H3K27me3 Drives Constricted Migration-Induced 3D Genome Rewiring in Melanoma

Repeated exposure of neoplastic cells to mechanical stresses during metastasis can drive stable morphological and physiological changes. We previously showed that A375 human melanoma cells subjected to 10 rounds of constricted migration ("Bottom-10 cells") became significantly more migratory and exhibited H3K9me3 relocalization, transcriptional remodeling, and chromatin compartment changes relative to naïve parental A375 cells. Because these phenotypes persist across cell divisions, we hypothesized they are driven and maintained by specific epigenetic mechanisms. We profiled candidate histone modifications by CUT&RUN and applied a computational model to identify marks that explain compartment switches. In parental cells, H3K9me3 and H3K4me1 strongly predict locus stability in the B and A compartments, respectively. In contrast, changes in H3K27me3 dynamically predict A-to-B compartment switching after constricted migration. Consistently, altered H3K27me3 enrichment correlates with both differentially regulated genes and chromatin compartment changes between Bottom-10 and parental populations. We therefore tested whether inhibiting H3K27me3 deposition alters migratory potential and/or the "memory" of the increased migratory phenotype. Inhibiting EZH1/2 reduced migration efficiency overall, with a stronger effect on constricted than unconstricted migration. This reduction was accompanied by decreased genomic H3K27me3 enrichment and reversal of some compartment changes in Bottom-10 cells. However, acute treatment did not durably reverse the highly migratory Bottom-10 phenotype; cells returned to their initial state after drug removal. Chronic inhibition across many sequential rounds of constricted migration dramatically reduced the fraction of cells able to migrate. Notably, cells that migrated despite chronic EZH inhibition appeared to escape treatment, acquiring many typical Bottom-10 H3K27me3 and compartment changes even though acute inhibition tends to prevent them. Overall, these results highlight H3K27me3 as a key contributor to establishing and maintaining constricted migration-induced 3D genome changes.