Abbas Equipment Supplement

University Of Virginia, Charlottesville VA

Investigators

Linked publications & trials

Abstract

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.