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 哺乳动物细胞已经发展出多种非重叠机制，以确保DNA复制启动 从复制的起源一次，在每个分区周期中仅一次；失去对这些机制的控制 诱导基因组不稳定性，这是恶性转化的重要驱动力。越来越多的证据表明 较高的真核生物中的起源利用和激活受表观遗传因素的影响，但是确切的机制 在很大程度上不确定。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引起基因组 不稳定。组蛋白甲基转移酶SET8正在成为复制起始的关键调节剂 哺乳动物细胞通过其单甲基转移酶在组蛋白H4K20上的活性。细胞周期调节的酶 对于细胞周期的G1阶段中的起源许可是必不可少的，但在S期中蛋白水解降解。阻塞 此步骤触发在同一细胞周期或重新复制中的重复复制起始。 set8和 然而，H4K20Me也参与转录抑制和DNA双链断裂的修复 （DSB），但是这些看似独立的活动在复制启动还是重复复制中起作用 不知道。最重要的是，重新复制的性质或特征几乎一无所知 积累有缺陷的set8降解的细胞中的产品，也没有有关在哪里 基因组重新复制发生，或者是否有某些基因组区域更容易重新复制 就职。我们的新结果表明，由于set8降级而导致的重新复制不是随机的 很少有基因组位点的过程表现出大而显着的拷贝数增长，让人联想到基因组 在癌细胞中看到的扩增。其他初步研究表明，重新复制可能 起源于DNA双链断裂（DSB），可能在复制过程中自发出现，并且需要 转录沉默和DSB修复中涉及的基因活性。我们的创新初步 研究和实验方法旨在彻底检查这种重新复制的替代模型 就职。 AIM 1我们将确定重复的幅度（拷贝数增长）和基因组分布 在DSBS诱导后，在有缺陷的set8降解的细胞中复制的DNA。我们还将测试是否 这些参数在不同的癌细胞类型和癌症与非癌细胞中有所不同。 AIM 2将确定 异常稳定的set8和甲基化的H4K20的染色质占有率在有缺陷的细胞中 降解，以及这些是否与重新复制的DNA区域重叠。 AIM 3将定义 转录抑制和DSB修复蛋白重新诱导。成功执行 拟议的目标有望增加我们对调节复制启动机制的理解 哺乳动物细胞，并可以更好地理解这些机制的扰动 基因组不稳定性。