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Address correspondence to Mary-Lou Pardue, Dept. of Biology, 68-670, Massachusetts Institute of Technology, Cambridge, MA 02139. Tel.: (617) 253-6741. Fax: (617) 253-8699. E-mail: mlpardue{at}mit.edu
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Abstract |
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Key Words: intracellular targeting; HeT-A; TART; retrovirus; telomere
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Introduction |
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In addition to HeT-A and TART, which transpose only to telomeres, Drosophila contains other non-LTR retrotransposons that transpose into many parts of the genome, but not into HeT-A/TART telomere arrays. With a few exceptions, transposition of non-LTR elements does not appear to be targeted by specific DNA sequences at the insertion site. For example, HeT-A and TART have been found joined to many different sequences in "healing" broken chromosome ends (Biessmann et al., 1990, 1992; Sheen and Levis, 1994; Golubovsky et al., 2001). The lack of specific nucleotide sequence targets suggests that the targeting of the telomere elements may be governed by proteins associated with chromosome ends. These same proteins might serve to exclude nontelomeric elements from the terminal arrays.
The apparently random mixture of HeT-A and TART in telomere arrays suggests that the two elements have equivalent roles at the chromosome end. However, none of the D. melanogaster stocks studied have completely lost either element. The results presented here support the hypothesis that the two elements have a symbiotic relationship, with both elements contributing to their telomere-specific transposition.
Despite their role in forming telomeres, HeT-A and TART share characteristics of other retrotransposons. For example, TART has both the gag and pol coding regions typical of many retrotransposons. The pol region encodes reverse transcriptase. The sequence of this enzyme has been used to deduce phylogenetic relationships of retroelements. The analysis places TART into the jockey clade of insect non-LTR retrotransposons (Malik et al., 1999).
Surprisingly, HeT-A does not have a pol coding region and must obtain its reverse transcriptase activity from some other source. Whatever the source of this activity, HeT-A has been found to transpose much more frequently than TART (Savitsky et al., 2002). It is possible that TART provides the reverse transcriptase for HeT-A, but at this time, there is no evidence to support this suggestion. If TART does provide this activity for transposition, it raises the question of why HeT-A is more abundant than TART. Is HeT-A also supplying a necessary function?
In addition to their unique ability to transpose only to chromosome ends, HeT-A and TART also encode closely related Gag proteins (Pardue et al., 1996; Rashkova et al., 2002). This suggested that the Gag proteins might be involved in the telomere targeting, a suggestion supported by what is known of retroviral Gags, which are responsible for forming ribonucleoprotein particles that carry viral RNA through the cell. For example, retroviral Gag protein has been shown to be both necessary and sufficient to form a capsid localized to the appropriate region of the cell plasma membrane (for review see Swanstrom and Wills, 1997). Here, we explore a possibly analogous role for the Gag proteins of HeT-A and TART in positioning these elements at telomeres.
The hypothesis that Gag proteins have a role in the telomeric localization of HeT-A and TART is supported by evidence that the intracellular localization of these Gag proteins is significantly different from that of Gags of non-LTR elements that transpose only to nontelomeric sites in D. melanogaster chromosomes (Rashkova et al., 2002). The comparisons were performed by cytological localization of each protein in transiently transfected cultured Drosophila cells. Each Gag coding region was tagged with GFP. All proteins were expressed from the same promoter construct so that localization would be determined only by protein sequence, rather than by secondary factors such as promoter strength. The two telomeric transposon Gags were transported rapidly and efficiently into the nucleus. Gags of the nontelomeric retrotransposons had a very different localization. For two elements (Doc and I factor), essentially all of the proteins remained in the cytoplasm, whereas for the third element (jockey), only a small fraction reached the nucleus.
The efficient nuclear localization of HeT-A and TART Gags is consistent with the status of these elements as part of the cellular machinery (maintaining the chromosome ends) while the presumably parasitic elements are impeded in travel to the nucleus. The unexpected finding was that, inside the nucleus, HeT-A Gag and TART Gag had very different distributions. This raises the question of how their localization relates to the final transposition of these elements to telomeres. We now report further studies showing that HeT-A Gag is preferentially associated with chromosome ends. TART Gag does not associate with telomeres unless the two proteins are coexpressed. In such cells, HeT-A Gag efficiently redirects TART Gag to telomeres.
