Regulation of re-replication in mammalian cells

哺乳动物细胞再复制的调节

• 批准号: 10320029
• 负责人: TAREK A. ABBAS
• 金额: $ 31.45万
• 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/10320029
• 关键词: Affect; Bioinformatics; Catalytic Domain; Cell Cycle; Cell Line; Cells; Characteristics; Chromatin; Chromosomes; Complement; Complex; DNA; DNA Double Strand Break; DNA Repair; DNA Repair Gene; DNA biosynthesis; DNA replication origin; DNA-PKcs; DNA-dependent protein kinase; Development; Double Strand Break Repair; Ensure; Enzymes; Epigenetic Process; Eukaryota; Exhibits; Failure; Fire - disasters; Fluorescent in Situ Hybridization; G1 Phase; Gene Silencing; Generations; Genes; Genetic Engineering; Genome; Genomic Instability; Genomic Segment; Genomics; Goals; Histones; L3MBTL1 gene; Lead; Licensing; Malignant - descriptor; Malignant Neoplasms; Mammalian Cell; Mediating; Methylation; Methyltransferase; Mitosis; Modeling; Molecular; Nature; Nuclear; Pharmacology; Play; Population; Process; Proteins; Reader; Regulation; Replication Initiation; Replication Licensing; Replication Origin; Resistance; Role; S phase; Site; Stochastic Processes; Testing; Transcription Repressor; Transcriptional Regulation; Transferase; anti-cancer therapeutic; cancer cell; cell type; cytotoxic; design; epigenetic regulation; gene repression; genome sequencing; genomic locus; histone methyltransferase; improved; innovation; mutant; 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 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 certain genomic regions 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. In Aim 1, we will determine the magnitude (copy number gains) and genomic distribution of the re-replicated DNA in bulk and single cells with defective SET8 degradation and following the induction of DSBs at defined genomic sites. We will also test if these parameters vary in different cancer cell types and in cancer vs. non-cancer cells. In Aim 2, we will define the roles of histone H4K20 methylation, transcriptional silencing by the H4K20me reader L3MBTL1, and proteins involved in the repair of DSBs in effecting re-replication. 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激发基因组 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，人们对重新复制的性质或特征知之甚少。 产物在有缺陷的 SET8 降解的细胞中积累，也没有关于在何处的信息 基因组再复制发生，或者某些基因组区域更容易诱导再复制。我们的新 结果表明，由于 SET8 降解缺陷导致的重新复制不是一个随机过程，很少有 基因组位点表现出大量且显着的拷贝数增益，让人想起基因组扩增 见于癌细胞。其他初步研究表明，重新复制可能源于 DNA 双倍 复制过程中可能自发出现的链断裂（DSB），需要相关基因的活性 转录沉默和 DSB 修复。我们的创新性初步研究和实验 方法旨在彻底检查这种再复制诱导的替代模型。在目标 1 中，我们 将确定大量重新复制的 DNA 的大小（拷贝数增益）和基因组分布， SET8 降解有缺陷的单细胞，并在确定的基因组位点诱导 DSB 后。我们 还将测试这些参数在不同癌细胞类型以及癌症细胞与非癌细胞中是否有所不同。在目标 2 中， 我们将通过 H4K20me 阅读器定义组蛋白 H4K20 甲基化和转录沉默的作用 L3MBTL1 和参与 DSB 修复以影响再复制的蛋白质。的成功执行 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，并导致更好地理解这些机制的扰动如何引起 基因组不稳定。