Sequence-specific Binding to Telomeric DNA by CEH-37, a Homeodomain Protein in the Nematode Caenorhabditis elegans*

Seung Hyun Kim {ddagger} §, Soon Baek Hwang {ddagger} §, In Kwon Chung ¶ and Junho Lee {ddagger} ¶ ||

From the {ddagger}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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Caenorhabditis elegans can serve as a model system to study telomere functions due to its similarity to higher organisms in telomere structures. We report here the identification of the nematode homeodomain protein CEH-37 as a telomere-binding protein using a yeast one-hybrid screen. The predicted three-dimensional model of the homeodomain of CEH-37, which has a typical helix-loop-helix structure, was similar to that of the Myb domain of known telomere-binding proteins, which is also a helix-loop-helix protein, despite little amino acid sequence similarity. We demonstrated the specific binding of CEH-37 to the nematode telomere sequences in vitro by competition assays. We determined that CEH-37 binding required at least 1.5 repeats of TTAGGC and that the core sequence for binding was GGCTTA. We found that CEH-37 had an ability to bend telomere sequence-containing DNA, which is the case for other known telomere-binding proteins such as TRF1 and RAP1, indicating that CEH-37 may be involved in establishing or maintaining a secondary structure of the telomeres in vivo. We also demonstrated that CEH-37 was primarily co-localized to the chromosome ends in vivo, indicating that CEH-37 may play roles in telomere functions. Consistent with this, a ceh-37 mutation resulting in a truncated protein caused a weak high incidence of male phenotype, which may have been caused by chromosome instability. The identification of CEH-37 as a telomere-binding protein may represent an evolutionary conservation of telomere-binding proteins in terms of tertiary protein structure rather than primary amino acid sequence.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Telomeres, the specialized nucleoprotein complexes at the ends of linear eukaryotic chromosomes, are essential for the maintenance of chromosome integrity since they provide protection from exonucleolytic degradation and prevent fusion between chromosome ends (14). Telomeres in most eukaryotes are composed of tandem repeats of short sequence elements, typically 5–8 bp in length. The strand of the telomere that contains the 3' terminus is usually rich in G residues, and it extends beyond the complementary C-rich strand to terminate as a single-stranded 3' overhang in several evolutionarily divergent organisms. The integrity and proper functions of telomeres rely upon associations between the telomeric repeats and specific binding proteins. Among such proteins, a group of proteins specifically bind to the double-stranded repeats. In mammalian cells, two homologous double-stranded telomere-binding proteins, TRF1 and TRF2, have been identified (58). The Schizosaccharomyces pombe Taz1 protein is proposed to be a functional homolog of the human TRF proteins (9, 10). In plants, Myb domain-containing proteins have been identified as telomere-binding proteins (11, 12). The Saccharomyces cerevisiae Rap1 protein is also a double-stranded telomere-binding protein (13). Rap1p contains two Myb-like domains, and TRF1, TRF2, and Taz1p each contain a single Myb-like domain at the C terminus. It is worth noting that proteins recently added to the list of telomere-binding proteins were initially identified as having Myb-like domains (for example, 11,12).

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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Yeast One-hybrid Screening—For one-hybrid screening, we used (TTAGGC)26 repeats placed upstream of the HIS3 reporter gene in pAS2-KSH as a bait and a random-primed C. elegans cDNA library (provided by R. Barstead) as the source of prey. Positive clones were selected on SD/–His/–Trp/–Leu medium containing 33 mM 3-aminotriazole.

Gel Shift Assay—We 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 5–8% 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 Derivatives—To 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 (1st–37th amino acids of CEH-37), the homeodomain (38th–94th amino acids), and the C-term (C-terminal region) domain (95th–278th 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 Assay—We 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 Assay—We 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 {gamma}-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 Vivo—To 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 Interference—Full-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. 100–200 µg/ml double-stranded RNA was injected into wild-type or mutant L4 stage animals.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Identification of CEH-37 as the Nematode Telomere-binding Protein—We first cloned C. elegans telomeric repeat sequences by amplifying genomic DNA with telomere sequences as primers. We obtained clones containing 6, 26, 60, and 120 repeats of the sequence TTAGGC. We constructed a bait plasmid that contains 26 telomere repeats and screened 3.6 x 106 yeast colonies after transformation of a prey library. We identified two positive clones from this screen, which had an identical 1.3-kb cDNA insert that encodes a previously predicted protein gene ceh-37. ceh-37 had been identified by its sequence homology to homeobox proteins (16). The CEH-37 protein had highest homology to the cone rod homeobox protein (CRX) and the OTX2 protein (Fig. 1, A and B). However, the homology between CEH-37 and these homeobox proteins did not extend outside the homeobox region. Specifically, although 42 of the 60 amino acid residues of the homeodomain in CEH-37 are identical to those of CRX, the glutamine-rich domain, the basic domain, and the tail domain, all of which are conserved in OTX family proteins, are not conserved in CEH-37. Furthermore, sequences conserved among OTX proteins, SIWSPASESP, SYFSG, LSPM, and LDYKDQ, are not conserved in CEH-37. This discrepancy in the primary structures of CEH-37 and other OTX proteins indicates that the functions of the remaining region of CEH-37 differ from those of the OTX proteins. Comparison of CEH-37 with known telomere-binding proteins TRF1, TRF2, and Taz1p showed no apparent homology from the standpoint of amino acid sequences. Although the known telomere-binding proteins have an N-terminal dimerization domain consisting of nine {alpha} helices (17), CEH-37 apparently lacks this typical dimerization domain. Furthermore, the TRF proteins have a Myb domain at their C-terminal region as the DNA binding domain, whereas CEH-37 has a homeodomain in its N-terminal region.



