High-throughput sequencing of retrotransposon integration provides a saturated profile of target activity in Schizosaccharomyces pombe

Abstract

The biological impact of transposons on the physiology of the host depends greatly on the frequency and position of integration. Previous studies of Tf1, a long terminal repeat retrotransposon in Schizosaccharomyces pombe, showed that integration occurs at the promoters of RNA polymerase II (Pol II) transcribed genes. To determine whether specific promoters are preferred targets of integration, we sequenced large numbers of insertions using high-throughput pyrosequencing. In four independent experiments we identified a total of 73,125 independent integration events. These data provided strong support for the conclusion that Pol II promoters are the targets of Tf1 integration. The size and number of the integration experiments resulted in reproducible measures of integration for each intergenic region and ORF in the S. pombe genome. The reproducibility of the integration activity from experiment to experiment demonstrates that we have saturated the full set of insertion sites that are actively targeted by Tf1. We found Tf1 integration was highly biased in favor of a specific set of Pol II promoters. The overwhelming majority (76%) of the insertions were distributed in intergenic sequences that contained 31% of the promoters of S. pombe. Interestingly, there was no correlation between the amount of integration at these promoters and their level of transcription. Instead, we found Tf1 had a strong preference for promoters that are induced by conditions of stress. This targeting of stress response genes coupled with the ability of Tf1 to regulate the expression of adjacent genes suggests Tf1 may improve the survival of S. pombe when cells are exposed to environmental stress.

Figure 1.

The distribution of Tf1 integration in the genome of S. pombe. (A) The numbers of independent insertion events from the Hap_Mse_2 experiment are shown within 1 kb intervals of the three chromosomes of S. pombe. The positions of centromeres are indicated by blue triangles. The total number of independent integration events in each chromosome are labeled. (B) The distribution of Tf1 integrations within10-kb intervals of the S. pombe genome is shown for the Hap_Mse_2 experiment. Also shown are the distribution of the random control (MRC of Hap_Mse_2, magenta) and the Poisson distribution (green) based on the mean of the integration data.

Figure 2.

The distance from Tf1 integration sites to the nearest ORF. The x coordinate is the distance from the 5′ and 3′ ends of ORFs. The y coordinate shows the number of integration events within bins of 100 bp. Insertions closer to the 5′ end of an ORF were plotted upstream of the ORF (green arrow), while insertions closer to the 3′ end of an ORF were plotted downstream of ORF. Insertions within ORFs were tabulated within 15 bins of equal proportion. The percentage of the independent integrations in ORF was labeled. (A) Hap_Mse_2; (B) MRC for Hap_Mse_2; (C) Dip_Mse.

Figure 3.

Venn diagrams showing the number of intergenic regions that had integration events in both the Hap_Mse_1 and Hap_Mse_2 experiments. Under the name of each experiment is listed the total number of intergenic regions that had at least one insertion. (A) Comparison of Hap_Mse_1 and Hap_Mse_2; (B) Comparison of Hap_Mse_2, Dip_Mse and Dip_Hpy.

Figure 4.

Comparison between two experiments (Hap_Mse_2 and Dip_Mse) of the number of insertions within each intergenic region. Each unit of the surface represents a group of intergenic regions. The x coordinate shows the number of integrations/intergenic region in the Hap_Mse_2 data. The y coordinate shows the number of integrations/intergenic region in the Dip_Mse data. The z coordinate has a log scale and shows the number of intergenic regions with the same x and y coordinates. The colors of the surface and the associated key represent values of the z coordinate.

Figure 5.

The ranking of intergenic regions based on their number of insertions detected in the Hap_Mse_2 experiment. (A) All intergenic regions of S. pombe were plotted on the x-axis in order of their number of insertions (blue). (Magenta) A distribution based on the random control data of MRC Hap_Mse_2. (B) The divergent intergenic regions were plotted on the x-axis in order of their number of insertions (blue). (Magenta) The corresponding random control MRC Hap_Mse_2. (C) The tandem intergenic regions were plotted on the x-axis in order of their number of insertions (blue). (Magenta) The corresponding distribution of random control MRC Hap_Mse_2. (D) The convergent intergenic regions were plotted on the x-axis in order of their number of insertions (blue). (Magenta) The random control MRC Hap_Mse_2.

Figure 6.

All ORFs of S. pombe ranked by the number of insertions in the Hap_Mse_2 experiment. The ORFs were plotted on the x-axis in order of their number of insertions (blue). (Magenta) The distribution of the corresponding random control MRC Hap_Mse_2 increased in number to have the same number of insertions in ORFs as in the Hap_Mse_2 data.

Figure 7.

Promoters of stress-induced genes are preferred targets of integration. The intergenic regions were ranked by numbers of insertion and then placed into bins of 500. (A–E) The numbers of promoters induced by various environmental stresses in each bin were tabulated. The total number of promoters induced by each stress (N) is listed. (F) The number of promoters induced when cells are starved for nitrogen. (G) As a control, the bins were tabulated for the numbers of promoters they contained from a set of 500 promoters that were selected at random.