The 3D nuclear position and compartmentalization of genes prime their response to mechano-confinement.

Cells continuously receive mechanical inputs from their environment. For example, during migration in narrow spaces, or in solid tumors, cells and their nuclei experience confinement which they sense and respond to. Emerging evidence identifies the genome as a central mediator of these responses, dynamically reorganizing its three-dimensional (3D) structure to regulate both short- and long-term gene expression programs, enabling cellular adaptation to mechanical constraints. However, the mechanisms underlying such responses, especially those linking the 3D genome and transcriptome, are unknown. Here, we utilize controlled cell confinement followed by Hi-C and transcriptome analyses to map and model temporal responses to mechano-confinement in the 3D genome. We identify clusters of genes (termed "TECs") exhibiting coordinated temporal transcriptional responses tied to their differential radial positioning within the nucleus. Additionally, we uncover a genome-wide, partially reversible response, wherein chromosomes reposition under confinement to enhance select genome compartments, aligning with temporal gene regulation patterns. Specifically, one such cluster, TEC7, is linked to 3D genome restructuring and nuclear translocation of NF-κB, driving cytokine expression and secretion. Our findings reveal how 3D genome reorganization and transcriptional programs can drive mechanoresponses, opening up for new views into the mechanisms of mechanogenomic regulation in health and disease.