Methylation of histone H4 lysine 20 by PR-Set7 ensures the integrity of late replicating sequence domains in Drosophila. Drosophila melanogaster

The methylation state of lysine 20 on histone H4 (H4K20) has been linked to cell cycle progression, Origin Recognition Complex (ORC) binding, and replication origin regulation. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7, which is essential for genome integrity and cell cycle progression. PR-Set7 depletion in mammalian cells results in defective S-phase progression and the accumulation of DNA damage, which could be partially attributed to a defect in pre-Replication Complex (pre-RC) formation and origin activity. However, these studies were limited to a handful of mammalian origins, and it remains unclear how PR-Set7 and H4K20 methylation impact the replication program on a genomic scale. Using Drosophila Kc167 cells, we employed genetic, cytological, and genomic approaches to better understand the role of PR-Set7 and H4K20 methylation in regulating DNA replication and governing genome stability. We find that depletion of Drosophila PR-Set7 and loss of H4K20me1 result in the accumulation of DNA damage and an ATR-dependent cell cycle arrest. The cell cycle arrest occurs during the second S-phase following loss of PR-Set7 activity, suggesting that accumulation of nascent H4K20 is recalcitrant to the DNA replication program. Deregulation of H4K20 methylation had no impact on origin activation throughout the genome; instead, we found that the DNA damage marker, phosphorylated H2A.v (γ-H2A.v), accumulated specifically in late replicating domains in the absence of PR-Set7. This suggests that the molecular basis for the cell cycle arrest and accumulation of DNA damage resulting from loss of PR-Set7 is stochastic fork collapse within late replicating domains. Overall design: Examination of BrdU enrichment in the presence of hydroxyurea to map activation of early replication origin under control, PR-Set7, ATR, and ATR/PR-Set7 RNAi treatment conditions in duplicates using Illumina HiSeq and Miseq respectively; map early origin in 6 other conditions using Illumina HiSeq; examination of H4K20me1 in early or late S-phase with two different experimental methods, using Hiseq; examination of a histone variant with control or PR-Set7 RNAi treatment, in duplicates, using Hiseq.

组蛋白H4上赖氨酸20（H4K20）的甲基化状态，与细胞周期进程、起源识别复合物（Origin Recognition Complex, ORC）结合以及复制起始位点调控密切相关。H4K20的单甲基化修饰（H4K20me1）由受细胞周期调控的组蛋白甲基转移酶PR-Set7介导，该酶对基因组完整性与细胞周期进程至关重要。在哺乳动物细胞中敲低PR-Set7会导致S期进程异常以及DNA损伤积累，这一现象可部分归因于复制前复合物（pre-Replication Complex, pre-RC）形成缺陷与起始位点活性异常。然而，现有研究仅针对少数哺乳动物复制起始位点展开，目前仍不清楚PR-Set7与H4K20甲基化如何在全基因组层面影响复制程序。本研究以果蝇Kc167细胞为模型，采用遗传学、细胞学与基因组学手段，深入探究PR-Set7及H4K20甲基化在调控DNA复制与维持基因组稳定性中的作用。我们发现，果蝇PR-Set7敲低以及H4K20me1缺失会导致DNA损伤积累，并引发依赖于ATR的细胞周期阻滞。该细胞周期阻滞发生在PR-Set7活性丧失后的第二个S期，这提示新生H4K20的积累难以适配DNA复制程序。H4K20甲基化失调并不会影响全基因组范围内的复制起始位点激活；相反，我们发现在PR-Set7缺失的情况下，DNA损伤标志物磷酸化H2A.v（γ-H2A.v）仅在晚期复制区域中积累。这表明，PR-Set7缺失引发的细胞周期阻滞与DNA损伤积累的分子基础，是晚期复制区域内发生的随机性复制叉崩溃。实验整体设计：分别利用Illumina HiSeq与Illumina MiSeq平台，对对照组、PR-Set7 RNA干扰组、ATR RNA干扰组以及ATR/PR-Set7联合RNA干扰组细胞在羟基脲（hydroxyurea）处理下的5-溴脱氧尿苷（Bromodeoxyuridine, BrdU）富集情况进行重复检测，以定位早期复制起始位点；另外通过Illumina HiSeq平台完成6种其他条件下的早期复制起始位点定位；采用两种不同实验方法，利用HiSeq平台检测早S期与晚S期细胞中的H4K20me1水平；利用HiSeq平台对对照组与PR-Set7 RNA干扰组细胞的一种组蛋白变体进行重复检测。