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

全基因组加倍（Whole genome doubling, WGD）是人类癌症中反复出现的事件，其可促进染色体不稳定性与非整倍体的获得。然而，WGD细胞中染色质的三维组织形式及其对致癌表型的贡献目前仍不明确。本研究显示，在p53缺陷细胞中，WGD可诱导染色质分离丧失（Loss of Chromatin Segregation, LCS），其特征为长短染色体、A/B亚区室以及相邻染色质结构域之间的分离程度降低。LCS的发生是由绕过四倍体检查点激活的细胞中CCCTC结合因子（CTCF）与组蛋白H3赖氨酸9三甲基化（H3K9me3）的下调所驱动。纵向分析结果表明，LCS可使WGD细胞中的基因组区域具备亚区室重新定位的潜能，最终导致源自WGD细胞的肿瘤中出现与致癌基因激活相关的染色质与表观遗传改变。值得注意的是，亚区室重新定位事件在很大程度上独立于染色体改变，提示这是促进肿瘤发生与进展的互补机制。总体而言，LCS可启动染色质构象改变，最终导致致癌性的表观遗传与转录修饰，表明染色质进化是WGD驱动型癌症的标志性特征。

实验整体设计：为评估WGD即刻后的染色质组织，我们对RPE TP53-/-、CP-A TP53-/-（克隆3与克隆19）及K562细胞的WGD前和WGD后样本开展了染色质构象捕获（Hi-C）实验。我们采用了不同的WGD诱导方案，以排除是实验处理本身而非WGD导致了染色质水平的改变。此外，我们纳入了仅表现出染色体不稳定性但无WGD的细胞（仅CIN），以验证CIN与WGD是否会引发相同的表型。我们还对CP-A TP53-/-细胞中检测到的自发高倍体细胞进行了分选，并通过Hi-C进行检测。鉴于我们推测LCS是由WGD后四倍体检查点缺陷导致的CTCF与H3K9me3表达不足所引起，我们对经CDK4/6抑制剂处理以模拟p53诱导的四倍体检查点相关G1期延长的CP-A TP53-/- WGD细胞，以及CP-A p53野生型WGD细胞均开展了Hi-C实验——因为这两种模型中CTCF与H3K9me3的表达水平均得到了恢复。随后，我们对具备致瘤特性的长期WGD后细胞进行了检测。我们分别通过两种方式培养细胞：一是将RPE TP53-/- 20周WGD后细胞（T1、T2、T3）接种于NSG小鼠体内成瘤；二是在体外通过软琼脂克隆培养，培养源自CP-A TP53-/-克隆3 6周WGD后细胞（C1、C2）与CP-A TP53-/- 20周WGD后细胞（C1、C2）的克隆。如前文所述，每项实验均设置了生物学重复。源自不同WGD诱导方案的样本在样本登记表中被标记为"experiment 1/2"。所有实验组均与其对应的对照组进行比较。