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Long terminal repeat (LTR) retrotransposons are closely related to retroviruses and, as such, are important models for the study of viral integration and target site selection. The transposon Tf1 of Schizosaccharomyces pombe integrates with a strong preference for the promoters of polymerase II (Pol II)-transcribed genes. Previous work in vivo with plasmid-based targets revealed that the patterns of insertion were promoter specific and highly reproducible. To determine which features of promoters are recognized by Tf1, we studied integration in a promoter that has been characterized. The promoter of fbp1 has two upstream activating sequences, UAS1 and UAS2. We found that integration was targeted to two windows, one 180 nucleotides (nt) upstream and the other 30 to 40 nt downstream of UAS1. A series of deletions in the promoter showed that the integration activities of these two regions functioned autonomously. Integration assays of UAS2 and of a synthetic promoter demonstrated that strong promoter activity alone was not sufficient to direct integration. The factors that modulate the transcription activities of UAS1 and UAS2 include the activators Atf1p, Pcr1p, and Rst2p as well as the repressors Tup11p, Tup12p, and Pka1p. Strains lacking each of these proteins revealed that Atf1p alone mediated the sites of integration. These data indicate that Atf1p plays a direct and specific role in targeting integration in the promoter of fbp1.
To guarantee their proliferation, retrotransposons and retroviruses integrate into the chromosomes of their hosts. Long terminal repeat (LTR) transposons have propagated extensively throughout the evolution of eukaryotes, and this has allowed them to impact greatly the organization of the eukaryotic genome. The cDNA copies of these elements are integrated by the retroelement-encoded integrase (IN). The insertion sites exhibit target specificities that are often unique to different viruses and transposons. Human immunodeficiency virus type 1 (HIV-1) integrates with a bias in favor of genes that are actively transcribed by polymerase II (Pol II) (34, 37). There are both structural and biochemical data which show that HIV-1 IN interacts with the host factor LEDGF (4, 8-10, 27, 35), and there is evidence that this interaction directs where the viral DNA integrates (11, 14, 26, 35). In the case of LTR retrotransposons, the disruption of essential sequences is avoided by directing the integration of cDNA to “safe havens.” In the budding yeast Saccharomyces cerevisiae, LTR retrotransposons Ty1 and Ty3 integrate preferentially upstream of genes transcribed by RNA Pol III (5, 6, 12, 23), whereas Ty5 insertions occur within regions of heterochromatin (38). The LTR retrotransposon of Schizosaccharomyces pombe, Tf1, integrates with a strong preference for sequences upstream of RNA Pol II-transcribed genes (1, 3, 36). This specificity protects the coding capacity of S. pombe from being disrupted by Tf1 and, in turn, maintains the fitness of the host. Previous work with plasmid-based targets demonstrated that the majority of Tf1 insertions cluster in promoters in reproducible patterns that are specific for each gene (25).
To identify which promoters are targets of Tf1 integration, a genome-wide profile of 73,125 insertion sites was obtained (18). Because high-throughput pyrosequencing was used to sequence the insertions from multiple experiments, this work generated a reproducible profile of integration levels for each intergenic sequence in the genome of S. pombe. The overwhelming majority of the inserts occurred in just 31% of the promoters. These strong targets are enriched for promoters that respond to environmental stress.
The promoter of fbp1 is a site of Tf1 integration when placed in a target plasmid (25). This promoter has two cis-acting elements, upstream activating sequence 1 (UAS1) and upstream activating sequence 2 (UAS2), that are each capable of contributing to the activation of fbp1 transcription (30). UAS1 has a CRE (cyclic AMP response element)-like element which is the binding site for a heterodimer of the basic leucine zipper (b-Zip) proteins Atf1p and Pcr1p (30). UAS2 resembles a Saccharomyces cerevisiae stress response element (STRE) that is thought to be bound by the transcriptional activator Rst2p (19). The Tup1p-like corepressors Tup11p and Tup12p repress transcription of fbp1 by promoting the assembly of repressive chromatin in the fbp1 promoter (21, 22).
