Retrotransposon profiling of RNA polymerase III initiation sites

Abstract

Although retroviruses are relatively promiscuous in choice of integration sites, retrotransposons can display marked integration specificity. In yeast and slime mold, some retrotransposons are associated with tRNA genes (tDNAs). In the Saccharomyces cerevisiae genome, the long terminal repeat retrotransposon Ty3 is found at RNA polymerase III (Pol III) transcription start sites of tDNAs. Ty1, 2, and 4 elements also cluster in the upstream regions of these genes. To determine the extent to which other Pol III-transcribed genes serve as genomic targets for Ty3, a set of 10,000 Ty3 genomic retrotranspositions were mapped using high-throughput DNA sequencing. Integrations occurred at all known tDNAs, two tDNA relics (iYGR033c and ZOD1), and six non-tDNA, Pol III-transcribed types of genes (RDN5, SNR6, SNR52, RPR1, RNA170, and SCR1). Previous work in vitro demonstrated that the Pol III transcription factor (TF) IIIB is important for Ty3 targeting. However, seven loci that bind the TFIIIB loader, TFIIIC, were not targeted, underscoring the unexplained absence of TFIIIB at those sites. Ty3 integrations also occurred in two open reading frames not previously associated with Pol III transcription, suggesting the existence of a small number of additional sites in the yeast genome that interact with Pol III transcription complexes.

Figure 1.

High-throughput sequencing of de novo Ty3 integration sites. Step 1: Expression of Ty3-ppt-HIS3 containing two unique 60-bp tags for amplification of integration joints. Step 2: Alternative reverse transcription products, dependent on position of ppt. Step 3: Restriction of genomic DNA with one of three restriction enzymes (Csp6I, HpyCH4V, and HaeIII) and circularization. DNA fragments flanking de novo Ty3 integration sites were selectively amplified by inverse PCR (iPCR) using primers that annealed to the Ty3 U5-end LTR and Ty3 tag-1. Primers contained Illumina adaptor sequences and the Ty3 LTR primer contained the Illumina sequencing primer sequence. Step 4: The iPCR products from separate reactions were combined and processed for sequencing using the Illumina GAIIx system. Ty3 transcription begins at the 5′ edge of R and continues through U5 into the internal domain and the 3′ U3 and R regions. Reverse transcription regenerates the complete U3-R-U5 LTR at each end of the cDNA. (Vertical dashed lines) Digestion/ligation sites; (red arrow) HIS3 ORF; (bent arrows) iPCR primers. Arrows indicate primers: (light blue) Ty3 U5-end LTR; (pink) tag-1; (gray) upstream Illumina adaptor with sequencing primer; (black) downstream Illumina adaptor.

Figure 2.

Alignment of sequencing reads to yeast genomic sequence. (A) Sequence of Ty3 integration joint. High-throughput sequencing reads began with 17 nt from the U5 end of the Ty3 LTR and continued into genomic sequence. Informative reads aligned uniquely with ∼19 nt of target site DNA in the reference genome. Each unique junction defined an integration event. A sequencing read representing a collection of events at tK(CUU)F is shown. (B) Analysis of target loci. Reads captured by events separated by 10 nt or less were combined into clusters (shaded box). The distribution of reads (hits) among events clustered at tK(CUU)F is shown. (Dashed lines) Ty3 LTR sequences. Depending on the orientation in which Ty3 integrated, sequencing reads aligned with either the Watson or Crick strand of the genomic sequence of yeast reference strain S288C. (Right column) The numbers of reads for the same integration joint (event densities). The most-sequenced junctions in the two strands were offset by the 5-bp stagger characteristic of Ty3 insertion sites (triangles).

Figure 3.

Targets with preexisting Ty3 LTRs were used at a slightly greater frequency in the presence of RAD52. Cluster densities determined in RAD52 (x-axis) and rad52Δ (y-axis) strains are shown as the fraction of total cluster densities per experiment in log scale (inset in linear scale). Clusters at targets with preexisting Ty3 LTRs (sigma; solid squares, lower line and description) and clusters at unoccupied sites (not sigma; dots, upper line and description) were plotted.

