Retrotransposon Tf1 is targeted to pol II promoters by transcription activators (Leem, Ripmaster et al. 2008).

Many transposable elements protect the coding capacity of their host by directing integration to nonessential regions of the genome. The preference of Tf1 for integrating upstream of genes is a mechanism that protects the coding sequences of S. pombe. This preference may be due to interactions of IN with chromatin factors. This possibility is supported by the presence in the IN of a CHD that may bind histones with specific modifications (Malik and Eickbush 1999). Alternatively, the integration of Tf1 upstream of ORFs could be due to interaction with chromatin remodeling complexes or transcription factors. To identify what determinants specify the positions of integration we developed a plasmid-based targeting assay in cells induced for Tf1 transposition.

1. A plasmid based targeting assay for Tf1 transposition identified a 160 bp window of integration upstream of ade6.

To identify what factors determine the insertion sites of Tf1 integration we developed a plasmid based targeting assay that measured the integration activity of specific sequences. A strain of S. pombe was generated that contained a plasmid for the expression of Tf1 and a plasmid for target sequences. Tf1 was marked with neo so that integration would result in resistance to G418. After transcription of Tf1-neo was induced, the expression plasmid was removed by counter selection and cells with insertion events were selected on medium containing G418. Target plasmids extracted from individual patches were introduced into bacteria by selecting for resistance to ampicillin. Due to the presence of neo in Tf1, target plasmids with insertions resulted in colonies that were resistant to both ampicillin and kanamycin. These plasmids were readily identified and the position of their insertions sequenced.

To test the integration preferences of Tf1 a target plasmid was created that contained a potential target consisting of the ORF of ade6, its upstream intergenic region, and a portion of the adjacent ORF, bub1 (Fig. 1). The ade6 and bub1 genes were in divergent orientation so the 405 bp integenic sequence contained two promoters. This particular segment of the pombe genome contains chromatin structure that is maintained when placed on a plasmid. The only other portion of the target plasmid that contained a promoter was LEU2d, a selectable gene from S. cerevisiae that has a damaged promoter. The target plasmid was introduced into a strain of S. pombe and individual patches of cells were induced for the expression of Tf1-neo. No selection was placed on the function of ade6. The patches were then replica printed to medium containing G418 to select for cells with integration events. Target plasmids were isolated by preparing DNA from each G418R patch and electroporating the DNA into bacteria. Colonies resistant to ampicillin and kanamycin contained target plasmids with insertions of Tf1-neo. Of 43 independent insertions of Tf1 isolated in the plasmid, 41 (95%) occurred in the intergenic region between bub1 and ade6 (Fig. 1). These results demonstrated that the preference of Tf1 for integration upstream of ORFs was reconstituted within the context of a plasmid. All 41 insertions in the intergenic region occurred within a 160 bp window. Interestingly, the insertions clustered at specific nucleotides where no preferences for orientation were observed.

To identify which sections of bub1-ade6 were important for targeted integration, we made a series of deletions and tested their impact on the frequency of integration (Fig. 2). These data revealed that the 160 bp target window was the only portion of bub1-ade6 that was important for efficient integration. To test whether the insertion window was sufficient to be an integration target, a plasmid was created that contained just the 160 bp window. These experiments showed that just 173 bp of the promoter sequence functioned as a highly specific target for integration.

2. The preferred sites of integration in the intergenic region of bub1-ade6 correspond to specific chromatin structures.

The targeting of Tf1 to the region of the ade6 and bub1 promoters suggests that the transcription of these two genes may play a critical role in integration. This possibility was tested by comparing the amounts of mRNA produced by the deletion plasmids to their efficiency of targeted integration. Two deletions in the promoter sequences exhibited sharp reductions in the transcription from both promoters and yet both plasmids supported efficient targeting. This indicates that the transcription activity of the promoters per se did not play an important role in targeted integration.

The result that active transcription was not important for efficient integration into the promoters of bub1-ade6 suggests that features such as DNA binding proteins may be recognized by Tf1. To determine whether nucleosomes or DNA-bound transcription factors were positioned at integration sites we mapped the chromatin at the promoters of bub1-ade6 with micrococcal nuclease. These experiments revealed four supersensitive sites clustered together within the intergenic sequence. The intensity of these bands and their close spacing within the promoter region suggests they were created by transcription factors bound to regulatory sequences. Of the five major sites of insertion, four corresponded closely with positions of micrococcal sensitivity. This close association suggests the promoter binding proteins responsible for the micrococcal sites played a role in recruiting IN to the sites of integration. This is consistent with the finding that 160 bp of the bub1-ade6 promoters was the only sequence required for targeted integration. Unfortunately, the transcription factors that bind the promoters of bub1-ade6 are unknown. As a result, we chose to study the integration activity of a promoter for which the transcription factors are known.