When fbp1 is present within a target plasmid, the integration of Tf1 occurs at two prominent sites, 30 and 40 bp downstream of UAS1 (25). This pattern of integration requires the Atf1p binding site at UAS1 and the Atf1p protein. Importantly, other promoters in target plasmids have patterns of integration that do not rely on Atf1p. To identify the key determinants responsible for targeting integration in the fbp1 promoter, we conducted an extensive study of the promoter sequences and the factors that promote transcription. Here we report that two discrete target windows close to UAS1 are the only sequences in the promoter required for the pattern of integration. Although Atf1p was necessary for directing integration to target window 1 (TW1), other known factors that mediate fbp1 transcription, i.e., Pcr1p, Rst2p, and Tup11p/Tup12p, did not contribute to integration. While UAS2 was not a target of integration, we found that it did promote efficient transcription of fbp1. In addition, we found that a synthetic promoter induced by LexA fused to an activator, VP16, was not a target of Tf1 integration. These data indicate that Atf1p plays a direct and specific role in targeting integration to TW1 of the fbp1 promoter.
S. pombe strains were transformed with various plasmids by use of lithium acetate (16). Selective medium contained Edinburgh minimal medium (EMM) (29) and 2 g of dropout mix, similar to that of Rose et al. (32), except that the adenine level was increased to a final concentration of 250 μg/ml. A liter of rich medium (YES) contained 5 g Difco yeast extract, 30 g glucose, and 2 g of dropout powder. Strains deleted for tup12 required S. pombe minimal glutamate (PMG) medium for growth. PMG was identical to EMM, but the nitrogen source was monosodium glutamate (3.74 g/liter) in place of ammonium chloride (13). Where indicated, 10 μM vitamin B1 was added to repress the nmt1 promoter. EMM/PMG-5-FOA plates contained 1 mg/ml 5-fluoroorotic acid (F5050; U.S. Biologicals, Swampscott, MA). YES-5-FOA-G418 contained 500 μg/ml (corrected for purity) of Geneticin (11811-031; Life Technologies, Rockville, MD) and 1 mg/ml 5-FOA.
Table Table11 shows the yeast strains used for this study, and Table Table22 lists the plasmids produced. The pcr1, sty1, rst2, tup11, tup12, and pka1 genes were deleted from either YHL1101 or YHL1102 by using the drug resistance marker nat1, encoding nourseothricin acetyltransferase (33). A fragment of DNA containing nat1 flanked by 80 nucleotides (nt) of homologous sequence was created by PCR, using pCR2.1-nat (pHL2621) as the template. Primers used for PCRs are listed in Table S1 in the supplemental material. To induce homologous recombination between the genes to be deleted and the nat1-containing fragment, 5 μg of the nat1 DNA was introduced into yeast strains by lithium acetate transformation (16). Nourseothricin (NAT)-resistant colonies were isolated by growing the transformants overnight on YES medium and then replica printing them onto YES plates containing 100 μg/ml of NAT (Werner BioAgents, Germany). DNA blots and PCR were performed to confirm the deletion of the desired genes. A double-knockout strain deleted for tup11 and tup12 was created by crossing haploid strains individually lacking tup11 and tup12.
To isolate strains with stable chromosomal integration of the LexA DNA-binding domain [LexA(DBD)]-VP16 and LexA(DBD) cassettes, YHL1024 was transformed with BsiWI-digested pHL2821 and pHL2822, respectively. Stable integrants were selected on EMM lacking histidine. Next, the resulting stable his+ integrants (YHL9806 and YHL9811) were individually mated with YHL1101, and after sporulation, spores with the ura− leu− his+ genotype were selected. The presence of the LexA(DBD)-VP16 and LexA(DBD) expression cassettes in YHL9807 (YHL1101 and YHL9806 mating spores) and YHL9813 (YHL1101 and YHL9811 mating spores) was verified by colony PCR and Southern hybridization.
