Regulation of re-replication in mammalian cells

项目摘要 哺乳动物细胞进化出多种不重叠的机制，以确保DNA复制启动 在每个分裂周期中只有一次复制的起源；失去对这些机制的控制 导致基因组不稳定，这是恶性转化的重要驱动因素。越来越多的证据表明 高等真核生物的起源、利用和激活受到表观遗传因素的影响，但确切的机制 在很大程度上是不确定的。我们的长期目标是阐明控制复制的基础机制 在哺乳动物细胞中的启动并理解这些机制的扰动如何引发基因组 不稳定。组蛋白甲基转移酶Set8正在成为复制启动的关键调节因子 哺乳动物细胞通过其对组蛋白H4K20的单甲基转移酶活性。细胞周期调节酶 在细胞周期的G1期对起源许可是必不可少的，但在S期被蛋白质降解；阻断 这一步骤触发了相同细胞周期内的重复复制启动或再复制。集8和 然而，H4K20me也参与转录抑制和DNA双链断裂的修复 (DSB)，但这些看似独立的活动是否在复制启动或重新复制中发挥作用 不知道。最重要的是，人们对重新复制的性质或特征知之甚少 在有缺陷的Set8降解的单元中积累的产品，也没有关于 如果某些基因组区域更容易引起重新复制，就会发生基因组重新复制。我们的新产品 结果表明，由有缺陷的Set8降解引起的重新复制不是一个随机过程，很少 基因组位置显示出巨大而显著的拷贝数增加，这让人想起基因组扩增 在癌细胞中可见。更多的初步研究表明，重复复制可能起源于DNA Double 在复制过程中可能自发产生的链断裂(DSB)，需要相关基因的活性 在转录沉默和DSB修复中。我们创新的初步研究和实验 设计的方法是彻底检查这种替代的再复制诱导模型。在目标1中，我们 将决定批量和批量复制的DNA的数量(拷贝数增加)和基因组分布 有缺陷的Set8降解的单个细胞，以及在特定基因组位置诱导DSB之后。我们 还将测试这些参数在不同类型的癌细胞以及癌症和非癌症细胞中是否有所不同。在目标2中， 我们将通过H4K20me阅读器确定组蛋白H4K20甲基化、转录沉默的作用 L3MBTL1，以及参与DSB修复的蛋白质。成功地执行了 提出的AIMS有望增加我们对 并有助于更好地理解这些机制的扰动是如何引发 基因组不稳定。

Project Summary Mammalian cells have evolved multiple non-overlapping mechanisms to ensure that DNA replication initiates from origins of replications once and only once in each division cycle; loss of control over these mechanisms induces genomic instability, an important driver of malignant transformation. Increasing evidence suggests that origin utilization and activation in higher eukaryotes is influenced by epigenetic factors, but exact mechanisms are largely undefined. Our long-term goals are to elucidate the underpinning mechanisms that control replication initiation in mammalian cells and to understand how perturbations of these mechanisms provokes genomic instability. The histone methyltransferase SET8 is emerging as a key regulator of replication initiation in mammalian cells through its mono-methyltransferase activity on histone H4K20. The cell cycle regulated enzyme is essential for origin licensing in G1 phase of the cell cycle, but is proteolytically degraded in S-phase; blocking this step triggers reiterative replication initiation within the same cell cycle or re-replication. Both SET8 and H4K20me, however, are also involved in transcriptional repression and in the repair of DNA double strand breaks (DSBs), but whether these seemingly independent activities play a role in replication initiation or re-replication is not known. Most importantly, little to nothing is known about the nature or characteristics of the re-replication products that accumulate in cells with defective SET8 degradation, nor is there information on where in the genome re-replication occurs or if certain genomic regions are more prone to re-replication induction. Our new results show that re-replication resulting from defective SET8 degradation is not a stochastic process, with few genomic sites exhibit large and significant copy number gains, reminiscent of genomic amplifications that are seen in cancer cells. Additional preliminary studies suggest that re-replication may originate from DNA double strand breaks (DSBs) that may spontaneously arise during replication, and requires the activity of genes involved both in transcriptional silencing and in DSB repair. Our innovative preliminary studies and experimental approaches are designed to thoroughly examine this alternative model of re-replication induction. In Aim 1, we will determine the magnitude (copy number gains) and genomic distribution of the re-replicated DNA in bulk and single cells with defective SET8 degradation and following the induction of DSBs at defined genomic sites. We will also test if these parameters vary in different cancer cell types and in cancer vs. non-cancer cells. In Aim 2, we will define the roles of histone H4K20 methylation, transcriptional silencing by the H4K20me reader L3MBTL1, and proteins involved in the repair of DSBs in effecting re-replication. The successful execution of the proposed aims promises to increase our understanding of the mechanisms regulating replication initiation in mammalian cells, and lead to a better understanding of how perturbations of these mechanisms provokes genomic instability.