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Results and discussion |
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To deposit the Het-dots in a single plane and spread them over a larger area, we have used a cytocentrifuge to flatten or break the nuclei. These preparations show the same numbers of Het-dots as seen by optical sections of dropped cells. However, the centrifuged cells reveal a clear difference in stability between Het-dots and TART Gag clusters (Fig. 2, C and D). Het-dots withstand spinning, whereas TART Gag clusters break down and the protein spreads through the nucleus. Apparently, protein associations in TART Gag clusters are not strong enough to withstand centrifugation.
In addition to differences in stability between Het-dots and TART Gag clusters, there is one unusual aspect of HeT-A Gag localization never seen with TART Gag or other retrotransposon Gags. About a third of the cells with Het-dots have a large smooth-edged body of cytoplasmic HeT-A Gag protein, usually well removed from the nucleus. We refer to this structure as the Het-body (Fig. 2 A). It is never seen in cells that do not have nuclear HeT-A protein. Cells with Het-bodies can still divide; we have observed them in telophase. In these cells, there was only a single Het-body and it is not clear how the material is eventually distributed between the daughters. We assume these bodies reflect overexpression of Gag, but if so, this overexpression shows that the cells treat this protein differently from other excess Gag protein. Other presumably overexpressed proteins form multiple aggregates, associated with more diffuse material, broadly distributed over the cytoplasm.
Het-dots are preferentially associated with chromosome ends
The number and localization of Het-dots fit the expectation for structures associated with chromosome ends. To test this hypothesis we looked for, but did not find, association of the dots with metaphase chromosomes. Instead, we found that both HeT-A and TART Gags diffuse throughout the cell at metaphase (Rashkova et al., 2002) with a few streaks of aggregated protein remaining. This behavior is similar to that reported for the chromatin protein, HP1, in Drosophila (Kellum et al., 1995) and for several sequence-specific transcription factors in human cells (Martinez-Balbas et al., 1995). Nuclear associations of HeT-A and TART Gags appear to reform during telophase; thus, testing the relation of Het-dots to telomeres requires a marker that can identify chromosome ends in interphase nuclei.
A number of telomere-associated proteins have been characterized for mammals and for yeast, but Drosophila telomereassociated proteins are still relatively unknown. One protein, HOAP, has been shown to associate predominantly with telomeres in Drosophila polytene chromosomes (Shareef et al., 2001). Polytene nuclei are interphase nuclei; however, we now find that HOAP remains on the chromosome throughout the cell cycle and can be detected on metaphase chromosomes in the cultured cells. The major sites of anti-HOAP antibody binding are the telomeres (Fig. 3). HOAP is found on all telomeres, although some chromosome ends stain less heavily than others. The relative staining level is similar on sister chromatids, suggesting that the amount of HOAP present may be characteristic of specific ends.
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These studies of metaphase chromosomes show that HOAP staining serves as a marker for chromosome ends through the cell cycle. Thus, the relation between Het-dots and telomeres in interphase nuclei can be analyzed by comparing the distribution of Het-dots with that of HOAP dots. To minimize nonspecific overlap in these small nuclei, we have done the analyses on centrifuge-flattened cells and broken nuclei. We find many of the Het-dots, ranging from 60 to 90% for different cells, overlap with HOAP dots. On visual inspection of spread nuclei, it is clear that Het-dots and HOAP dots associate closely (Fig. 4, A and C).
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Even if the two proteins were directly complexed at telomeres, one should not necessarily expect HOAP and Het-dots to be completely associated. Some telomeres might not have associated Het-dots because there is no reason to suppose that every telomere should have associated Het-dots in any given cell cycle. In addition, HOAP at centromeres should not have corresponding Het-dots. Furthermore, sample preparation could disrupt some associations between Gag, HOAP, and/or other components of the complexes at the chromosome ends.