To create a target plasmid with wild-type (WT) fbp1 (pHL2679), a SpeI and SbfI fragment was removed from pHL2410 (25), and a 2.9-kb fragment containing the promoter sequence and the open reading frame (ORF) of fbp1 was inserted. The deletions in the WT fbp1 plasmid were constructed by PCR cloning techniques. The plasmids and oligonucleotides used are indicated in Table Table22 and in Table S1 in the supplemental material, respectively. Plasmids deleted for target window 2 (TW2 del [pHL2796]) and for the fbp1 open reading frame (ORF del [pHL2798]) were constructed by fusion PCR with two overlapping oligonucleotides containing the desired deletion and two flanking oligonucleotides that amplified the final PCR product, with unique restriction sites located on either end. The PCR fragment for TW2 del was cloned into pHL2410 at unique PacI and BseYI sites, and that for ORF del was cloned at NgoMIV and BsrGI sites. To build a TW1 del (pHL2795) construct, a 625-bp fragment was amplified with primers HL1658 and HL1667 and ligated into pHL2679 digested with SpeI-NgoMIV. For construction of a UAS2 del plasmid, a 1,270-bp fragment was amplified with primers HL1658 and HL1660 and ligated into pHL2679 digested with SpeI-BglII. A TW1 miniconstruct was created by inserting a 258-bp PCR product containing TW1 into the Sbf1 and SpeI sites of pHL2410. Similarly, a TW2 miniplasmid was created by inserting a 70-bp fragment containing TW2 into the SbfI and SpeI sites of pHL2410.
To build the UAS2 (fbp1) miniplasmid pHL2799, the plasmid pHL2679 was digested and the resulting 6.9-kb blunt-ended fragment was circularized.
For construction of lacZ reporter plasmids, plasmid pCH150 (30) was digested with XhoI. SbfI linkers were added to the resulting fragment, which was then digested and ligated into pHL2679 and pHL2799 digested with NgoMIV-SbfI to build plasmids pHL2826 (fbp1-lacZ) and pHL2827 [UAS2 (fbp1)-lacZ], respectively.
Plasmid pHL2821 expressed the LexA(DBD)-VP16 expression cassette from the pREP41X version of nmt1 (15). The LexA(DBD)-VP16 cassette was amplified from pBSLexA::VP16_SV40 (24) and was ligated into pHL1767 digested with XhoI-XmaI. The nmt1-LexA(DBD)-VP16 cassette was then amplified from this intermediate plasmid and ligated into pHL1570 digested with EagI and SpeI.
Plasmid pHL2822 expressed the LexA DBD from the nmt1 promoter. The LexA(DBD) cassette was amplified from pHL2753 and ligated into pHL1767 digested with XhoI-XmaI. The nmt1-LexA(DBD) cassette was then amplified from this intermediate plasmid and ligated into pHL1570 digested with EagI and SpeI.
To build the LexA target plasmid pHL2820, eight tandem copies of the lexA operator placed upstream of the lacZ ORF were amplified as a 4.1-kb insert from pHL1426 and ligated into leu2D-marked pHL1425 digested with PstI and XmaI.
Target plasmids were introduced into S. pombe by transformation with lithium acetate followed by selection on EMM lacking leucine (EMM−Leu). These strains were then transformed with the Tf1 plasmid pHL2541 by selection on EMM−Leu−Ura. Strains were arranged on EMM−Leu−Ura+B1 plates in 1-cm2 patches. Following 48 h of incubation, these plates were replica printed to EMM−Leu−Ura−B1 to induce transposition. After 4 days of incubation, the strains were replica printed to EMM−Leu+FOA+B1 to select against the Tf1 donor plasmid. Following 2 days of incubation, the plates were again replica printed to EMM−Leu+FOA+B1. To select for transposition events, the strains were then replica printed to EMM−Leu+FOA+G418. After 2 days of growth, the patches were washed in 0.5 ml distilled H2O, resuspended in 0.5 ml citrate-phosphate-sorbitol buffer with Zymolyase 100T (50 mM Na2HPO4, 50 mM citric acid, 1.2 M sorbitol [pH 5.6], 2 mg/ml Zymolyase 100T [Seikagaku]), and incubated at 37°C for 1 h. After the incubation, the cells were resuspended in 100 μl TE (10 mM Tris-HCl, pH 7.9, 1 mM EDTA). Twelve microliters of 10% SDS was added, and the cells were incubated at 65°C for 5 min. Thirty-three microliters of 5 M potassium acetate was then added, and the samples were mixed well and placed on ice for 30 min. The cells were then centrifuged at 4°C for 10 min at 14,000 rpm in a microcentrifuge. The supernatants were ethanol precipitated, and the pellets were resuspended in 100 μl of 1× TE. These samples were phenol extracted and ethanol precipitated. This material was then electroporated into bacteria. All of the target plasmid constructs were assayed with pHL2541, a Tf1 plasmid that lacked an Ampr cassette. The insertions in the target plasmids were derived from two independent target plasmid transformants. Sixteen patches, each with an independent transformant from a donor plasmid, were assayed for each strain, and they gave similar patterns of integration for the specific strains.
