DNA methylation maintains integrity of higher order genome architecture (WGBS)

DNA replication timing and 3D chromatin organisation are associated with epigenomic changes across large domains during human differentiation and cancer progression. However, it is unclear if epigenome changes, in particular cancer-associated DNA hypomethylation, is a consequence or cause of changes observed in higher order genome architecture. Here, we compare replication timing profiles and three dimensional (3D) genome organisation, using Hi-C and single cell Repli-Seq in the DNMT1 and DNMT3B DNA methyltransferases double knockout hypomethylated DKO1 colorectal cancer cell line and its parental HCT116 cell line. We find that the hypomethylated cells show a profound loss of replication timing precision, gain of single cell replication timing heterogeneity and loss of chromatin conformation integrity. Discrete regions, that undergo a large change in replication timing in the hypomethylated cells, are associated with a loss of allelic replication timing and shrinking of late replicating Partially Methylated Domain (PMD) boundaries. In contrast, conservation of replication timing after DNA methylation depletion at PMDs is associated with the formation of new H3K9me3/H3K4me3 bivalent domains which may serve to prevent ectopic transcription and maintain cell viability. Together our results show that a loss of global methylation, a common hallmark of cancer, directly impacts on the precision of replication timing and contribute to deregulation of the 3D chromatin architecture. Overall design: Repli-Seq, single cell Repli-Seq, Hi-C, WGBS and Nanopore sequencing was conducted in HCT116 human colorectal cancer cell line and the same cell line with knockout of DNMT1 and DNMT3B (DKO1) (Rhee et al. 2002).

DNA复制时序与三维染色质构象在人类分化与癌症进展过程中，与大片段基因组区域的表观基因组改变紧密相关。然而目前仍未明确，表观基因组变化——尤其是癌症相关的DNA低甲基化——究竟是高阶基因组结构改变的结果，还是其诱发因素。本研究借助Hi-C与单细胞Repli-Seq技术，对比了DNA甲基转移酶DNMT1与DNMT3B双基因敲除的低甲基化结直肠癌细胞系DKO1及其亲本HCT116细胞的复制时序图谱与三维基因组构象。研究发现，低甲基化细胞出现了复制时序精准性的显著丧失、单细胞复制时序异质性的升高，以及染色质构象完整性的破坏。在低甲基化细胞中复制时序发生大幅改变的离散区域，与等位复制时序的丧失以及晚复制区域部分甲基化结构域（Partially Methylated Domain, PMD）边界的收缩密切相关。与之相反，在PMD区域的DNA甲基化缺失后仍保留复制时序保守性的区域，会形成新的H3K9me3/H3K4me3二价结构域，这可能有助于抑制异位转录并维持细胞活力。综上，本研究结果表明，作为癌症常见特征的全局甲基化丧失，会直接影响复制时序的精准性，并参与三维染色质构象的失调。整体实验设计：本研究对人类结直肠癌细胞系HCT116及其DNMT1与DNMT3B敲除细胞系DKO1（Rhee等，2002）开展了Repli-Seq、单细胞Repli-Seq、Hi-C、全基因组亚硫酸氢盐测序（WGBS）以及纳米孔测序（Nanopore sequencing）实验。