The three-dimensional structure of chromosomes plays an important role in gene expression regulation and also influences the repair of radiation-induced DNA damage. Genomic aberrations that disrupt chromosome spatial domains can lead to diseases including cancer, but how the 3D genome structure responds to DNA damage is poorly understood. Here, we investigate the impact of DNA damage response and repair on 3D genome folding using Hi-C experiments on wild type cells and ataxia telangiectasia mutated (ATM) patient cells. We irradiate fibroblasts, lymphoblasts, and ATM-deficient fibroblasts with 5 Gy X-rays and perform Hi-C at 30 minutes, 24 hours, or 5 days after irradiation. We observe that 3D genome changes after irradiation are cell type-specific, with lymphoblastoid cells generally showing more contact changes than irradiated fibroblasts. However, all tested repair-proficient cell types exhibit an increased segregation of topologically associating domains (TADs). This TAD boundary strengthening after irradiation is not observed in ATM deficient fibroblasts and may indicate the presence of a mechanism to protect 3D genome structure integrity during DNA damage repair.

The three-dimensional structure of chromosomes plays an important role in gene expression regulation and also influences the repair of radiation-induced DNA damage. Genomic aberrations that disrupt chromosome spatial domains can lead to diseases including cancer, but how the 3D genome structure responds to DNA damage is poorly understood. Here, we investigate the impact of DNA damage response and repair on 3D genome folding using Hi-C experiments on wild type cells and ataxia telangiectasia mutated (ATM) patient cells. Fibroblasts, lymphoblasts, and ATM-deficient fibroblasts were irradiated with 5 Gy X-rays and Hi-C was performed after 30 minutes, 24 hours, or 5 days after irradiation. 3D genome changes after irradiation were cell type-specific, with lymphoblastoid cells generally showing more contact changes than irradiated fibroblasts. However, all tested repairproficient cell types exhibited an increased segregation of topologically associating domains (TADs). This TAD boundary strengthening after irradiation was not observed in ATM deficient fibroblasts and may indicate the presence of a mechanism to protect 3D genome structure integrity during DNA damage repair.2 Translocations, deletions, and other genomic aberrations that may follow DNA damage can lead to cancer by directly mutating genes or altering their regulation 7, 8 . Recently, it has become clear that the disruption of 3D genome domains can also be oncogenic 9 . Does the process of DNA repair protect the 3D folding of the genome as well as the linear DNA sequence? Certain cell types are considered to be more radiosensitive than others, but little is known about what contributes to their radiosensitivity. The possibility remains that cell type specific chromosome positioning, along with initial epigenetic chromatin and folding states can influence which translocations occur and how well DNA is able to repair after exposure to IR, helping to explain why certain cell types are more sensitive to radiation 8, 10, 11. Previous studies suggest that DNA repair efficiency may differ for heterochromatin and euchromatin 12,13 . Heterochromatic regions may be more mobile and move to DNA repair sites, where they decondense 14,15 . Condensation or decondensation of specific chromatin regions may not be determined by their preexisting histone modifications 14 , suggesting that other factors may contribute to changes in 3D genome structure after DNA damage. One previous study demonstrated spatial clustering of DSBs in active genes by inducing specific breaks and measuring their interactions with Capture Hi-C 16 . This suggests that changes in the structure of local genome domains may happen at a broader scale after IR. Additionally, CCCTC-binding factor (CTCF) and cohesin have been shown to be early responders to DNA damage induced by IR 17,18,19,20 . These proteins have also been recently demonstrated to play significant roles in chromosome folding 21,22,23,24 , contributing to the formation of topologically associating domains (TADs). These genomic domains intera...

To spread from a localized tumor, metastatic cancer cells must squeeze through constrictions that cause major nuclear deformations. Since chromosome structure affects nucleus stiffness, gene regulation, and DNA repair, here, we investigate the relationship between 3D genome structure and constricted migration in cancer cells. Using melanoma (A375) cells, we identify phenotypic differences in cells that have undergone multiple rounds of constricted migration. These cells display a stably higher migration efficiency, elongated morphology, and differences in the distribution of Lamin A/C and heterochromatin. Hi‐C experiments reveal differences in chromosome spatial compartmentalization specific to cells that have passed through constrictions and related alterations in expression of genes associated with migration and metastasis. Certain features of the 3D genome structure changes, such as a loss of B compartment interaction strength, are consistently observed after constricted migration in clonal populations of A375 cells and in MDA‐MB‐231 breast cancer cells. Our observations suggest that consistent types of chromosome structure changes are induced or selected by passage through constrictions and that these may epigenetically encode stable differences in gene expression and cellular migration phenotype.

To spread from a localized tumor, metastatic cancer cells must squeeze through constrictions that cause major nuclear deformations. Since chromosome structure affects nucleus stiffness, gene regulation and DNA repair, here we investigate how confined migration affects or is affected by 3D genome structure. Using melanoma (A375) cells, we identify phenotypic differences in cells that have undergone 10 rounds of constricted migration. These cells display a stable increase in migration efficiency, elongated morphology, and an abnormal distribution of Lamin A/C and heterochromatin. Using Hi-C, we observe changes in chromosome spatial compartmentalization specific to constricted cells and related alterations in expression of genes associated with migration and metastasis. These cells also show increased nuclear deformations when cultured in a 3D collagen matrix and altered behavior when co-cultured with fibroblasts in organoids. Our observations reveal a relationship between chromosome structure changes, metastatic gene signatures, and the abnormal nuclear appearance of aggressive melanoma.