Abbas Equipment Supplement

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，并了解这些机制的扰动如何诱导基因组 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，人们对重新复制产品的性质知之甚少 在Set8降解缺陷的细胞中积累，也没有关于基因组重新定位的信息。 如果有某些基因组区域更容易再次复制诱导，就会发生复制。我们的新产品 结果表明，再复制不是一个随机的过程，只有少数基因组位点表现出较大的 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。其他内容 研究进一步表明，重新复制可能起源于复制过程中自发产生的DSB， 并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旨在彻底检验这一替代模式 再复制诱导。在目标1中，我们将通过以下方式绘制再复制起始点的基因组分布 进行全基因组染色质免疫沉淀(ChIP)研究异常稳定的Set8和 甲基化H4K20。我们将在FACS中使用重新复制的DNA的全基因组测序 单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 确定重新复制的DNA的位置和/或性质是否在不同的细胞类型和 癌症和非癌症细胞之间的关系。在目标2中，我们将阐明集合8被招募到 重新复制起始点，并确定转录抑制和DSB修复蛋白通过 使用了一种新颖的单部位Set8-DNA系留模块。成功执行拟议的AIMS承诺 为了增加我们对哺乳动物细胞复制启动的调控机制的理解，以及铅 更好地理解这些机制的扰动是如何引起基因组不稳定的。

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.