Quantitative measurements of transposition frequencies were performed as previously described (7). In brief, strains were grown on EMM agar plates (with the dropout recipe described above) that lacked thiamine. These cells were then resuspended to an optical density at 600 nm (OD600) of 5.0 and used to make a series of 10-fold dilutions, starting with 108 cells/ml and finishing with 104 cells/ml. One-hundred-microliter aliquots of the cells from the three lowest dilutions were then spread onto 5-FOA plates, and 0.1-ml aliquots of cells from the three highest dilutions were spread onto YES plates containing 5-FOA and G418. The transposition frequency was calculated as the percentage of colonies resistant to 5-FOA (FOAr) that were also resistant to G418 (G418r) [(number of FOAr colonies − number of G418r colonies)/number of FOAr colonies].
A large swatch of cells was patched onto EMM+Leu−Ura−B1 plates and allowed to grow until patches were dense (48 h). A wad of cells was scooped up and resuspended in 1 ml distilled H2O. The OD600 was measured. One-tenth of a milliliter of cells was added to 0.9 ml Z-buffer (28). Cells were permeabilized by adding 0.1% SDS and chloroform and were vortexed. Two-tenths of a milliliter of 4-mg/ml o-nitrophenyl-β-d-galactopyranoside (ONPG) substrate was added, and the reaction was timed until the solution turned yellow. The reaction was stopped by adding 0.5 ml of 1 M sodium carbonate. The OD420 was measured, and activities are reported in modified Miller units [Miller units = (1,000 × A420)/(T × A600), where A420 is the optical density read from the reaction mixture, A600 is the initial cell density read, and T is the time of reaction (in minutes) before adding sodium carbonate] (28).
When fbp1 sequences are placed in a plasmid, integration of Tf1 occurs at two prominent sites, 30 and 40 bp downstream of UAS1 (25). To determine which sequences of fbp1 are required for this pattern, we generated a series of sequential deletions of 1.7 kb that contained UAS1, UAS2, and the ORF of fbp1 (Fig. (Fig.1A).1A). These target plasmids were then subjected to integration using the target assay. Tf1-neo was expressed from a second plasmid, and the resulting integration events generated cells resistant to G418. Target plasmids extracted from these cells were introduced into bacteria, and copies of the target plasmids with Tf1-neo inserts were selected on medium containing kanamycin. These plasmids were then sequenced to determine the positions of Tf1 integration. Insertions mediated by Tf1 IN produce signature duplications of 5 bp at the target sites. All insertions included in our analyses resulted in 5-bp duplications.
The integration assay of the intact fbp1 gene showed insertions clustered at coordinates 3090 and 3100, the same two positions, 30 and 40 bp downstream of UAS1, that were the prominent sites in our previous study (Fig. (Fig.1B).1B). Insertions were also detected at positions 3170 and 3206, which together with positions 3090 and 3100 constituted a region of insertion we designated TW1. A second cluster of insertions, termed TW2, was detected at positions 2816, 2840, and 2874 (Fig. (Fig.1B).1B). This cluster was detected previously, but it was not as prominent as in the current study (25). Differences in the backbone sequence and copy number of the plasmid used here may account for the large number of insertions in TW2. Nevertheless, Tf1 recognized two regions of the fbp1 promoter: TW1, which is adjacent to the Atf1p binding site in UAS1, and TW2, which is located upstream of UAS1 (Fig. (Fig.1A1A).
