just the FAT domain of FAK not only brought down nuclear levels of paxillin but also caused a large immobile fraction of paxillin to be present at focal adhesions, as demonstrated by fluorescence recovery after photobleaching (FRAP) studies. Taken together, focal adhesion turnover and FAT domains regulate the nuclear localization of paxillin, suggesting a possible role for transcriptional control, through paxillin, by focal adhesions.

Maximal Fluctuations of Confined Actomyosin Gels: Dynamics of the Cell Nucleus.

We investigate the effect of stress fluctuations on the stochastic dynamics of an inclusion embedded in a viscous gel. We show that, in nonequilibrium systems, stress fluctuations give rise to an effective attraction towards the boundaries of the confining domain, which is reminiscent of an active Casimir effect. We apply this generic result to the dynamics of deformations of the cell nucleus, and we demonstrate the appearance of a fluctuation maximum at a critical level of activity, in agreement with recent experiments [E. Makhija, D. S. Jokhun, and G. V. Shivashankar, Proc. Natl. Acad. Sci. U.S.A. 113, E32 (2016)PNASA60027-842410.1073/pnas.1513189113].

Coupling between chromosome intermingling and gene regulation during cellular differentiation.

In this article, we summarize current findings for the emergence of biophysical properties such as nuclear stiffness, chromatin compaction, chromosome positioning, and chromosome intermingling during stem cell differentiation, which eventually correlated with the changes of gene expression profiles during cellular differentiation. An overview is first provided to link stem cell differentiation with alterations in nuclear architecture, chromatin compaction, along with nuclear and chromatin dynamics. Further, we highlight the recent biophysical and molecular approaches, imaging methods and computational developments in characterizing transcription-related chromosome organization especially chromosome intermingling and nano-scale chromosomal contacts. Finally, the article ends with an outlook towards the emergence of a functional roadmap in setting up chromosome positioning and intermingling in a cell type specific manner during cellular differentiation.

Chromosome intermingling-the physical basis of chromosome organization in differentiated cells.

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.