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Corresponding author: Scott E. Devine, Emory University School of Medicine, 4133 Rollins Research Center, 1510 Clifton Rd. NE, Atlanta, GA 30322., sedevin{at}emory.edu (E-mail)
Communicating editor: S. SANDMEYER
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Retroviruses and their relatives, the long terminal repeat (LTR) retrotransposons, carry out complex life cycles within the cells of their hosts. We have exploited a collection of gene deletion mutants developed by the Saccharomyces Genome Deletion Project to perform a functional genomics screen for host factors that influence the retrovirus-like Ty1 element in yeast. A total of 101 genes that presumably influence many different aspects of the Ty1 retrotransposition cycle were identified from our analysis of 4483 homozygous diploid deletion strains. Of the 101 identified mutants, 46 had significantly altered levels of Ty1 cDNA, whereas the remaining 55 mutants had normal levels of Ty1 cDNA. Thus, approximately half of the mutants apparently affected the early stages of retrotransposition leading up to the assembly of virus-like particles and cDNA replication, whereas the remaining half affected steps that occur after cDNA replication. Although most of the mutants retained the ability to target Ty1 integration to tRNA genes, 2 mutants had reduced levels of tRNA gene targeting. Over 25% of the gene products identified in this study were conserved in other organisms, suggesting that this collection of host factors can serve as a starting point for identifying host factors that influence LTR retroelements and retroviruses in other organisms. Overall, our data indicate that Ty1 requires a large number of cellular host factors to complete its retrotransposition cycle efficiently.
A relative of vertebrate retroviruses, Ty1 is a long terminal repeat (LTR) retrotransposon in the yeast Saccharomyces cerevisiae (
The cDNA replication step of the retrotransposition cycle is thought to occur mainly within virus-like particles that are assembled in the cytoplasm (
After the Ty1 cDNAs are fully replicated, they are imported into the nucleus where they are integrated into chromosomal DNA. This final integration step of the retrotransposition cycle occurs with a high degree of specificity in the yeast genome, and new retrotransposition events are targeted preferentially to tRNA genes and other genes that are transcribed by RNA polymerase III (RNA pol III;
Since the Ty1 retrotransposition cycle is complex and spans several intracellular compartments, it could be envisioned to involve a wide range of cellular host factors. Our goal was to take advantage of the excellent genomic resources that have become available in S. cerevisiae to systematically identify such host factors. To achieve this goal, we performed a functional genomics screen for host genes that influence Ty1 using the recently completed collection of gene deletion strains generated by the Saccharomyces Genome Deletion Project (
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plasmids and strains:
The Ty1-HIS3 donor plasmid pAR100 was constructed by first cloning a BamHI/NotI fragment carrying a Gal-Ty1-neo insert (
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Haploid deletion strains were constructed from the yeast strain BY4741 (MATa ura3
0 leu20 his31 met150) using a LEU2 cassette generated by PCR as described (
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Ty1 retrotransposition assay:
The complete set of homozygous gene deletion strains (release 2) was obtained from Research Genetics (Huntsville, AL). A complete list of the genes in the collection can be viewed at the Research Genetics website (ftp://ftp.resgen.com/pub/deletions/Homo_diploids_041902.txt). All strains in plates 301–349 were transformed individually with the pAR100 test plasmid in batches of 96 following the order established by the Saccharomyces Genome Deletion Project using a lithium acetate method adapted to 96-well culture boxes (
GAL1-lacZ, HIS3, and YPG secondary screens:
Mutants that were positive in the initial screen were retested in three separate secondary assays to eliminate mutants that affected our assay rather than Ty1 retrotransposition itself. First, all mutants were tested in a GAL1-lacZ reporter assay to identify host genes that influenced the GAL1 promoter used to induce retrotransposition from the Ty1 test plasmid. Positive and negative controls were used in all of the GAL1:lacZ assays to define the range of the assay. In each case, three controls were used: (i) the wild-type BY4743 strain containing the GAL1:lacZ plasmid (which served as a strong positive control), (ii) an isogenic gal4 strain containing the GAL1:lacZ plasmid (which served as a weak positive control), and (iii) the BY4743 strain lacking the GAL1:lacZ plasmid (which served as a negative control). Each mutant was compared in triplicate to these controls, and mutants with reduced ß-galactosidase activity were eliminated. Mutants with weak, moderate, and strong reductions in ß-galactosidase activity were identified and eliminated with this approach. Only a small fraction of the mutant candidates affected the GAL1 promoter as judged by the X-gal assay (
ura30/ura30 leu20/leu20 his31/his31 met150/MET15 lys20/LYS2) were eliminated from further analysis.