HeT-A Gag recruits TART Gag to specific locations
TART Gag does not associate preferentially with chromosome ends. We see no significant coincidence between TART Gag and HOAP in nuclei that have not been centrifuged. The association cannot be studied in centrifuged cells because the TART Gag clusters break down and the protein is spread over most of the nucleus around the nucleolus (Fig. 4 B).
This distribution of TART Gag changes dramatically when the protein is coexpressed with HeT-A Gag. For these experiments, TART Gag was tagged with YFP and HeT-A Gag with CFP because these two fluorochromes can be detected separately in the same preparation. In single transfections, YFP- and CFP-tagged Gag proteins behaved exactly as did their GFP-tagged counterparts.
When HeT-A Gag is coexpressed with TART Gag, the two proteins colocalize completely (Fig. 5 A). The localization is controlled by HeT-A Gag. TART Gag is seen in Het-dots and also in Het-bodies. The association between the two proteins is strong enough to withstand centrifugation. Preliminary experiments with deletion derivatives of the proteins (unpublished data) have shown that the association is dependent on amino acid sequences in the region of the zinc knuckles of both proteins (Pardue et al., 1996).
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Conclusions
Drosophila is remarkable for adapting two non-LTR retroelements to maintain its telomere arrays (Fig. 1). In this paper, we show that Gag proteins encoded by these elements have the potential to target their transposition intermediates to chromosome ends. Our finding that HeT-A Gag overrides the localization of TART Gag in cotransfections leads to an intriguing speculation about the roles of each of these elements in forming Drosophila telomeres. HeT-A does not encode a reverse transcriptase but TART does. TART may provide this activity for both elements, whereas HeT-A may be responsible for the final targeting of both retrotransposons to the telomere. This role in targeting can explain why HeT-A, the element lacking its own reverse transcriptase, is so abundant. The colocalization suggests that these two telomeric transposons may have coevolved into symbiotes, with TART supplying the reverse transcriptase and HeT-A the nuclear targeting.
Like other metazoa, Drosophila has many kilobases of DNA in its telomere arrays. Little is known about rates of turnover and replacement on normal telomeres in metazoa; however, studies on yeasts and other organisms reveal that telomere sequences are in dynamic flux, with sequence gains and losses that are influenced by genetic background, by growth conditions, by cell type, and by developmental stage (Blackburn, 2001). The experiments described here study the behavior of overexpressed proteins, but they reveal a mechanism of retrotransposon localization that has the flexibility to maintain the dynamic telomeres suggested by the yeast studies. This system is efficient; almost nothing is left behind, arguing that even a small amount of expressed Gag protein would get to a telomere. This system is also robust because it can accommodate a significant amount of protein before formation of the cytoplasmic Het-body, which appears to represent an overload of the system. Such a mechanism has the capacity to respond rapidly to the need to change telomere length; an important adaptive mechanism for the cell.
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Materials and methods |
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Cell culture and transfection
Drosophila SL2 cells were maintained in DME supplemented with 10% FCS, 0.5% lactalbumin hydrolysate, and 10 mM nonessential amino acids. For transfection, 2.5 x 106 cells/ml in 5 ml of DME were incubated at 25°C for 1820 h. Transfections were performed in 2.5 ml of serum-free DME using a Cytofectene Transfection Reagent Kit (Bio-Rad Laboratories) and 510 µg of plasmid DNA purified with a Plasmid Midi Kit (QIAGEN). Medium containing DNA was replaced after 6 h with DME plus 100 µg/ml penicillin and 100 µg/ml streptomycin. Cells were analyzed at 24 and 48 h.
Slide preparation
Transfected SL2 cells were dropped onto slides 48 h after transfection and allowed to settle for 20 min, or diluted 10-fold with 1xPBS and spun onto slides for 3 min in a cytocentrifuge at 1,600 rpm. Cells were fixed with 3.7% formaldehyde in PBT (1xPBS; 0.1% Tween 20) for 30 min, washed three times for 5 min in PBT, and stained with 0.2 µg/ml DAPI in 20 mM Tris-HCl, pH 7.4, for 1 min. Slides were mounted in 1xPBS, 50% glycerol. For anti-HOAP staining, nontransfected cells were treated with 5 µg/ml colchicine for 3 h, diluted 10-fold with 0.5% sodium citrate for 5 min, spun onto slides in a cytocentrifuge, and fixed as described above. Slides were incubated 30 min at RT with blocking solution (10% FCS in PBT) for 2 h with a 1:2,000 dilution of rabbit anti-HOAP antibody (Shareef et al., 2001) with Cy3-secondary antibody (Jackson ImmunoResearch Laboratories), and then stained with DAPI.
