From the
National Research Laboratory and the
¶Molecular Aging Research Center, Department of
Biology, Yonsei University, 134 Shinchon, Seodaemun-ku, Seoul 120-749,
Korea
Received for publication, March 3, 2003 , and in revised form, April 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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The idea that proteins implicated in telomere protection are evolutionarily conserved at the functional level rather than the amino acid sequence level has gained momentum (for example, Ref. 14). We wished to extend the understanding of universal mechanisms of telomere protection and functions using the nematode Caenorhabditis elegans as a model system. C. elegans can serve as a model system to study telomere functions because it has typical telomeric repeats ranging from 4 to 9 kb (15), and it has many advantages in molecular genetic approaches such as its complete genome, short life cycle, and many molecular tools available. However, no telomere-binding protein has been identified in C. elegans. As a first step toward the studies of telomere functions in the nematode, we identified a telomere-binding protein in C. elegans. We undertook the yeast one-hybrid approach to avoid any bias toward proteins that have amino acid similarity to known telomere-binding proteins. The nematode telomeric DNA consists of TTAGGC repeats, a sequence motif that differs from that of mammals and plants. A protein identified in this screen, CEH-37, has a structural domain similar to the homeodomain but lacks a Myb-like domain. We show that the homeodomain of CEH-37 is structurally similar to the Myb domain of known telomere-binding proteins despite their primary sequence dissimilarities. We also show that CEH-37 specifically binds nematode double-stranded telomere DNA and bends telomere-containing DNA. We demonstrate that CEH-37 is primarily localized to the chromosome ends in vivo. Finally, we show that CEH-37 is required for chromosome stability in vivo, acting together with mrt-2, a checkpoint protein gene acting on the telomeres.
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EXPERIMENTAL PROCEDURES |
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Gel Shift AssayWe prepared probes from a (TTAGGC)6 fragment excised from pGEM-TE and from various repeats of the telomere oligomer. To reduce nonspecific binding, in vitro translated recombinant proteins were preincubated with 1 µg of poly(dI-dC) and 0.5 µg of nonspecific double-stranded DNA. Radioactive probe was added, and the mixtures were incubated for 15 min at room temperature. Total binding mixtures were loaded on 58% nondenaturing polyacrylamide gels and subjected to electrophoresis in 0.5x TBE (54 mM Tris borate, pH 8.3, 1 mM EDTA). The binding reaction was monitored by autoradiography and by analysis with a Fuji phosphorimaging device.
Construction and Expression of CEH-37 DerivativesTo perform domain studies of CEH-37, we generated deletion derivatives of CEH-37 by PCR. We designed the PCR primers so that they could amplify the N-term1 (N-terminal region) domain (1st37th amino acids of CEH-37), the homeodomain (38th94th amino acids), and the C-term (C-terminal region) domain (95th278th amino acids of the protein). The following oligonucleotides were used as primers for PCR: D1 (5'-GGGATCCGACCAGCTATAGTTACTTCAC-3'), D2 (5'-GGGATCCTCGGAAGAATCGTCGCGAACG-3'), D3 (5'-GGAAGCTTAAGTACCGCCTCCAAGAG-3'), D4 (5'-GGAAGCTTTCAGATCATTGCTGCATTG-3'), D5 (5'-GCTCGAGGTTGCTTCATTTTGGTTTTTG-3'), and D6 (5'-GCTCGAGTTACAAATTATTTCCATTTG-3'). The PCR products were subcloned into the pRSET vector (Invitrogen). The subclones containing the full-length and deletion derivatives of the ceh-37 cDNA (N, N-H, N-C, and H) were translated using the TNT T7-coupled reticulocyte lysate system (Promega). In the case of coexpression of the full-length and N-terminal domains, the two templates were mixed and co-translated. For yeast one-hybrid assays, the PCR fragments were cloned into the pACT vector (Clontech).
