Local features determine Ty3 targeting frequency at RNA polymerase III transcription start sites

Abstract

Retroelement integration into host genomes affects both genome structure and function. A goal of a considerable number of investigations is to elucidate host features that influence insertion site selection. The retrotransposon Ty3 of Saccharomyces cerevisiae inserts proximal to the transcription start sites (TSS) of genes transcribed by RNA polymerase III (RNAP3). In this study, differential patterns of insertion were profiled genome wide using a random barcode-tagged Ty3. Saturation transposition showed that tRNA genes (tDNAs) are targeted at widely different frequencies even within isoacceptor families. Ectopic expression of Ty3 integrase (IN) showed that it localized to integration targets independent of other Ty3 proteins and cDNA. IN, RNAP3 and transcription factor Brf1 were enriched at tDNA targets with high frequencies of transposition. To examine potential effects of target DNA on transposition, targeting frequency was reconstituted on high-copy plasmids within 75 bp of 5' flanking sequence plus the tDNA. Weighting of insertions according to frequency identified an A/T-rich sequence followed by C as the site of strand transfer. This site lies immediately adjacent to the adenines previously implicated in RNAP3 TSS motif (CAA). In silico DNA structural analysis around the TSS showed that targets with reduced DNA curvature coincide with elevated integration. We propose that activity of the Ty3 intasome complex (IN and cDNA) is modulated by a combination of host factor occupancy and insertion site architecture, resulting in the wide range of Ty3 targeting frequencies.