Microscopy
Fluorescence miscroscopy used an Eclipse microscope (model E600; Nikon) equipped with a CCD camera. Images were taken using Spot RT v3.0 software and processed with Adobe Photoshop® 5.5.
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Footnotes |
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Acknowledgments |
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This work has been supported by grants GM50315 and GM57006 from the National Institutes of Health (to M.L. Pardue) and NIH grant GM59765 (to R. Kellum).
Submitted: 6 May 2002
Revised: 5 September 2002
Accepted: 9 September 2002
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References |
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Biessmann, H., K. Valgeirsdottir, A. Lofsky, C. Chin, B. Ginther, R.W Levis, and M.-L. Pardue. 1992. HeT-A, a transposable element specifically involved in "healing" broken chromosome ends in Drosophila melanogaster. Mol. Cell. Biol. 12:39103918.[Abstract]
Eickbush, T.H. 2002. R2 and related site-specific non-long terminal repeat retrotransposons. Mobile DNA II. N. Craig, R. Craigie, M. Gellert, and A. Lambowitz, editors. American Society for Microbiology, Washington, D.C. 813835.
Golubovsky, M.D., A.Y. Konev, M.F. Walter, H. Biessmann, and J.M. Mason. 2001. Terminal retrotransposons activate a subtelomeric white transgene at the 2L telomere in Drosophila. Genetics. 158:11111123.
Kellum, R., J.W. Raff, and B.M. Alberts. 1995. Heterochromatin protein 1 distribution during development and during the cell cycle in Drosophila embryos. J. Cell Sci. 108:14071418.
Luan, D.D., M.H. Korman, J.L. Jakubczak, and T.H. Eickbush. 1993. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell. 72:595605.[Medline]
Malik, H.S., W.D. Burke, and T.H. Eickbush. 1999. The age and evolution of non-LTR retrotransposable elements. Mol. Biol. Evol. 16:793805.[Abstract]
Pardue, M.-L., and P.G. DeBaryshe. 1999. Telomeres and telomerase: more than the end of the line. Chromosoma. 108:7382.[CrossRef][Medline]
Pardue, M.-L., and P.G. DeBaryshe. 2002. Telomeres and transposable elements. Mobile DNA II. N. Craig, R. Craigie, M. Gellert, and A. Lambowitz, editors. American Society for Microbiology, Washington, D.C. 870887.
Rashkova, S., S.E. Karam, and M.-L. Pardue. 2002. Element-specific localization of Drosophila retrotransposon Gag proteins occurs in both nucleus and cytoplasm. Proc. Natl. Acad. Sci. USA. 99:36213626.
Savitsky, M., O. Kravchuk, L. Melnikova, and P. Gregoriev. 2002. Heterochromatin protein 1 is involved in control of telomere elongation in Drosophila melanogaster. Mol. Cell. Biol. 22:32043218.
Shareef, M.M., C. King, M. Damaj, R.K. Badagu, D.W. Huang, and R. Kellum. 2001. Drosophila heterochromatin protein (HP1)/origin recognition complex (ORC) protein is associated with HP1 and ORC and functions in heterochromatin-induced silencing. Mol. Biol. Cell. 12:16711685.
Sheen, F.-M., and R.W. Levis. 1994. Transposition of the LINE-like retrotransposon, TART, to Drosophila chromosome termini. Proc. Natl. Acad. Sci. USA. 91:1251012514.
Swanstrom, R., and J.W. Wills. 1997. Synthesis, assembly, and processing of viral proteins. Retroviruses. J.M. Coffin, S.H. Hughes, and H.E. Varmus, editors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 263334.