Yeast One-hybrid AssayWe transformed each ceh-37 deletion derivative constructed in pACT (Clontech) into the yeast strain CG-1945 (Clontech) transformed previously with the bait construct. Equivalent numbers of transformants were spread on selective media (SD/His/Trp/Leu containing 25 mM 3-aminotriazole) or nonselective media (SD/Trp/Leu). The ratio of the number of colonies on selective and on nonselective media was taken as a measure of the viability of each deletion derivative under selection for the expression of the HIS3 reporter. We regarded this viability rate as reflecting the strength of the telomere binding activity of each deletion derivative.
DNA Bending AssayWe generated three probes of the same size
but containing telomere repeats in different locations by PCR using pGEM-TE
with (TTAGGC)6 repeats as a template and the following
-32P end-labeled primers: Tel L1
(5'-GCCGCGGGAATTCGAT-3'), Tel L2
(5'-TGCTTCCGGCTCGTATG-3'), Tel M1
(5'-GTTGTAAAACGACGGG-3'), Tel M2
(5'-TTAGGTGACACTATAGAATAC-3') Tel R1
(5'-CTTCGCTATTACGCCAGC-3'), Tel R2
(5'-ATTCACTAGTGATTAAGC-3'). Probes were purified from 10%
polyacrylamide gels. Gel shift assays with in vitro translated CEH-37
were carried out as described above.
Subcellular Localization of CEH-37 in VivoTo determine the subcellular localization of CEH-37, we substituted the full-length ceh-37 ORF with the histone 2B ORF in the plasmid pJH4.52. pJH4.52, a plasmid that contains the pie-1 regulatory region, which drives GFP expression in early embryos, was kindly provided by Geraldine Seydoux. We then microinjected the construct mixed with PstI-digested N2 genomic DNA into mut-7 (pk204) mutants to avoid the germline suppression of transformed DNA and to prolong GFP expression over several generations. The transgenic animals containing this reporter construct were observed by a fluorescence microscope (Carl Zeiss). 4,6-diamidino-2-phenylindole was used to visualize chromosomes.
RNA InterferenceFull-length mrt-2 cDNA was obtained by RT-PCR and inserted into pPD 129.36. single-stranded RNAs were generated with the RiboMax in vitro transcription kit (T7, Promega). After phenol: chloroform treatment and EtOH precipitation, single-stranded RNA products were denatured at 70 °C for 10 min and allowed to anneal by cooling to room temperature. 100200 µg/ml double-stranded RNA was injected into wild-type or mutant L4 stage animals.
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RESULTS |
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Since CEH-37 shares sequence similarity with homeodomain proteins, which are generally transcription factors, whereas most identified double-stranded telomere-binding proteins contain Myb domains, we wished to compare the three-dimensional structure of CEH-37 with that of other telomere-binding proteins. Because both domains are composed of a helix-turn-helix structure, it is conceivable that the homeodomain of CEH-37 would be structurally similar to the Myb domain. We utilized the SWISS MODEL software (www.expasy.org/swiss mod) to model the CEH-37 homeodomain. We were unable to use the solved TRF1 Myb domain structure as a template because there is little primary amino acid sequence similarity between TRF1 and CEH-37. Instead, we used the Engrailed homeodomain structure since it contains a homeodomain similar to that of CEH-37. For structural comparison, we fitted the solved three-dimensional structure of the Myb domain of TRF1 to the three-dimensional model of the homeodomain of CEH-37 using the SPDBV software (www.expasy.org/swissmod). We found that the two proteins have very similar tertiary structures (Fig. 1C). We also found that the modeled three-dimensional structure of the S. pombe Taz1 protein, a TRF ortholog (9), is similar to that of the TRF1 Myb domain and the CEH-37 homeodomain except that it lacks the third helix. We found little, if any, similarity between the CEH-37 structure and that of RAP-1, a telomere-associated protein identified in S. cerevisiae and humans (data not shown). Consistent with this, it was recently reported that the NMR structure of hTRF1 bound to DNA is different from that of Rap1p bound to DNA (18). The structural similarity between CEH-37 and hTRF1 implies that the nematode telomere-binding protein may represent an evolutionary adaptation in terms of tertiary protein structure rather than primary amino acid sequence.