3. The promoter of fbp1 was a target of Tf1 IN.

To test whether Tf1 integration was directed by transcription factors, we studied the insertion activity of a promoter that has been characterized extensively. The promoter of fbp1 is highly regulated and contains an upstream activating sequence (UAS1) that consists of an eight bp binding site for the transcription activator Atf1p (Neely and Hoffman 2000). In addition, UAS2 consists of a short binding sequence for the transcription activator Rst2p as well as other factors (Neely and Hoffman 2000; Higuchi, Watanabe et al. 2002).

We tested whether the promoter of fbp1 was a target for Tf1 integration using the target plasmid assay. Eighty-six percent of the insertions in the plasmid occurred within the promoter region of fbp1. Thirteen ( 62%) of the 21 insertions isolated in the plasmid occurred at the two dominant positions, 4923 and 4933 (Fig. 3A). These positions were just 30 and 40 bp downstream of UAS1. The lack of any insertions in the open reading frame and the clustering of inserts upstream of the open reading frame indicated that the insertions were specific for the promoter of fbp1. The role of transcription factors in integration was further supported by the concentration of integrations that occurred adjacent to the binding site of Atf1p at UAS1.

4. Targeted integration at UAS1 of fbp1 required a functional binding site for Atf1p.

The integration activity of fbp1 and the published information about UAS1 allowed us to ask whether the clustering of inserts in the promoter region was the result of UAS1 function. We changed the sequence of all eight nucleotides in UAS1 of the fbp1 plasmid and used the target assay to ask whether the pattern of integration was changed. In the plasmid with the mutated UAS1, just three of 42 insertions (7%) occurred at positions 4923 and 4933 (Fig. 3B). Thus, the mutated UAS1 resulted in a 9-fold reduction in integration at the two dominant positions of insertion. The defect in the ability of UAS1 to be recognized by Tf1 allowed the secondary targets 5183 and 5149 to become major sites of integration. This result demonstrated that a functional binding site for the transcription factor Aft1p plays an important role in the targeting of Tf1 integration to the two major insertion sites in the fbp1 promoter.

The integration of Tf1 in the promoter of fbp1 may result from a tethering of IN to UAS1 by Aft1p. An interaction of Atf1p with IN could be direct or through proteins that bind Aft1p, such as the coactivator Pcr1p. Immunoprecipitation of Atf1p from cell extracts revealed that IN was in a complex with Atf1p. Further experiments tested whether Atf1p itself contributed to integration in the promoter of fbp1. Results from plasmid target experiments revealed that in the strain lacking Atf1p, insertions were no longer directed to the fbp1 promoter. Together, these results indicate that Atf1p mediates integration of Tf1 in fbp1 by tethering IN to UAS1. Whether integration at other promoters is due to Aft1p or other transcription factors remains to be tested.

5. Integration into ade6 disrupted the promoter but stimulated transcription.

The relationship between transposable elements and their hosts allows for integration of the transposon that does not impair the fitness of the host. The insertion of Tf1-neo introduces approximately 6 kb of DNA into the promoter region of genes. This form of DNA damage might be expected to disrupt promoter function. For example, one deletion we made in the promoter of ade6 revealed key promoter elements were upstream of all the major sites of integration. We tested whether the integration of Tf1-neo at the primary site of insertion, nucleotide 4573, disrupted the function of the ade6 promoter. Target plasmids with Tf1-neo insertions were introduced into S. pombe and the levels of ade6 mRNA were measured on RNA blots. The ade6 mRNA was quantified relative to the amounts of actin mRNA. The copy number of the plasmids in each transformant was determined on DNA blots and these values were used to adjust the levels of ade6 mRNA. Surprisingly, insertion of Tf1-neo actually stimulated ade6 transcription. Insertion in the left orientation at nucleotide 4573 increased ade6 mRNA by 2.1-fold while insertion in the right orientation increased ade6 expression by 3.6-fold. Even though the insertions were downstream of essential promoter elements, ade6 expression was not reduced. The absence of defects in the expression of ade6 together with the increases observed suggests the intriguing possibility that Tf1 carries promoter elements that are capable of repairing the promoters disrupted by the transposon.

We tested directly whether Tf1 was capable of activating a damaged version of the ade6 promoter that lacked key promoter elements. A deletion that removed the sequences between the target site 4573 and the ATG of bub1 reduced levels of ade6 mRNA 4.3-fold. An identical construct except that it contained Tf1-neo inserted at nucleotide 4573 produced 7.2 and 2.9 times more ade6 mRNA depending on the orientation of Tf1-neo. These data demonstrate that Tf1-neo introduced promoter elements that substituted for the key transcription components located upstream of nucleotide 4573.
The insertion of Tf1 sequences could cause the target promoters to adapt the regulatory properties of Tf1 transcription. If Tf1 expression is similar to a closely related element Tf2, conditions of oxidative stress would induce a wave of transposition that in turn could cause cellular genes to become regulated by stress. This process could provide a selective advantage for the host by inducing genes that could increase survival. This would be an evolutionary strategy for creating populations of cells expressing different genes in response to stress that would improve the chances of survival for the species.