Dilution assays:
Retrotransposition levels were measured in triplicate for each mutant by plating serial dilutions of cells that had been induced for retrotransposition on medium that was selective for retrotransposition events (SC-H) and on two control media (SC and SC-U). Cells were scraped from the SC plus 5-FOA patches into water and diluted to an OD600 of 1. Twofold dilutions were prepared in 96-well microtiter dishes and then plated on all three media using a multichannel pipettor. The SC plate served as a control for adjusting the cells to an OD600 of 1, whereas the SC-U plate served as a control to ensure that the test plasmid had been eliminated successfully on the 5-FOA step. The number of cells growing at each dilution on the SC-H plate was compared to similar dilutions prepared from the wild-type strain and the fold change was estimated (rounding to the nearest twofold dilution). The three independent measurements were averaged to produce the final fold-change value reported (Fig 2C and supplemental Table 1 at http://www.genetics.org/supplemental/).
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Targeting assays:
The plasmid-based assay for tRNA gene targeting was performed as described previously using a Gal-Ty1-neo donor plasmid and the SUF16 target plasmid pSD546 (
-32P]ATP using T4 polynucleotide kinase. The PCR then was performed with the SD609 and SD610 primers using the following cycling parameters: (i) 94° for 4 min; (ii) 28 cycles of 94° for 30 sec, 60° for 30 sec, and 72° for 30 sec; and (iii) a final extension at 72° for 10 min. A total of 150 His+ colonies were independently pooled in duplicate for each mutant and analyzed using genomic DNA templates prepared from the pools. Following PCR, the reactions were precipitated with ethanol, washed with 70% ethanol, and resuspended in 10 µl water plus 10 µl of deionized formamide loading buffer. The samples were heated to 100° for 3 min and analyzed on a denaturing 6% polyacrylamide urea gel. The gel was dried and exposed to XAR5 film overnight at -80°.
Ty1 cDNA analysis:
Ty1 cDNA was measured by Southern hybridization analysis after a 48-hr induction in medium containing galactose. DNA was isolated from duplicate pAR100 transformants and analyzed as follows. After measuring the DNA concentration of each sample with a spectrophotometer, 10 µg of DNA was digested with the restriction endonuclease AflII (which cuts 2472 bp from the right end of Ty1-HIS3 cDNA) and run on a 1% agarose gel. The DNA was transferred to a nylon membrane (Osmonics, Minnetonka, MN) and then hybridized to a 1.4-kb probe that spanned the full HIS3 gene. Using this strategy, cDNA originating from the pAR100 donor plasmid was detected, but cDNA arising from genomic Ty1 copies was not detected. The HIS3 probe also hybridized to the linearized donor plasmid pAR100 and the his31 allele in the BY4743 strain background, thereby generating two additional bands in each lane (at 13 and 5 kb, respectively). These bands served as loading controls to ensure that equal amounts of DNA were analyzed in each lane. The prehybridization/hybridization buffer contained: 6x SSC, 0.01 M EDTA (pH 8.0), 5x Denhardt's solution, 0.5% SDS, and 100 µg/ml sheared, denatured salmon sperm DNA. The prehybridization, hybridization, and final wash steps were carried out at 65°. The washed membranes were exposed to XAR5 film and analyzed with a Fujix BAS1000 phosphoimager after exposing the membranes to phosphoimaging screens. Ty1 cDNA was measured in the duplicate samples by digital analysis of the scanned images, and the duplicates were averaged to obtain the final values reported. The Ty1 cDNA levels were considered to be altered from wild type if the average of the duplicate measurements was <50%, or >200%, of wild-type control cDNA levels.