Figure 4.

Ty3 integration targets known Pol III–transcribed genes. Ty3 integration sites (filled triangles); Pol III promoter elements, TBP binding site (TATA), box A (unfilled), box B (dark gray), and box C (light gray). TSS are shown only for genes that are transcribed as precursors (bent arrow). TSS is not available for iYGR033C.

Figure 5.

Quantitative Ty3 transposition at tDNAs with high and low DNA sequencing reads. (A) Distribution of sequencing reads from five top decile tDNA clusters and five bottom decile tDNA clusters. Yeast strains YMA1322 and YMA1356 transformed with Ty3-HIS3 expression plasmid pKN3050 were induced for Ty3 expression for 24 h. Insertions into tDNA target clusters were ranked by number of reads, and example top and bottom decile genes (shown) were selected. ETC1 was not detected by sequencing and was used as a negative test case. Bars show reads in which Ty3 and target are transcribed in the same (open) and divergent (gray) orientations and the total (black) is shown. (B) YMA1322 transformed with Ty3-ppt expression plasmid pKN3097 was induced for Ty3-ppt expression as described in A, and Ty3 integrations at reporter target loci were measured using qPCR (Methods). Measurements were within the linear range as standardized using plasmid versions of the Ty3 insertions at each locus. Bars represent technical triplicates of biological duplicates. (C) TFIIIB occupancy of reporter Ty3 target loci. Yeast strain YKN1692 expressing TFIIIB subunit Brf1 tagged with 3HA and transformed with empty URA3 vector plasmid YCplac33 was grown under inducing conditions for 24 h, and Brf1 occupancy at reporter loci was measured using ChIP. ChIP values were determined relative to an ACT1 control. The fold of enrichment for tDNA reporter tV(AAC)G3 to ACT1 was 73:1. The data are expressed relative to the fold enrichment at tV(AAC)G3 set at 100%. (D) Ty3 integration frequencies at tDNA reporter loci and flanking sequence. YMA1322 transformed with Ty3-ppt plasmid pKN3097 and vector pXP622 containing reporter target loci and flanking sequence (120 bp upstream and downstream of target gene) was induced to express Ty3 for 36 h, and integrations were quantified by qPCR. Data are expressed as described in B. (E) Based on target activity (D), promoter elements in strong target loci [tK(CUU)F, tA(UGC)E, tT(UGU)G1, tG(CCC)D, and tG(GCC)F2] and weak target loci [tG(GCC)G2, tV(AAC)G1, tD(GUC)K, tQ(UUG)D1, and tV(AAC)G3] were analyzed for consensus using logo sequence analysis software (http://weblogo.berkeley.edu).

Figure 6.

Ty3 targeting of novel loci requires Pol III promoter element. (A) Box A–box B pairs at iSCS3 and iYIL100W target loci. (Top row) Approximate 20-nt spacing between integration sites (i) and box A and the consensus box A and box B sequence of fungal tDNAs, with an average spacing of 29 bp (Marck et al. 2006). Lowercase indicates divergence from consensus. (Right column, box B mut.) Box B mutations described in B and C. (B) Target activity of novel Ty3 target iYIL100W. YMA1322 transformed with pKN3050 and plasmids containing 500-bp iYIL100W-flanking sequence and a box B mutant derivative was induced to express Ty3 for 36 h. Integration was monitored by PCR (+/−). (Triangles) Insertion sites; (double line) chromosomal DNA; (TATA) upstream TATA-box consensus; (m) mutated box B (mutation sequence listed in A). (C) Target activity of iSCS3. As described in B, 500-bp iSCS3-flanking sequence and variants were tested for targeting. (Filled triangles) Insertion sites determined from sequence analysis; (empty triangles) those recovered from target plasmids; (lm and rm) mutated box B in loci shown; (lh and rh) half truncation as shown.