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.

项目概要 哺乳动物细胞已经进化出多种不重叠的机制来确保 DNA 复制的启动 在每个分裂周期中从复制起点一次且仅一次；失去对这些机制的控制 诱导基因组不稳定，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 很大程度上是未定义的。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 正在成为复制起始的关键调节因子。 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 对于细胞周期 G1 期的来源许可至关重要，但在 S 期会被蛋白水解降解；阻塞 此步骤触发同一细胞周期内的重复复制启动或重新复制。 SET8 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尚不清楚 不知道。最重要的是，人们对再复制产品的性质知之甚少，甚至一无所知。 SET8 降解有缺陷的细胞中积累，也没有关于基因组中何处重新聚集的信息。 复制发生或者如果存在某些基因组区域更容易诱导再复制。我们的新 结果表明，重新复制不是一个随机过程，并且只有少数基因组位点表现出大 显着的拷贝数增加，让人想起在癌细胞中看到的基因组扩增。额外的 研究进一步表明，再复制可能源自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旨在彻底检验这种替代模型 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位点的基因组分布图： 对异常稳定的 SET8 进行全基因组染色质免疫沉淀 (ChIP) 研究 甲基化 H4K20。我们将在 FACS 分选中使用重新复制的 DNA 的全基因组测序 (WGS) 单细胞来确定再复制产物和由此形成的连接的性质。我们还将 确定重新复制的 DNA 的位置和/或性质在不同细胞类型之间是否存在差异，以及 癌症与非癌细胞之间。在目标 2 中，我们将阐明招募 SET8 的机制 再复制起始位点并通过以下方式定义转录抑制和 DSB 修复蛋白的作用 使用新型单位点 SET8-DNA 束缚模块。成功执行拟议目标有望 增加我们对哺乳动物细胞复制起始调节机制的理解，并导致 更好地理解这些机制的扰动如何引起基因组不稳定。