CEH-37 Specifically Binds to C. elegans Telomere Sequences in VitroTo examine whether the CEH-37 protein specifically binds to the C. elegans telomere, we performed competition assays using the nematode, human, and rice telomere sequences as cold competitors (Fig. 2). Although the nematode cold telomere sequence was able to efficiently compete with the binding ability of CEH-37, neither the human nor the rice telomere sequence was able to compete with CEH-37 (Fig. 2A). To examine whether CEH-37 binds to other sequences than the telomere sequence of the nematode genome, we performed competition assays with the consensus CRX binding motifs. We found that the CRX binding motif sequences did not compete with the CEH-37 binding, indicating that CEH-37 probably binds only to the nematode telomere sequence (Fig. 2B). Gel retardation assays with different numbers of the telomeric repeats showed that CEH-37 binding required at least 1.5 repeats of TTAGGC, that is, TTAGGCTTA was sufficient to bind CEH-37 (Fig. 2C). We found that mutations in any single nucleotide from the 4th G through the 9th A in an oligomer of TTAGGCTTAGGC abolished CEH-37 binding ability, indicating that the GGCTTA motif forms the core of the CEH-37 binding site (Fig. 2D). CEH-36, a possible paralog of CEH-37, did not bind the telomere at all (data not shown), indicating that this closely related C. elegans homeodomain protein does not bind to telomeric sequences.
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To define the roles of the domains of CEH-37 in telomere binding, we examined various CEH-37 derivative proteins. By in vitro binding assays, we found that the C-term was dispensable for telomere binding because the truncated protein lacking the C-terminal region was able to bind the telomeric DNA (Fig. 3A). We also found that the N-term of the protein and the homeodomain were both necessary and sufficient for binding to telomeric DNA (Fig. 3A). Supporting this, in the yeast one-hybrid assay, only the colonies that contained N-H domains, but not N-term, C-term, or H-C domains, survived in media lacking histidine at a rate comparable with that of colonies containing the full-length CEH-37 (Table I). From these in vitro and in vivo domain study results, we suggest that the homeodomain is directly involved in contacting DNA and that the N-term domain may be involved in dimerization of the protein units. Consistent with this, the N-term domain competitor efficiently inhibited the binding of CEH-37 to telomeric sequences, indicating that the N-term domain acts in a dominant negative manner (Fig. 3B). However, we cannot rule out other possible reasons for the competition by the N-term domain because the competition assay was not a direct evidence of dimerization. The idea that the N-term of CEH-37 may be required for dimerization parallels known telomere-binding proteins despite the lack of amino acid sequence homology.
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CEH-37 Bends DNA by Binding to Telomeric DNA in VitroA telomere-binding protein, TRF1, but not TRF2, is known to bind and bend telomere sequences (19). This bending may be important for forming loop structures at the telomeric ends. We thus examined whether CEH-37 could bend DNA. Three probes were used in this assay: a 200-bp probe with six telomere sequence repeats at the left end (indicated as L in Fig. 4A); a probe identical to the left side except for an internal telomere sequence (indicated as M in Fig. 4A); and a third probe containing a telomere sequence at the right end (indicated as R in Fig. 4A). The mobility of the CEH-37-M complex is slower than that of the CEH-37-L or CEH-37-R complexes (Fig. 4B, left panel). This result clearly shows that CEH-37 bends the DNA when bound to telomere DNA sequences. Interestingly, the CEH-37 protein lacking the C-terminal domain could not bend DNA despite its ability to bind telomeric DNA (Fig. 4B, right panel), indicating that the C domain is important for the proper function of CEH-37. The fact that CEH-37 can bend telomere repeat-containing DNA raises the possibility that CEH-37 may have roles in establishing or maintaining a secondary structure of telomeres in vivo.