A deletion in the fbp1 promoter that removed UAS1 and the sequence up to but not including UAS2 was called TW1 del. Integration in this plasmid occurred primarily at positions 2840 and 2874, the two dominant insertion sites in TW2 (Fig. (Fig.1C).1C). A deletion that left UAS1 intact but removed TW2 and all upstream sequences was termed TW2 del. The insertions in this plasmid occurred predominantly at position 3100, the principal site of insertion of TW1 (Fig. (Fig.1D).1D). These results showed that TW1 and TW2 function as target regions independent of each other.
Deletion of UAS2 and the sequence down to but not including the ORF of fbp1 resulted in a plasmid called UAS2 del. This plasmid accumulated insertions in TW1 and TW2, in the same dominant positions as those detected in the intact plasmid (Fig. (Fig.1E).1E). A plasmid that lacked the ORF of fbp1 was called ORF del, and this deletion caused no significant changes in the pattern of integration (Fig. (Fig.1F).1F). Thus, UAS2 and the coding sequences of fbp1 made no observable contributions to the pattern of integration.
To determine which sequences were sufficient for TW1 and TW2 to function as specific windows of integration, we generated plasmids with the individual target windows and a minimal amount of flanking sequence. The plasmid TW1 Mini contained a 258-bp (positions 2885 to 3142; coordinates from the WT plasmid) sequence that encompassed TW1. The two dominant positions of insertion in this plasmid corresponded to the two major sites of insertion in TW1 of the intact plasmid, positions 3090 and 3100 (Fig. (Fig.2A).2A). The miniplasmid TW2 Mini contained TW2 within a sequence of just 70 bp (positions 2811 to 2880; coordinates from the WT plasmid). In this minimal plasmid, the dominant positions of integration corresponded to positions 2840 and 2874, the same two major sites of integration in TW2 as those in the context of the intact plasmid (Fig. (Fig.2B).2B). The results from the assays with the miniplasmids revealed that the sequences of the target windows and short lengths of flanking base pairs were sufficient to direct integration at the same nucleotide positions that were targeted when the intact promoter was present.
The previous study of integration in fbp1 in the context of a target plasmid revealed that integration in the promoter is highly dependent on Atf1p, a transcription factor that binds UAS1 (25). To test whether Atf1p contributed to the pattern of integration in the version of the fbp1 plasmid used in the current study, we conducted a target assay with a strain that lacked atf1 (Fig. (Fig.2C).2C). In the absence of Atf1p, we saw a significant reduction of integration in TW1. No insertions were detected in the principal positions of TW1, positions 3090 and 3100. However, insertions were detected at the two key positions of TW2, positions 2840 and 2874. To test whether integration at TW2 was mediated by Atf1p, a target assay was performed with TW2 Mini in the strain lacking Atf1p. The large number of insertions in TW2 showed clearly that the target activity of this window was independent of Atf1p (Fig. (Fig.2D).2D). Taken together, these data confirmed the previous finding that Atf1p mediates integration at TW1 but, interestingly, does not contribute to integration at TW2.
To stimulate the transcription of genes, Atf1p must be phosphorylated by the mitogen-activated protein kinase Sty1p (17). We therefore tested whether phosphorylation of Atf1p was required for the integration pattern in TW1 of fbp1. In an integration assay performed with a strain that lacked sty1, only 2 of 14 insertions were positioned in TW1. Of the remaining 12, 3 were in TW2 and 9 were in the vector. The lack of sty1 caused a 2.4-fold reduction in integration in TW1 (compared to the wild type) (Fig. (Fig.1A).1A). The reduction in integration in TW1 caused by the deletion of sty1 was comparable to the level observed when Atf1p was absent (3.0-fold) (Fig. (Fig.2C2C).
