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Regulation of re-replication in mammalian cells

哺乳动物细胞再复制的调节

基本信息

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
项目类别：
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

项目摘要

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.

项目总结