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Systematic analysis of Ty1 retrotransposition in the gene deletion collection:
Our experimental strategy involved screening the collection of yeast deletion strains developed by the Saccharomyces Genome Deletion Project (
6200 yeast genes have been deleted in this single isogenic collection of knockout strains, allowing many genes to be tested in parallel for possible effects on a given process (in this case, Ty1 retrotransposition). Approximately 17% of the genes in yeast are "essential" and thus produce lethal phenotypes upon gene deletion (
A donor plasmid carrying the Ty1 element was introduced separately into 4483 homozygous deletion strains using a 96-well transformation protocol, and each strain was tested for its ability to support Ty1 retrotransposition (see MATERIALS AND METHODS). The modified Ty1 element used in these studies was placed under the control of the galactose-inducible GAL1 promoter and was used to test retrotransposition as described previously (1 genetic background of the knockout collection (
Using this plasmid-based assay, deletion strains with significantly altered levels of Ty1 retrotransposition were identified readily from the knockout collection (Fig 1B). In fact, 2.3% of the yeast genes tested showed a Ty1 retrotransposition phenotype, for a total of 101 mutants in the collection of 4483. Most of the mutants had decreased levels of retrotransposition (99/101 or 98%), whereas only 2/101 (2%) had increased levels (yml105c and yol159c). Retrotransposition mutants were independently confirmed by retransforming each strain with the Ty1 plasmid and retesting it along with the original transformants and frozen stocks of the original transformants. The results of these comparisons were remarkably consistent (Fig 2A).
Three classes of genes are expected to affect the Gal-Ty1 assay rather than affect retrotransposition itself: (i) genes that affect GAL1 promoter function, (ii) genes that affect marker gene function (in this case, HIS3), and (iii) genes that affect respiration and thus affect galactose utilization. Therefore, all initial mutant candidates were subjected to three secondary tests to eliminate host genes that affected these aspects of our assay rather than Ty1 retrotransposition itself (see MATERIALS AND METHODS). As expected, gal and his mutants were identified in these secondary screens, along with mutants that grew poorly on YPG medium. Growth on YPG medium was tested to identify mutants that do not support respiration and thus might not utilize galactose efficiently in our retrotransposition assay. Although all of these mutants represented unwanted by-products of our genomic screen, they were fully expected to affect our assay and thus served as excellent internal controls for the accounting system of the knockout collection. No other sources of background are known to affect the specific Gal-Ty1 assay used in these studies. Therefore, the remaining 101 Ty1 host factor (thf) mutants that passed these secondary tests were considered to have Ty1 retrotransposition phenotypes. These 101 genes are known to participate in a wide range of cellular processes and could be classified into 10 major groups on the basis of the known functions of the encoded proteins (Table 1). The data for the chromatin mutants are shown in Fig 2 and the full set of data for all of the mutants in Table 1 can be viewed at our website database (http://biochemistry.emory.edu/ty1).
To more precisely measure the degree of change in each retrotransposition mutant, we performed quantitative retrotransposition assays on all of the mutants listed in Table 1 (see also MATERIALS AND METHODS). The results of these assays confirmed and extended the initial observations from the patch assays. Fifty of the mutants produced "moderate" (three- to eightfold) changes in retrotransposition levels and 51 mutants produced "strong" (greater than eightfold) changes in retrotransposition levels (supplemental Table 1 at http://www.genetics.org/supplemental/). An example of the assay is shown in Fig 2B and Fig C. Additional mutants also were identified, but strains with fewer than threefold changes were omitted from the final collection.