Atf1p may play a direct role in mediating integration in the promoter of fbp1, or it could be that by activating transcription, Atf1p induces subsequent steps of transcription that are responsible for integration. If the role of Atf1p in integration were indirect, other factors that promote fbp1 transcription would also influence integration at this promoter. Pcr1p forms a heterodimer with Atf1p, and together these proteins activate transcription of fbp1 by binding UAS1 (30). Interestingly, in a strain that lacked Pcr1p, the pattern of integration was unchanged, with the bulk of the integration occurring at the principal sites in TW1 (3090 and 3100) and TW2 (2840 and 2874) (Fig. (Fig.3A).3A). Rst2p is a transcription activator that promotes the transcription of fbp1 from UAS1 and UAS2 (19). The pattern of integration was also unchanged in a strain lacking Rst2p (Fig. (Fig.3B).3B). Tup11p and Tup12p are orthologs of the global repressor TUP1 of S. cerevisiae and are redundant repressors of transcription in S. pombe that inhibit histone remodeling in promoter sequences (21). These factors inhibit transcription of fbp1 by as much as 100-fold (22). Lack of Tup11/12 function might be expected to broaden the positions of integration in the promoter of fbp1. However, the strain lacking Tup11p and Tup12p showed no significant change in the pattern of integration (Fig. (Fig.3C).3C). Therefore, Tup11p and Tup12p did not contribute to the pattern of integration in the promoter of fbp1. In separate assays that quantified the overall levels of Tf1 integration genome-wide, we found that strains lacking Pcr1p, Atf1p, Rst2p, Tup11p, and Tup12p produced similar amounts of transposition to those of wild-type strains (Table (Table33).
While deletion of the pcr1, rst2, tup11, and tup12 genes did not change the pattern of integration in the promoter of fbp1, the deletions might have altered the efficiency of integration. To quantify the efficiency of integration into the target plasmids, the batches of plasmid DNA from strains of S. pombe that were introduced into bacteria for isolating insertions were also introduced into bacteria to count the fraction of plasmids that contained Tf1-neo. The efficiency of integration in each target plasmid was calculated as the number of plasmids with inserts (Kanr colonies) divided by the total number of plasmids (Ampr colonies). Table Table44 lists the efficiencies of integration for the intact fbp1 plasmid and shows that deletions in pcr1 and rst2 caused small changes in the efficiency of integration compared to that of the wild-type strain. Despite the modest levels of these changes, chi-square analyses indicated that the differences from wild-type efficiencies were statistically significant (P < 1 × 10−6). In order to test the efficiency of the strain lacking both tup11 and tup12, a medium containing glutamate as the nitrogen source had to be used because media containing ammonium chloride did not support growth of strains lacking Tup12p. With the glutamate medium, the integration efficiency of the strain lacking tup11 and tup12 was 3-fold higher than that of the wild-type strain (Table (Table4).4). Here, too, the difference in efficiency from the wild type was statistically significant (chi-square test; P < 1 × 10−6).
The finding that Atf1p was the only transcription factor required to position integration at TW1 of fbp1 suggests that Atf1p plays a direct role in targeting integration. However, it remained possible that any factor regulating fbp1 transcription could promote integration and that the reason the factors Pcr1p, Rst2p, Tup11p, and Tup12p did not contribute to integration at TW1 was because these factors did not promote fbp1 transcription in the context of a high-copy-number plasmid. To address this possibility, we replaced the ORF of fbp1 with the ORF of lacZ. This allowed us to measure the transcription levels of the fbp1 promoter in strains that lacked the transcription regulators (Fig. (Fig.4A).4A). The strains lacking the activators atf1, pcr1, and rst2 expressed significantly less lacZ activity than did wild-type cells (Fig. (Fig.4B).4B). In the cases of pcr1Δ and atf1Δ strains, the levels of lacZ activity were similar to that of a strain with a plasmid that lacked lacZ (Fig. (Fig.4B).4B). These data indicate that even in the context of the high-copy-number plasmid, these factors did promote transcription of the fbp1 promoter.
