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

Abstract

Retroelement integration into host genomes affects chromosome structure and function. A goal of a considerable number of investigations is to elucidate features influencing insertion site selection. The Saccharomyces cerevisiae Ty3 retrotransposon 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 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 cis-acting DNA features on transposition, targeting was tested on high-copy plasmids with restricted amounts of 5' flanking sequence plus tDNA. Relative activity of targets was reconstituted in these constructions. Weighting of genomic insertions according to frequency identified an A/T-rich sequence followed by C as the dominant site of strand transfer. This site lies immediately adjacent to the adenines previously implicated in the RNAP3 TSS motif (CAA). In silico DNA structural analysis upstream of this motif showed that targets with elevated DNA curvature coincide with reduced integration. We propose that integration mediated by the Ty3 intasome complex (IN and cDNA) is subject to inputs from a combination of host factor occupancy and insertion site architecture, and that this results in the wide range of Ty3 targeting frequencies.

Figure 1.

Genome-wide Ty3 insertion mapping using barcode-tagged elements. (A) Ty3 insertion sites are plotted with aligned mature tRNA-coding sequence (tDNA) = 0th bp and insertion site defined as the tDNA proximal end of the 5-bp TSD. Each dot represents the normalized unique barcodes of insertions starting at the strand transfer site proximal to the tDNA per 10,000 hits: (red) hot; (black) average; (blue) cold. Note that classification into hot, average, and cold was based on subsequently aggregating insertions per a 50-bp upstream window of the tDNA. (B) Insertions from A are binned across a 50-bp window upstream of the tDNA (colored as in A). (C) Circos tracks display chromosomes, tDNAs, Ty3 LTRs, and Ty3 insertion frequency: (outside to inside) colored as in A; (Ty3 LTRs) peach.

Figure 2.

Basal transcription factors, RNAP3, and IN at Ty3 targets. (A) ChIP-seq analysis of N-FLAG-tagged Brf1, Rpc34, and IN. Heatmaps showing normalized fold enrichment (FE) for RNAP3-transcribed genes relative to flanking sequence; rows ordered according to Ty3 transposition frequency top to bottom, high to low, as indicated by the colored bar. Position 0 refers to the nucleotide encoding the 5′ end of the mature tRNA. Above the heatmaps is an expanded view of FE for Brf1, Rpc34, and IN for hot and cold genes compared to untagged Brf1 and Rpc34 strains and empty vector strain for IN with standard deviation represented in lower intensity. (B) Peak analysis averaged for all tDNAs and traced over a 1-kb surrounding window. Lightened haze around each line indicates standard deviation. WT refers to the untagged parent strain of N-FLAG Brf1 and N-FLAG Rpc34; WT + empty vector (pKP3915) control for N-FLAG IN (WT + pKP4010). The dotted vertical line represents the average Ty3 insertion site position. (C) ROC analysis comparing hot versus all other tDNA, hot versus cold, and cold versus all other tDNAs. Legends for each plot show the AUC with 99% confidence intervals (CI). As a positive control for each plot, Ty3 insertion frequency (used to define hot and cold) is included to show perfect association between phenotype classifier and FE predictors (black line).

Figure 3.

Ty3 insertion frequency into high-copy-number target plasmids. (A) Diagram of tDNA target assay. Measurement of insertion frequency of Ty3 into tDNA hot and cold target plasmids by qPCR described in Methods. (B) Frequency of insertion (%) into tDNA target plasmids. Biological replicates N = 4–6. (C) Diagram of hot tQ(CUG)M and cold tQ(UUG)B target tDNAs and sequence swaps. (D) Sequence upstream of tDNA mature coding region influences Ty3 insertion frequency. Measurement of insertion frequency into hot and cold tDNA targets with and without sequence swaps described in C. Insertion frequency expressed as arbitrary units (AU) normalized to native tDNA targets. Biological replicates N = 10–12. The diamond (◊) indicates a data point (14,124) excluded from the box plot. (E) Transcription levels from tDNA target plasmids. Box plots show transcription from tQM and tQB (left), or tQMt target plasmids with 25 or 75 bp of upstream sequence from tQBt (middle) and vice versa (right). White boxes represent transcription from genomic SUP61, an intron containing tRNA where nascent pre-tRNA was measured as a proxy for transcription. Values expressed as normalized arbitrary units (AU).

Figure 4.

Ty3 insertion site analysis upstream of tDNA genes. (A) WebLogo analysis of the 11 bp comprised of Ty3 5-bp target site duplication (TSD) and ±3 bp flanking sequence. Each TSD was weighted by the total number of Ty3 insertions at that site. Brackets indicate the corresponding nucleotide positions (top row of numbers) assigned to each dinucleotide bin (bottom row of numbers). (B) Plots of dinucleotide frequency determined from sequences shown in A. Dinucleotide starts at position indicated (“0” = dinucleotide at positions 0 and 1 of TSD, etc.). YR/RY (top) and YY/RR (bottom) plots of TSD and flanking sequence shown in A. Vertical dashed lines mark the dinucleotide bins representing the borders of the TSD. Horizontal dashed line represents the random frequency of the YR dinucleotide in the S. cerevisiae genome (23.25%). Random frequency of all dinucleotide bins in the S. cerevisiae genome are 23.25% (YR), 23.25% (RY), 26.71% (YY), and 26.79% (RR). (C) WebLogo analysis of conserved motifs within a 23-bp window upstream of all tDNA genes by MEME Suite. All four DNA nucleotides occur at roughly the same frequency at position 6. (D) Distance analysis of Ty3 TSD to MEME-predicted and empirically determined TSS upstream of tDNAs. From top to bottom: distance between MEME-predicted TSS and TSS of 29 tDNAs empirically determined by Yukawa et al. (2011); distance between Ty3 TSD and empirical TSS; distance between Ty3 TSD and MEME-predicted TSS of all tDNAs in this study categorized by hot, average, and cold phenotypes. For all comparisons, distance is measured from the fifth base of the TSD to the first conserved “C” nucleotide in both MEME-predicted motifs and empirically determined motifs (see text for detailed explanation). The first, second, and third quartiles of each data set are denoted by white lines on each violin plot.

Figure 5.

Role of curvature in target site determination. (A) Plot of curvature as determined by bend.it analysis (Vlahovicek et al. 2003) of 100 bp upstream of tDNA for hot (tQM) and cold (tQB) targets. (B) Linear regression of curvature versus insertion frequency. (C,D) Plots of curvature of hot and cold plasmid targets and swapped sequences as described in legend of Figure 3C.

Figure 6.

Modeled comparison of RNAP3 TSS and Ty3 integration TSD. (A) RNAP3 complex is recruited by the TFIIIB complex containing Brf1, Spt15, and Bdp1 that binds ∼20–40 bases upstream of the tDNA. RNAP3 subunit Rpc34 is positioned near the active site of the TSS and transcription bubble (Wu et al. 2012). (B) Suggested model of Ty3 integration into target DNA at or near the TSS. Ty3 integration complex is recruited to target sites via interactions with Brf1. Ty3 cDNA integration may require bending of the host DNA into the integrase complex active site to facilitate strand transfer with Ty3 cDNA ends. Model suggests that flexibility of this region contributes to integration, whereas stiffness corresponding to curvature does not.