(A-31) Genomic regulation of epithelial cells during early crowding

Introduction:: Epithelial cell crowding occurs as the population expands within a confined space. Signaling cues from crowding in turn regulate cell behavior and fate changes during diverse biological processes, including organogenesis, wound healing, and cancer progression. While the late phase of epithelial crowding, known as jamming transition, has been well-characterized, we know little about the regulation of cells during the initial phase of crowding, in which both biomechanical and biochemical cues readily emerge. In this work, we decipher the upstream control and downstream effects of gene expression changes by first systematically characterizing the transcriptional trajectory of Madin-Darby Canine Kidney (MDCK) cells in actively expanding tissues during the early phase of crowding.


Materials and Methods:: MDCK cells were used to model the 2D monolayer jamming transition, in which we validated the transition by confirming cell cycle arrest and a decline in motility. To acquire gene expression data, we performed bulk RNA sequencing at 15 timepoints throughout crowding. To quantify the biomechanical and biochemical inputs, we measure the evolution in cell morphology and E-cad distribution, respectively. We then identified potential pathways responsible for the observed transcriptional responses by immunostaining histone marks and YAP.


Results, Conclusions, and Discussions:: By performing a time course transcriptome analysis, we observed a biphasic trajectory, marking the early and late phases of crowding (Fig. 1A). We found ST6GALNA2 (regulating cell-substrate interactions) and KRT27 (encoding intermediate filaments) upregulated and downregulated, respectively in early crowding. We further found that the geometric constraint evolution and E-cad junction formation can be correlated with the genomic signatures of the early and late phases of crowding, respectively (Fig. 1B). To delineate the mechanism that shapes the transcriptional trajectory, we next investigated epigenetic modifications and transcription factor activities (Fig. 1C). We found that the H3 level decreases throughout crowding suggesting that the geometric constraint could cause transcriptional responses via epigenetic modifications in early crowding (Fig. 1D). In summary, by combining transcriptional trajectory measurements with quantitative confocal image analyses, we defined gene expression and epigenetic alterations in the early phase of epithelial crowding. Our results suggest substantial epigenetic alterations, which lead to a specific transcriptional response, occur as early as the cell-cell junctions are formed. In future work we aim to perform ATAC-Seq to characterize the evolution of the epigenetic landscape of epithelial cells in response to crowding. We also aim to identify the governing mechanism of H3K27me3 downregulation by investigating proteosome degradation.


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