Another observation suggesting that Atf1p has a specific function capable of promoting integration at fbp1 was that insertions clustered at UAS1 and did not occur in the vicinity of UAS2 (Fig. (Fig.1B).1B). To test whether UAS2 induced fbp1 transcription from a high-copy-number plasmid, we removed UAS1 (nucleotides 2545 to 3600) from the plasmid that contained the fbp1 promoter fused to lacZ (Fig. (Fig.4A).4A). As expected from studies of fbp1 in its chromosomal context (30), UAS2 in the reporter plasmid UAS2(fbp1)-lacZ supported approximately 50% of the expression produced by the intact promoter (Fig. (Fig.4B).4B). This result is significant because it shows that although UAS2 was capable of inducing expression comparable to that produced by UAS1, UAS2 was not a target of integration (Fig. (Fig.1B).1B). One simple explanation for why insertions did not occur at UAS2 is that when both enhancers were present in the same plasmid, UAS1 may have outcompeted UAS2 as a target for the insertions. This possibility was tested by asking whether insertions would occur in UAS2 if UAS1 and all upstream sequences were absent. The target plasmid UAS2 Mini contained fbp1 with the same deletion of UAS1 and upstream sequences as that used in the lacZ-containing plasmid UAS2(fbp1)-lacZ. When UAS2 Mini was used as the target plasmid in an integration experiment, no insertions occurred in the promoter (Fig. (Fig.4D).4D). Thus, the same UAS2 promoter sequence that exhibited high levels of expression did not function as a target of integration.
In another effort to test whether UAS2 had the potential to mediate integration, we conducted integration assays with a strain that lacked the cyclic AMP-dependent protein kinase catalytic subunit (Pka1p). Without Pka1p, transcription mediated by UAS1 and UAS2 is fully derepressed (30). Because this disruption of the protein kinase A pathway increased fbp1 expression to the extent that the plasmid was not tolerated, we had to first delete the fbp1 ORF from the target plasmid (Fig. (Fig.4C).4C). Even in the absence of Pka1p, UAS2 did not function as a target when the intact promoter was present (Fig. (Fig.4C4C).
In an independent approach to test whether active transcription is sufficient to promote Tf1 integration, we created a synthetic promoter and tested it for integration activity. We generated a synthetic transcription activator that consisted of the DNA binding domain of LexA fused to the activation domain of VP16. The LexA-VP16 fusion was expressed by the nmt1 promoter from a single-copy locus (Fig. (Fig.5A).5A). The target plasmid contained a synthetic promoter that consisted of eight tandem copies of the lexA operator placed upstream of the lacZ ORF (Fig. (Fig.5B).5B). LacZ assays of this strain showed that the synthetic activator and promoter supported substantial expression that was dependent on the VP16 activation domain (Fig. (Fig.5C).5C). When cells were grown under identical conditions, the amount of lacZ expression induced by LexA-VP16 was comparable to expression levels of the intact promoter of fbp1 (data not shown). Despite its high levels of expression, the results of an integration assay revealed that the synthetic promoter was not a target of integration (Fig. (Fig.5D5D).
To understand what determines the position of Tf1 integration, we examined the pattern of insertion in the promoter of fbp1. The insertion sites clustered into two distinct windows. The context of fbp1 within a target plasmid and the analysis of separate patches of cells allowed us to determine that positions 2840 and 2874 of TW2 and positions 3090 and 3100 of TW1 had many independent integration events. Our systematic deletion of the fbp1 sequences revealed that the target windows functioned independent of each other. More importantly, each target window was sufficient for establishing a specific set of coordinates as sites of repeated integration. These data, together with the observation that integration into TW1 and not TW2 depended on Atf1p, indicate that integration into the two target windows relies on different factors.
The integration assays we conducted were based on plasmid-carried copies of the fbp1 sequence. The positions of nucleosomes on ura4 and ade6 are the same regardless of whether the genes are carried on plasmids (2). However, it is possible that the chromatin bound to the fbp1 promoter in the plasmid may be assembled differently from what exists in the chromosomal copy of fbp1. As a result, the integration patterns observed within plasmids might not accurately represent the patterns that occur in the chromosomal copies of genes. However, the data from the genome-wide study of Tf1 integration showed insertions in the promoters of fbp1 and ade6 that corresponded closely to the patterns observed for the plasmid versions of these genes (18, 25). In four independent sets of integration data from the genome-wide study, insertions in fbp1 clustered in TW1 and TW2. In TW1, three inserts occurred at position 3090 and two occurred at position 3100. In TW2, one insert was detected at position 2840 and four occurred at position 2874. This close correlation between the insertions observed in the chromosomal copies of fbp1 and ade6 and the patterns detected in plasmid copies indicates that the target plasmid assay can faithfully reproduce authentic integration patterns. There are a few sites in the backbone of the plasmid that are frequent sites for integration. These include positions 340 and 384 in the bacterial gene bla. Since these positions are in a bacterial sequence, they do not represent natural insertion events. However, position 340 is targeted regardless of which other sequences are present in the plasmid or which host genes are deleted. It is possible that this site was fortuitously recognized by a factor that directs integration or that the DNA sequence itself has a structure that is directly recognized by IN.
