DESCRIPTION (provided by applicant): LTR Retrotransposons are mobile genetic elements and major constituents of eukaryotic genomes, where they contribute to structural variation and epigenetic regulation. They are highly related to Retroviruses, and like all parasitic elements their evolutionary success depends on exploitation of critical cellular processes. Their activity can lead to chromosomal alterations that drive diseases like cancer. We have discovered that the LTR Retrotransposon Tf1 inserts near genomic replication fork barriers, and includes new replication fork barriers in its own genome. In this proposal we aim to characterize the interaction of LTR Retrotransposons and host DNA replication, and how it influences integration site selection, genome integrity and epigenetic transcriptional silencing. The overall HYPOTHESIS to be evaluated is that the replication fork is a point of cross-talk between the host and the LTR retrotransposon, by way of trans-acting factors that bind to the transposon insertion site and the LTR and control replication fork progression through chromatin remodeling. This results in managed Homologous Recombination and transcriptional silencing. We will use the LTR Retrotransposons of the fission yeast Schizosaccharomyces pombe as models for the involvement of DNA replication in LTR Retrotransposon biology. Specific aims: 1. To determine the mechanism of Tf1 insertion site selection and to ascertain the role of Sap1 in this process. Sap1 is a DNA binding factor that blocks progression of the replication fork and guides insertion of Tf1 to the blocked sites. We wil characterize the involvement of the replication fork in the insertion pathway and the possible tethering role of Sap1 in the homing mechanism. 2. To determine the influence of Retrotransposons on DNA replication and their consequences on DNA replication directionality and genome stability. The LTR are notoriously recombinogenic but the reasons are unknown. We will investigate the involvement of the replication fork barriers present in LTR in their recombinogenic potential, and their consequences on genome instability and the phenomenon of directional replication. 3. To determine the mechanism of transcriptional silencing of LTR retrotransposons, and the interaction between DNA replication and RNAi. Heterochromatic silencing is a universal feature of Transposable Elements. We have discovered the same factors that manage replication fork progression at the LTR determine a novel repressive heterochromatin structure that silences it. We will investigate the involvement of the DNA replication management factors on the mechanisms of transcriptional silencing of LTR retrotransposons. Significance: These studies will provide a novel and comprehensive model of host-retroelement interactions, and their consequences on genome regulation and stability.

PUBLIC HEALTH RELEVANCE: Project narrative: Transposable Elements are major drivers of genome variability and regulation, and they have been implicated in processes of genome instability that lead to cancer. This project addresses several aspects of Transposon-host interaction that have consequences in normal genome function and in disease, such as: the selection of insertion sites, the destabilizing effects of Transposon accumulation on genome integrity, and the host genome defense by Transposon eviction and transcriptional silencing. LTR retrotransposons are highly related to Retroviruses, with whom they share a replication mechanism, and therefore they are excellent models for a wide group of genomic parasites. The principles of Transposon-host interactions learned from this investigation will be broadly applicable and illuminate new aspects of genome function.