Whole genome doubling drives oncogenic loss of chromatin segregation [Hi-C]

Whole genome doubling (WGD) is a recurrent event in human cancers and it promotes chromosomal instability and acquisition of aneuploidies. However, the 3D organization of the chromatin in WGD cells and its contribution to oncogenic phenotypes are currently unknown. Here, we show that in p53 deficient cells WGD induces loss of chromatin segregation (LCS), characterized by reduced segregation between short and long chromosomes, A and B sub-compartments, and adjacent chromatin domains. LCS is driven by downregulation of CTCF and H3K9me3 in cells that bypassed activation of the tetraploid checkpoint. Longitudinal analyses revealed that LCS primed genomic regions for sub-compartment repositioning in WGD cells, which resulted in chromatin and epigenetic changes associated with oncogene activation in tumours ensuing from WGD cells. Importantly, sub-compartment repositioning events were largely independent of chromosomal alterations, indicating that these were complementary mechanisms contributing to tumour development and progression. Overall, LCS initiates chromatin conformation changes that ultimately result in oncogenic epigenetic and transcriptional modifications, suggesting that chromatin evolution is a hallmark of WGD-driven cancer. Overall design: For assessing chromatin organization immediately after WGD, chromatin conformation capture (Hi-C) was performed in pre- and post-WGD cells in RPE TP53-/-, CP-A TP53-/- (clone 3 and clone 19), and K562 cells. Distinct WGD inductions were used to exclude that the treatment itself and not WGD is responsible for changes observed at chromatin level. Additionally, cells exhibiting chromosomal instability only, but not WGD, (CIN-only) were considered to check whether CIN and WGD are leading to the same phenotype. Spontaneous high ploidy cells detected in the CP-A TP53-/- were sorted and assessed by Hi-C. Considering LCS was hypothesized to be caused by CTCF and H3K9me3 insufficiency due to defective tetraploidy checkpoint after WGD, Hi-C was performed in CP-A TP53-/- WGD cells were treated with CDK4/6 inhibitor to mimic the G1 extension assciated with a p53-induced tetraploidy checkpoint, as well as in CP-A p53 wild type WGD cells, since in both cases CTCF and H3K9me3 levels were rescued. Long-term post-WGD cells with tumorigenic properties were then assessed. Cells were grown both in vivo in NSG mice from RPE TP53-/- 20w pWGD cells (T1, T2, T3), and in vitro in soft agar as colonies, derived from CP-A TP53-/- clone 3 6w pWGD (C1, C2) and CP-A TP53-/- 20w pWGD (C1, C2). Each experiment was performed in replicates, as indicated. Samples derived from distinct WGD inductions are marked as "experiment 1/2" in the sample sheet. All conditions were compared to their respective controls.