DNA hypomethylation promotes UHRF1- and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states [RNA-Seq]. DNA hypomethylation promotes UHRF1- and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states [RNA-Seq]

Mono-ubiquitination of lysine 18 on histone H3 (H3K18ub), catalyzed by UHRF1, is a DNMT1 docking site that facilitates replication-coupled DNA methylation maintenance. Its functions beyond this are unknown. Here, we genomically map simultaneous increases in UHRF1-dependent H3K18ub and SUV39H1/H2-dependent H3K9me3 following DNMT1 inhibition. Mechanistically, transient accumulation of hemi-methylated DNA at CpG islands facilitates UHRF1 recruitment and E3 ligase activity towards H3K18. Notably, H3K18ub enhances SUV39H1/H2 methyltransferase activity and, in colon cancer cells, nucleates new H3K9me3 domains at CpG island promoters of DNA methylation-silenced tumor suppressor genes. Disrupting UHRF1 enzyme activity prevents H3K9me3 accumulation while promoting PRC2-dependent H3K27me3 as a tertiary layer of gene repression in these regions. In contrast, disrupting H3K18ub-dependent SUV39H1/H2 activity enhances the transcriptional activating and antiproliferative effects of DNMT1 inhibition. Collectively, these findings reveal roles for UHRF1 and H3K18ub in regulating a hierarchy of repressive histone methylation signaling and rationalize a combination strategy for epigenetic cancer therapy. Overall design: RNA-seq in RKO cells with SUV39H2 KO and dox-inducible SUV39H1 shRNA KD. There are four treatment groups: cells were treated with 1) DMSO control, 2) 20ng/mL doxycycline (SUV39H1 KD), 3) 1uM GSK3484862(DNMT1 inhibition), and 4) doxycycline+GSK3484862 (SUV39H1 KD + DNMT1 inhibition) for 5 days before RNA collection.

组蛋白H3赖氨酸18位点的单泛素化（Mono-ubiquitination, H3K18ub）由UHRF1催化，是DNA甲基转移酶1（DNMT1）的停靠位点，可促进复制偶联的DNA甲基化维持过程。除该功能外，其尚不明确的其他生物学功能尚未被阐明。本研究通过基因组学手段，绘制了DNMT1抑制后，UHRF1依赖的H3K18ub与SUV39H1/H2依赖的H3K9me3同时上调的基因组图谱。机制层面，CpG岛处的半甲基化DNA暂时性积累可促进UHRF1的招募，并增强其针对H3K18的E3泛素连接酶活性。值得注意的是，H3K18ub可增强SUV39H1/H2的甲基转移酶活性；在结肠癌细胞中，该修饰可在DNA甲基化沉默的抑癌基因的CpG岛启动子区域诱导形成全新的H3K9me3结构域。破坏UHRF1的酶活性可阻止H3K9me3的积累，同时会促进多梳抑制复合体2（PRC2）依赖的H3K27me3形成，以此作为这些区域基因沉默的第三重调控层级。与之相反，破坏H3K18ub依赖的SUV39H1/H2活性，则会增强DNMT1抑制所带来的转录激活与抗增殖效应。综上，本研究揭示了UHRF1与H3K18ub在调控抑制性组蛋白甲基化信号层级中的作用，并为表观遗传癌症治疗的联合策略提供了理论依据。总体实验设计：在携带SUV39H2敲除与多西环素诱导型SUV39H1短发夹RNA敲低的RKO细胞中开展RNA测序（RNA-seq）。共设置4组处理：1）二甲基亚砜（DMSO）对照组；2）20ng/mL多西环素处理组（诱导SUV39H1敲低）；3）1μM GSK3484862处理组（DNMT1抑制）；4）多西环素+GSK3484862联合处理组（SUV39H1敲低+DNMT1抑制）。所有处理均持续5天后收集细胞以进行RNA提取与测序。