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.

Orientation and repositioning of chromosomes correlate with cell geometry-dependent gene expression.

Extracellular matrix signals from the microenvironment regulate gene expression patterns and cell behavior. Using a combination of experiments and geometric models, we demonstrate correlations between cell geometry, three-dimensional (3D) organization of chromosome territories, and gene expression. Fluorescence in situ hybridization experiments showed that micropatterned fibroblasts cultured on anisotropic versus isotropic substrates resulted in repositioning of specific chromosomes, which contained genes that were differentially regulated by cell geometries. Experiments combined with ellipsoid packing models revealed that the mechanosensitivity of chromosomes was correlated with their orientation in the nucleus. Transcription inhibition experiments suggested that the intermingling degree was more sensitive to global changes in transcription than to chromosome radial positioning and its orientations. These results suggested that cell geometry modulated 3D chromosome arrangement, and their neighborhoods correlated with gene expression patterns in a predictable manner. This is central to understanding geometric control of genetic programs involved in cellular homeostasis and the associated diseases.

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.