Targeted integration:
The patch assays described above measure the overall levels of retrotransposition that occur in a given strain. Since Ty1 is known to integrate preferentially near tRNA genes, we also wanted to test whether any of the mutant strains in our collection affected the targeting step of the retrotransposition cycle. To do this, we needed to use an assay that would differentiate between mutants that affected any of the upstream steps of the retrotransposition cycle (and therefore did not produce integration events) vs. mutants that affected the integration step itself. Thus, we used a plasmid-based targeting assay to measure the frequency of Ty1 integration near a specific tRNA gene (SUF16) on a target plasmid. We previously found that the SUF16 tRNA gene served as a high-frequency target in this assay and that the resulting integration events were targeted to the region upstream of the SUF16 gene (
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It is important to note that these SUF16 targeting assays were performed exclusively with cells that contained at least one new retrotransposition event (i.e., grew on medium that was selective for new transposon insertions). As a consequence, retrotransposition events that were stalled at any of the upstream steps of the retrotransposition cycle were eliminated from this analysis. Each of the new transposon insertions that occurred in these cells was targeted to either the SUF16 plasmid or the yeast genome. If the new insertion was targeted to the SUF16 plasmid, then a new recombinant plasmid that carried a Ty1 element was formed. If, instead, the new insertion was targeted to the yeast genome, then the SUF16 plasmid remained unchanged. The SUF16 targeting frequency then was determined by rescuing all of the SUF16 target plasmids from the cells tested and by examining the fraction of target plasmids with new Ty1 insertions. This allowed us to directly measure whether the SUF16 plasmid was targeted at the normal frequency by Ty1 in a given strain.
We hypothesized that chromatin factors might affect tRNA gene targeting because they are physically associated with chromosomes. Such factors also are frequently involved in regulating the accessibility of DNA (
We next examined whether tRNA gene targeting was affected in any of the remaining mutants in our collection. We developed a more streamlined approach to rapidly examine all of the mutants in our collection using a PCR-based targeting assay that is similar to assays described previously (
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Ty1 generally integrates within an "integration zone" that begins 80 bp upstream of a tRNA gene and extends several hundred base pairs in the upstream direction (
All of the host factor mutants in our collection were tested for tRNA gene targeting using this PCR assay. The chromatin mutants first were reexamined, and, as expected, these mutants supported normal levels of targeting (Fig 3B, mutants 1–10). These results were consistent with the data obtained with the SUF16 plasmid-based assay (Table 2) and allowed us to calibrate the two assays. Although some variation in the number of PCR bands was observed between duplicate samples, approximately two to eight PCR bands were observed in most pools (Fig 3 and data not shown). These bands do not necessarily represent individual retrotransposition events. However, the heterogeneity in band patterns among different strains suggests that most bands probably do represent individual events. Almost all of the 101 host factor mutants examined in our collection had results that were equivalent to those shown in Fig 3B, and PCR products were detected in most of the mutants (Fig 3 and data not shown). However, two mutants, rit1 and ctk1, lacked new PCR products in both of the duplicate pools examined in three completely separate experiments (Fig 3, duplicate lanes 18 and 19, and data not shown). The two ubiquitous PCR bands present in all samples (and presumably caused by preexisting Ty1 copies) were present in rit1 and ctk1 (Fig 3). Therefore, technical failure of the PCR can be ruled out as a possible cause of the rit1 and ctk1 results. Thus, although the majority of the host factor mutants retained the ability to target Ty1 integration to tRNA genes, two mutants, rit1 and ctk1, displayed diminished levels of targeting. The Rit1 protein is an ADP ribosylase known to modify methionine tRNAs, whereas the Ctk1 protein is a protein kinase. These two proteins are likely to affect tRNA gene targeting through different mechanisms (see DISCUSSION).
The steady-state levels of Ty1 cDNA are altered in many of the host factor mutants:
We next determined whether the host factor mutants in our collection produced normal levels of Ty1 cDNA. Because double-stranded Ty1 cDNA is produced approximately midway through the retrotransposition cycle, it is a convenient measure of how far the retrotransposition cycle has progressed in a given mutant. Mutants with diminished levels of cDNA would be considered to affect the "early" steps of retrotransposition leading up to virus-like particle assembly and cDNA replication, whereas mutants with normal levels of cDNA would be considered to affect the "late" steps of retrotransposition that occur after cDNA production.
Interestingly, 9 of the 10 chromatin mutants examined were found to have significantly decreased levels of Ty1 cDNA compared to the wild-type BY4743 control strain (Fig 4A). Therefore, rather than affecting tRNA gene targeting, as we had originally postulated (Table 2), most of the chromatin mutants affected the production (or turnover) of Ty1 cDNA. Upon analyzing all of the mutants in our collection in duplicate by Southern analysis, we found a total of 44 strains with decreased levels of Ty1 cDNA (<50% of wild-type levels) and 2 mutants with increased levels of cDNA (>200% of wild-type levels; Fig 4 and Table 3). The remaining 55 mutants had normal levels of cDNA (between 50 and 200% of wild-type levels; Fig 4 and data not shown). Thus, almost half of the 101 mutants identified in our study affected the early steps of the Ty1 retrotransposition cycle leading up to the formation of virus-like particles and cDNA replication, whereas the remaining half affected the later steps that occur after cDNA replication.