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CEH-37 Is Mainly Localized to Telomeres in VivoTo determine the subcellular localization of the CEH-37 protein, we monitored fluorescence signals produced by a fusion of CEH-37 to a GFP reporter protein. For this experiment, we used the pie-1 promoter to express the CEH-37 fusion protein so that we could visualize embryonic chromosomes (20). We introduced the reporter gene into the mut-7 background, in which the germline suppression of transgenes is not maintained (21). We observed that the CEH-37 GFP fluorescence was primarily co-localized to the ends of the chromosomes at least in the metaphase (Fig. 5). From these results, we propose that CEH-37 binds telomeric DNA both in vitro and in vivo.
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ceh-37, Together with mrt-2, Is Required for Chromosome StabilityTo define the biological functions of CEH-37, we examined the phenotypes of a deletion mutant that lacks the C-terminal region, which was shown to be essential for DNA bending (Table II). This deletion mutant may represent a reduction-of-function mutation in ceh-37 since CEH-37 lacking the C domain did not bend telomeric DNA, which might be crucial for its function. Mutant animals showed low, but significant, embryonic lethality (0.01 < p < 0.025). We then examined whether the deletion mutant produced more males. A high incidence of males is a typical indicator of chromosome instability since the males can arise by abnormal chromosome segregation. The mutant animals indeed produced more males than wild-type animals (0.05 < p < 0.1).
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We then examined the genetic relationships between ceh-37 and mrt-2, which may function at C. elegans telomeres, by the RNA interference technique. mrt-2 was originally identified by a mutation conferring sterility due to telomeric instability (22); the wild-type allele was found to encode the telomeric checkpoint protein. We found that the frequency of males in the ceh-37 deletion mutants subjected to mrt-2 RNA interference was significantly larger than the simple sum of the frequency of the males in the ceh-37 mutant alone and the wild-type animals subjected to mrt-2 RNA interference (p < 0.005), indicating that ceh-37 may interact synergistically with mrt-2 with respect to telomeric functions (Table II).
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DISCUSSION |
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Most telomere-binding proteins identified so far rely upon a Myb domain for binding activity, but C. elegans utilizes a different motif, the homeodomain for CEH-37. An extensive data base search for homologs of the human TRF1, TRF2, and Rap1 proteins and of S. pombe Taz1p failed to uncover such a protein in the nematode.2 The absence of TRF homologs and the presence of a homeobox-containing telomere-binding protein raises the possibility that telomere-binding proteins may have independently evolved in the nematode. The nematode telomere DNA repeats have a sequence content that differs from that of other organisms. The mammalian telomere DNA repeat motif is TTAGGG, the plant motif is TTTAGGG, and the yeast sequence is altogether different. In the evolution of telomere sequences, the properties of telomere-binding proteins may have undergone changes as well. There are a few possible explanations as to why we identified a different type of protein from Myb domain-containing protein as a telomere-binding protein. One is that the nematode independently evolved the telomere-binding protein, but the structural limitations imposed by telomeres restricted the range of functional proteins to those with helix-loop-helix binding domains. A solution of the three-dimensional structure of CEH-37 bound to the telomere sequences may be relevant to this idea. A second possibility is that there may be as yet unidentified homeodomain telomere-binding proteins in other organisms. Homeodomain proteins have long been implicated in developmental programming, but there are many homeodomain-containing proteins in the human genome data base whose functions are not well established, and it would be interesting to examine whether any of these proteins binds human telomeres. On the other hand, because we used only one cDNA library in screening for the proteins binding to the telomere repeats, it is unlikely that our screen was saturated. It is conceivable that there are other proteins awaiting identification that specifically bind the nematode telomere DNA. It is of interest to identify more telomere-binding proteins and their functions.
The mechanisms by which telomere-binding proteins act may have undergone evolutionary diversification as well. For example, yeast RAP1p directly binds telomere sequences, whereas the human RAP1 homolog is recruited by TRF2 (23). The CEH-37 action may provide another example. The Myb-like domains of some telomere-binding proteins can bind alone to telomeres, but the CEH-37 homeodomain (without the N-terminal domain) cannot. This observation suggests that Myb domain-containing proteins and the homeodomain proteins may differ with respect to how they bind telomeres. The Myb domain of the rice telomere-binding protein binds the telomere, and proteins containing this domain bind as dimers (11, 19), indicating that this protein may first bind telomeres and then dimerize, whereas CEH-37 may dimerize before telomere binding.
