Regulation of re-replication in mammalian cells

哺乳动物细胞再复制的调节

• 批准号: 10387262
• 负责人: TAREK A. ABBAS
• 金额: $ 15.9万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10387262
• 关键词: Cell Cycle; Cells; Characteristics; Chromatin; DNA; DNA Double Strand Break; DNA Repair; DNA biosynthesis; Development; Double Strand Break Repair; Ensure; Enzymes; Epigenetic Process; Eukaryota; Exhibits; G1 Phase; Gene Silencing; Generations; Genes; Genome; Genomic Instability; Genomic Segment; Genomics; Goals; Histones; Lead; Licensing; Malignant - descriptor; Malignant Neoplasms; Mammalian Cell; Methyltransferase; Modeling; Molecular; Nature; Nuclear; Play; Process; Proteins; Regulation; Replication Initiation; Replication Origin; Role; S Phase; Site; Stochastic Processes; Testing; anti-cancer therapeutic; cancer cell; cell type; cytotoxic; design; gene repression; histone methyltransferase; innovation; whole genome

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 there are certain genomic regions that 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. Aim 1 we will determine the magnitude (copy number gains) and genomic distribution of the re- replicated DNA in cells with defective SET8 degradation and following the induction of DSBs. We will also test if these parameters vary in different cancer cell types and in cancer vs. non-cancer cells. Aim 2 will determine the chromatin occupancy of aberrantly stabilized SET8 and methylated H4K20 in cells with defective SET8 degradation, and whether these overlap with regions of re-replicated DNA. Aim 3 will define the role of transcriptional repression and DSB repair proteins in re-replication induction. 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.

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 启动在哺乳动物细胞，并了解如何扰动这些机制引起基因组 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，人们对再复制的性质或特征知之甚少 产品在有缺陷的SET 8降解的细胞中积累，也没有关于 发生基因组再复制，或者如果存在更倾向于再复制的某些基因组区域， 诱导我们的新结果表明，由缺陷性SET 8降解引起的再复制不是随机的， 具有很少基因组位点的过程表现出大的和显著的拷贝数增益，使人想起基因组 在癌细胞中看到的扩增。额外的初步研究表明，再复制可能 来源于DNA双链断裂（DSB），可能在复制过程中自发出现，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 研究和实验方法的目的是彻底检查这种替代模型的再复制 诱导目的1：我们将确定重组的大小（拷贝数增加）和基因组分布。 在具有缺陷性SET 8降解的细胞中以及在DSB诱导后复制DNA。我们还将测试 这些参数在不同的癌细胞类型中以及在癌细胞与非癌细胞中变化。目标2将决定 异常稳定的SET 8和甲基化H4 K20在SET 8缺陷细胞中的染色质占有率 降解，以及这些是否与重新复制的DNA区域重叠。目标3将确定以下方面的作用： 转录抑制和DSB修复蛋白在再复制诱导。的成功执行 提出的目标有望增加我们对调控复制起始的机制的理解， 哺乳动物细胞，并导致更好地了解这些机制的扰动如何引起 基因组不稳定性