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| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We have exploited a collection of gene deletion mutants developed by the Saccharomyces Genome Deletion Project (
Host factor mutants with altered levels of Ty1 cDNA:
Forty-six of the mutants identified in our screen had altered levels of Ty1 cDNA as measured by Southern hybridization analysis (Fig 4 and Table 3). Of the 46 mutants, 44 had decreased levels of cDNA, whereas 2 mutants had increased levels of cDNA. Since we eliminated mutants that affected the GAL1 promoter used in our Gal-Ty1 donor plasmid, none of the mutants is expected to affect the initial transcription step of the retrotransposition cycle in this system (Fig 5, step 1). However, several subsequent steps of the cycle must be completed before any Ty1 cDNA can be replicated, and mutants with diminished levels of cDNA could be deficient in any of these steps. Such steps include: (i) the initial processing of Ty1 mRNA in the nucleus (Fig 5, step 2), (ii) the export of Ty1 mRNA from the nucleus (Fig 5, step 3), (iii) the translation of Ty1 proteins on ribosomes (Fig 5, step 4), and (iv) the assembly of virus-like particles in the cytoplasm (Fig 5, step 5). The cDNA levels might also be affected by changes in the rate of cDNA replication or turnover.
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Of the 10 chromatin mutants examined in our study, 9 produced diminished levels of Ty1 cDNA compared to the BY4743 wild-type strain (Fig 4A). One possible model to explain these results would be that these chromatin factors normally play an important role in protecting the Ty1 cDNA from degradation by nucleases. In the absence of these chromatin factors, the Ty1 cDNA is more vulnerable to nuclease digestion, and thus Ty1 cDNA levels are decreased in such chromatin mutants. This model predicts the existence of an important chromatinized cDNA intermediate that is necessary for retrotransposition. An alternative model would be that these chromatin factors regulate the expression of other genes that, in turn, affect cDNA replication or turnover. Such genes might include some of the other "early" genes identified in our study (Table 1). Additional studies will be required to differentiate between these (and perhaps other) models.
A number of other mutants in our collection also displayed decreased levels of cDNA and thus appear to affect early steps of the retrotransposition cycle. Within the RNA metabolism group, for example, both the cbc1 and cbc2 mutants had reduced levels of Ty1 cDNA (Fig 4B). The Cbc1 and Cbc2 proteins form a "cap binding complex" that binds to the cap structure of cellular mRNAs (
Host factor mutants with normal levels of Ty1 cDNA:
We also identified 55 mutants that had normal levels of Ty1 cDNA (within a range of plus or minus twofold of the wild-type control levels) as judged by Southern analysis (Fig 4 and data not shown). These mutants are likely to affect one or more of the "late" steps of retrotransposition that occur after the production of cDNA. One of the first steps that must occur after cDNA replication is the nuclear localization of the newly replicated Ty1 cDNA and integrase. Although it is presently unclear as to how the 6-kb Ty1 cDNA enters the nucleus, Ty1 integrase has a nuclear localization sequence that is required for retrotransposition (
After entering the nucleus, the cDNA is integrated into chromosomal DNA, primarily near tRNA genes (Fig 5, step 8). Despite the large number of host factors identified in our screen, only two factors that affected tRNA gene targeting were identified (see below). A likely explanation for this seemingly small number of targeting mutants is that we examined only the nonessential yeast genes in our study. Because most of the RNA pol III transcription factors are encoded by essential genes, it is likely that we missed at least some targeting factors by focusing only on nonessential yeast genes. Therefore, additional screens (focused on essential genes) will be necessary to identify all of the host factors involved in targeting.