The functions of the single-stranded telomere-binding proteins have been conserved in evolution despite their apparent lack of high sequence homology. Pot1 was identified in S. pombe and in humans as a distant relative of the ciliate TEBPa protein (24). Recently, the structure of Cdc13 was shown to be similar to that of TEBPa (14). An emerging consensus is that single-stranded telomere-binding proteins may have different primary structures but that they have common structures, such as the OB fold, and common functions, such as telomere capping (25). This may also be true for double-stranded telomere-binding proteins. Mammalian TRF proteins and C. elegans CEH-37 have unrelated primary structures, but their tertiary structures and functions may be conserved.
It has been shown that a long stretch of mammalian double-stranded telomere DNA bends back on itself to form a large loop (t-loop) and that the 3' G-rich single-stranded overhang at the end of the t-loop invades an internal double-stranded telomeric region, producing a displacement loop (d-loop) (26, 27). These loops are proposed to mask telomere termini from cellular activities that can act on DNA ends. TRF2 has been shown to be critical in both loop formation and stabilization. In Oxytricha, Trypanosoma, and budding yeast, telomeres are reported to form loops (2830). However, it is not yet established whether these loops confer functional telomere protection. Instead, it appears that loops are limited to Oxytricha and Trypanosoma minichromosomes and that the telomeric loops in budding yeast are involved in the regulation of gene expression rather than telomere protection. Furthermore, the formation of telomeric loops has not been well established for other model species such as S. pombe and the nematode C. elegans. Our preliminary data showed that a minority of genomic DNA hybridized with telomere probe showed reduced mobility on a two-dimensional gel electrophoresis (data not shown), raising the possibility that the nematode telomeres may indeed form loops. It would be interesting to examine whether the nematode telomeres contain loops by more direct methods such as electron microscopy.
One critical question that awaits clearer answer is what the physiological functions of ceh-37 are in vivo. Our data suggest that ceh-37 may play important roles at the telomeres such as maintenance of chromosome stability. However, the deletion mutation that we examined in this study did not cause visible telomere-related phenotypes such as shortened telomere length or shortened life span (data not shown). One possibility is that the mutation we examined was a deletion mutation expected to result in a truncated protein with the N-term and the homeodomain intact, indicating that this mutation may not be a null mutation. It would be interesting to identify and examine a complete null mutation of ceh-37. There is another possibility in difficulties in interpreting the genetic data: the presence of redundancy among telomere-binding proteins. It would be important to identify other telomere-binding protein genes in C. elegans and examine their functions in conjunction with ceh-37. On the other hand, one cannot rule out the possibility that CEH-37 may have functions other than telomere binding in vivo. One example of the multifunctional telomere-binding protein is RAP1p. RAP1p in yeast was originally identified as a protein that binds both silencer and activator sequences (31), and later, it was reported to bind telomeres (13). Thus RAP1p in yeast is a multifunctional protein both in gene regulation and in telomere function. We found that CEH-37 was primarily co-localized to the ends of chromosomes, but we also noticed very faint fluorescence throughout the chromosomes, raising the possibility that this faint GFP signal might represent a biologically meaningful function of ceh-37.
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FOOTNOTES |
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Both authors contributed equally to this work.
|| To whom correspondence should be addressed. Tel.: 82-2-2123-2663; Fax: 82-2-312-5657; E-mail: Leej{at}yonsei.ac.kr.
1 The abbreviations used are: N-term, N-terminal; C-term, C-terminal; GFP,
green fluorescent protein; ORF, open reading frame; h, human.
2 S. H. Kim, S. B. Hwang, I. K. Chung, and J. Lee, unpublished
observation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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