Abbas Equipment Supplement

阿巴斯装备补充

• 批准号: 10799093
• 负责人: TAREK A. ABBAS
• 金额: $ 8.34万
• 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/10799093
• 关键词: Cell Cycle; Cells; Characteristics; DNA; DNA Double Strand Break; DNA Repair; DNA biosynthesis; Double Strand Break Repair; Enzymes; Epigenetic Process; Equipment; Eukaryota; Exhibits; G1 Phase; Gene Silencing; Genes; Genome; Genomic Instability; Genomic Segment; Genomics; Goals; Histones; L3MBTL1 gene; Licensing; Location; Malignant - descriptor; Malignant Neoplasms; Mammalian Cell; Maps; Methylation; Methyltransferase; Modeling; Nature; Proteins; Reader; Replication Initiation; Replication Origin; Role; S phase; Site; Sorting; Stochastic Processes; Testing; cancer cell; cell type; chromatin immunoprecipitation; design; gene repression; genome sequencing; genome-wide; histone methyltransferase; innovation; novel; recruit; 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 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.

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 哺乳动物细胞的启动，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，对重新复制产品的性质几乎一无所知 积聚在有缺陷的set8降解的细胞中，也没有关于基因组中的位置的信息 复制发生或某些基因组区域更容易重新诱导。我们的新 结果表明，重新复制不是随机过程，并且只有少数基因组位点显示大 显着的拷贝数增长，让人联想到癌细胞中看到的基因组扩增。额外的 研究进一步表明，重新复制可能源自在复制过程中自发出现的DSB， 并且需要参与转录沉默和DSB修复中涉及的基因活性。我们的创新性 初步研究和实验方法旨在彻底检查这种替代模型 重新诱导。在AIM 1中，我们将通过绘制重新复制启动位点的基因组分布 对异常稳定的SET8和 甲基化的H4K20。我们将使用FACS分类中重新复制的DNA的整个基因组测序（WGS） 单个细胞确定重新复制产物的性质和如此形成的连接。我们也会 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/或 在癌症与非癌细胞之间。在AIM 2中，我们将阐明SET8招募到的机制 重新恢复起始位点并通过转录抑制和DSB修复蛋白的作用 使用新型的单位点SET8-DNA螺旋模块。拟议的目标的成功执行承诺 为了增加我们对调节哺乳动物细胞复制起始的机制的理解，并引导 为了更好地理解这些机制的扰动如何引起基因组不稳定性。