Integration assays with S. pombe lacking atf1 confirmed the previous finding that Atf1p is required for the pattern of integration associated with UAS1 (25). In addition, our use of TW1 and TW2 miniplasmids revealed that Atf1p was specifically required for the integration at TW1 and did not contribute to the inserts in TW2. These data and the previous finding that the pattern of integration in the fbp1 promoter depends on the enhancer sequence of UAS1 suggest two types of models. Atf1p bound at its recognition sequence in UAS1 may simultaneously bind directly to integrase and direct integration to occur at the primary sites 30 nt and 40 nt downstream of UAS1. This possibility is supported by coimmunoprecipitation assays showing that IN is in a complex with Atf1p (25). Alternatively, integrase may be directed to UAS1 by any number of other transcription factors that rely on Atf1p simply because it initiates transcription. The result that other factors that modulate fbp1 transcription, such as Pcr1p, Rst2p, Tup11p/Tup12p, and Pka1p, did not contribute to the pattern of integration indicates that Atf1p plays a direct role in targeting integration. It is particularly telling that Pcr1p and Atf1p must bind UAS1 together as a heterodimer to stimulate transcription. The lacZ assays showed that deletion of pcr1 reduced expression of fbp1 to the level exhibited by the strain lacking atf1. Nevertheless, deletion of pcr1 did not alter the pattern of integration. This indicates that Atf1p plays a direct role in mediating integration in TW1. Interestingly, cells lacking tup11 and tup12 had three times more integration in the plasmid than did wild-type cells. It is known from micrococcal nuclease digestion that cells lacking tup11 and tup12 have reduced chromatin structure in the promoter of fbp1 (20, 21). This lack of structure may account for the corresponding increase in integration efficiency.
Factors other than Atf1p can direct integration. Cells lacking Atf1p accumulate insertions genome-wide, with frequencies similar to those of wild-type cells. The finding that integration in TW2 was independent of Atf1p also showed that factors other than Atf1p can direct integration.
Since it appears that factors other than Atf1p can direct integration, we asked whether any transcription activator bound to its enhancer could promote integration. The result showing that integration at fbp1 is directed to UAS1 and not to UAS2 indicates that binding of a transcription activator to its cognate UAS does not necessarily promote integration. The fact that UAS2 is an efficient enhancer of transcription in the context of our plasmid supports the conclusion that activated transcription is not sufficient to mediate integration. Additional support for this conclusion was that the transcription activator Rst2p promoted efficient transcription of fbp1 in the plasmid but did not contribute to integration. These results indicate that only specific transcription factors, such as Atf1p, are capable of mediating integration.
Further support for the model that only specific transcription factors are able to direct integration came from the study of an artificial promoter. By fusing the DNA binding domain of LexA to the activator domain of VP16, we were able to drive efficient transcription of a lacZ reporter that had eight upstream copies of the lexA binding site. Nevertheless, this artificial promoter was not a target of Tf1 integration, showing that the binding of a transcription activator and the initiation of transcription were not by themselves capable of mediating integration. This result also argues against the possibility that integration is mediated by a general component of the transcription machinery. The most likely explanation for how integration is targeted is that there is a specific set of transcription factors that include Atf1p and that these proteins have the specific abilities to recruit integrase to positions in promoters and to promote integration. Our future studies will focus on identifying this key set of transcription factors.
We thank Young-Eun Leem for generating the stain with the deletion of sty1.
This research was supported by the Intramural Research Program of the NIH from the Eunice Kennedy Shriver National Institute of Child Health and Human Development.
Published ahead of print on 27 October 2010.
†Supplemental material for this article may be found at http://jvi.asm.org/.