Abbas Equipment Supplement

项目摘要 哺乳动物细胞已经进化出多种非重叠机制，以确保DNA复制启动 从复制的起源一次，只有一次在每个分裂周期;失去控制这些机制 诱导基因组不稳定性，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 启动哺乳动物细胞，并了解如何扰动这些机制诱导基因组 不稳定组蛋白甲基转移酶SET 8正在成为复制起始的关键调节因子， 哺乳动物细胞通过其对组蛋白H4 K20的单甲基转移酶活性。细胞周期调节酶 在细胞周期的G1期对来源许可至关重要，但在S期被蛋白水解降解;阻断 该步骤触发相同细胞周期内的重复复制起始或再复制。SET 8和 然而，H4 K20 me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起始或再复制中发挥作用， 不知道。最重要的是，人们对复制产物的性质知之甚少， 在有缺陷的SET 8降解细胞中积累，也没有关于基因组中何处重新合成的信息。 复制发生，或者是否存在更倾向于再复制诱导的某些基因组区域。我们的新 结果表明，再复制不是一个随机过程，只有少数基因组位点表现出大的 显著的拷贝数增加，让人想起在癌细胞中看到的基因组扩增。额外 研究进一步表明再复制可能起源于复制过程中自发产生的DSB， 并且需要参与转录沉默和DSB修复的基因的活性。我们的创新 初步研究和实验方法的目的是彻底检查这种替代模型， 再复制诱导。在目标1中，我们将通过以下方法绘制再复制起始位点的基因组分布图： 对异常稳定的SET 8进行全基因组染色质免疫沉淀（ChIP）研究， 甲基化H4 K20。我们将使用全基因组测序（WGS）的重新复制的DNA在FACS分选 单个细胞，以确定再复制产物的性质和由此形成的连接。我们还将 确定再复制DNA的位置和/或性质是否在不同细胞类型之间变化，以及 癌细胞和非癌细胞之间的差异。在目标2中，我们将阐明SET 8被招募的机制， 重新复制起始位点，并通过转录抑制和DSB修复蛋白的作用， 使用新型单位点SET 8-DNA-系链模块。成功地实现拟议的目标， 增加我们对哺乳动物细胞复制起始调节机制的理解， 更好地理解这些机制的扰动如何引起基因组不稳定。

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 induce 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 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 there are certain genomic regions that are more prone to re-replication induction. Our new results show that re-replication is not a stochastic process, and that only a few genomic sites exhibit large significant copy number gains, reminiscent of genomic amplifications that are seen in cancer cells. Additional studies further suggest that re-replication may originate from DSBs that 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 map the genomic distribution of re-replication initiation sites by performing genome-wide chromatin-immunoprecipitation (ChIP) studies of the aberrantly stabilized SET8 and methylated H4K20. We will use whole genome sequencing (WGS) of the re-replicated DNA in FACS-sorted single cells to determine the nature of the re-replication products and the junctions thus formed. We will also determine whether the location and/or nature of the re-replicated DNA varies between different cell types and between cancer vs. non-cancer cells. In Aim 2, we will elucidate the mechanism by which SET8 is recruited to re-replication initiation sites and define the role of transcriptional repression and DSB repair proteins through the use of a novel single-site SET8-DNA-tethering module. 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 provoke genomic instability.