After cDNA integration, some level of DNA repair is likely to be required at the integration site, and perhaps at other sites in the yeast genome, to repair damaged DNA created during retrotransposition. Four DNA repair mutants were identified in our study. Three of the DNA repair mutants, mms22, rad52, and xrs2, had normal levels of cDNA (Fig 4B) and therefore affected late steps of the retrotransposition cycle. Such factors could be involved in repairing chromosomal DNA damage at integration sites or elsewhere in the genome. The remaining mutant, apn1, had significantly decreased levels of cDNA and thus affected an early step of the retrotransposition cycle (Fig 4B). The Apn1 protein is an apurinic/apyrimidinic endonuclease that cleaves DNA at abasic sites to facilitate DNA repair. One possible explanation for Apn1 function would be that it is involved in cDNA repair prior to integration. If the cDNA were not repaired properly in an apn1 mutant, perhaps the cDNA would be targeted for degradation.
Finally, most of the groups of genes listed in Table 1 contain both "early" and "late" mutants. Therefore, none of the groups appears to be devoted to a single step of the retrotransposition cycle. Nevertheless, some of the groups have a disproportionate number of mutants devoted to either early or late stages of the retrotransposition cycle. For example, six of the seven transcription elongation mutants (elp1, elp2, elp3, elp4, elp6, and kti12) were found to affect the late stages of retrotransposition. All six of these "late" transcription elongation mutants, in principle, could affect retrotransposition by affecting the transcription of even a single "late" gene. Thus, our screen may have identified groups of genes involved in other processes (such as transcription elongation) that are necessary for retrotransposition. This might help to account for the large number of mutants identified in our study. Additional secondary screens and assays will be necessary to identify these groups and to determine how such factors work together to influence retrotransposition.
Diminished tRNA gene targeting in rit1 and ctk1 mutants:
Although most of the mutants identified in our study retained the ability to target Ty1 integration to tRNA genes, two of the mutants, rit1 and ctk1, had diminished levels of tRNA gene targeting in our PCR assay (Fig 3). The Rit1 protein, which is an ADP ribosylase, is known to modify the methionine tRNA that serves as a primer for Ty1 strong-stop synthesis during cDNA replication (
We also observed a decrease in tRNA gene targeting in the ctk1 mutant (Fig 3B). Ctk1p is a protein kinase known to regulate RNA polymerase II (RNA pol II) activity by phosphorylating the largest subunit of RNA polymerase II, Rpo21p (
A comparison of studies using Gal-Ty1 vs. chromosomal donor elements:
haploid strain background, whereas the mutants in our genomic screen were constructed in a diploid strain background.
Other studies also suggest that these experimental variables have an impact on retrotransposition experiments. For example, two previous studies reported seemingly contradictory results on the effects of rad52 mutations on Ty1 retrotransposition (
We found that a rad52 mutation led to a modest decrease in Ty1 retrotransposition, in agreement with the
Additional comparisons with previous Ty1 host factor screens:
A number of additional host factors have been identified that affect the Ty1 retrotransposition cycle (
As expected, we identified the dbr1 gene in our screen and observed a decrease in retrotransposition that was similar to the decrease reported previously (
Potential Ty1 host factor homologs in other organisms:
Potential Ty1 host factor homologs were identified in a number of other organisms by performing BLAST searches of the public databases with the host factors identifed in our study (
In conclusion, our study has increased the number of host factors known to affect Ty1 and, together with previous studies, provides a more complete picture of the relationship between Ty1 and its yeast host. Our study provides a foundation for many future studies on the retrotransposition cycle. Functional genomics screens such as ours, using the complete yeast knockout collection, will be very useful for identifying the nonessential genes involved in a given cellular process and will be invaluable for dissecting complex biological processes.
| FOOTNOTES |
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1 These authors contributed equally to this work. 
| ACKNOWLEDGMENTS |
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We thank Shari Corin for helpful advice and critical review of the manuscript. We thank Danny Reines for the pGAL1-lacZ plasmid used in this study and Melissa Geddie for constructing some of the deletion strains used in the studies presented in Table 2. We also thank Andy Bennett for helpful discussions. This work was supported by University Research Council grant no. 2-80302 (S.E.D.) and Research Scholar grant no. RSG-01-173-01-MBC from the American Cancer Society (S.E.D.).
Manuscript received November 11, 2002; Accepted for publication March 17, 2003.
| LITERATURE CITED |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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