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FIG. 1.
Identification of CEH-37 as a telomere-binding protein. A, genomic structure of ceh-37. The ceh-37 gene is composed of five exons. The region deleted in ceh-37(ok272) is indicated. The coding region for the homeodomain is marked as shaded boxes, separated by an intron. B, sequence alignment of CEH-37 with its homologs. The accession numbers of the GenBankTM sequences are CEH-37 (Q93356 [GenBank] ); CEH-36 (Q93352 [GenBank] ); human CRX (O43186 [GenBank] ); and human OTX2 (AAG16243 [GenBank] ). The homeodomain is underlined. CRX-specific amino acids, which are not conserved in CEH-37, are marked by line boxes. As shown in C, a three-dimensional model of CEH-37 homeodomain was similar to the solved structure of hTRF1 DNA binding domain. The upper left panel shows a three-dimensional model of CEH-37 homeodomain generated by SWISS-MODEL using Engrailed as a structural modeling template. The predicted structure of CEH-37 homeodomain was superimposed using the "magic fit" command of the SPVDB software. The upper right panel shows the solved structure of the hTRF1 Myb domain. The lower left panel shows a predicted tertiary structure of Taz1p, an S. pombe telomere-binding protein. For modeling Taz1p, hTRF1 was used as a template, and the third helix of Taz1p does not appear in the predicted structure, either due to a lack of primary sequence homology or due to a lack of structural conservation. The three-dimensional model of the CEH-37 homeodomain is virtually indistinguishable from that of the hTRF1 Myb domain. The lower right panel shows the merged image of CEH-37, hTRF1, and Taz1p structures.

 

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 Vitro—To 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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FIG. 2.
CEH-37 binds specifically to the C. elegans telomere in vitro. As shown in A, gel shift assays were performed using the in vitro translated CEH-37 and a radiolabeled C. elegans telomere probe with the cold C. elegans telomere sequence (lanes 5–7), the cold human telomere sequence (lanes 8–10), and the cold rice telomere sequences as competitors (lanes 11–13). Although the nematode telomere efficiently competed with CEH-37 binding, neither the human nor the rice telomere sequences could compete with CEH-37. Lane 1, a control with the rabbit reticulocyte extract alone; lanes 2–4, with 5, 15, and 25 pmol of CEH-37 translates, respectively. All other experiments were with 5 µl of CEH-37 translates. As shown in B, CEH-37 does not bind to CRX recognition sequences. Gel shift assays were performed using in vitro translated CEH-37 and a radiolabeled C. elegans telomere probe with the cold CRX recognition sequence as competitors. The consensus CRX binding site is (T/C)TAAT(C/T). The sequences of the competitors were: A, TTAATC (lane 4); B, CTAATC (lane 5); C, TTAATT (lane 6); and D, CTAATT (lane 7). None of the sequences were competitive for CEH-37 binding. Lane 1, no CEH-37 translate; lane 2, no competitor; lane 3, the nematode telomere sequence as the competitor. As shown in C, CEH-37 requires at least 1.5 repeats of TTAGGC for its binding. Gel shift assays were performed using radiolabeled probes consisting of different copy numbers of TTAGGC. The numbers of repeats in the probes are indicated. (–) and (+) indicate the absence or presence of CEH-37 in each reaction. As shown in D, GGCTTA is the core sequence of CEH-37 binding. Lanes 1 and 2 are with wild-type probes. Lane 1, without CEH-37 translate; lane 2, with CEH-37 translate. Lanes 3–14 contained probes with single nucleotide mutations in each position of TTAGGC repeats. The single nucleotide mutations were introduced such that A was changed to C, G was changed to T, C was changed to A, and T was changed to G. Any one nucleotide change in the 4th G to the 9th A position abolished CEH-37 binding.

 

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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FIG. 3.
Domain studies of CEH-37. As shown in A, the N-term and homeodomain are sufficient for telomere binding. Although the homeodomain alone or the N-term domain alone did not bind the telomere, the protein lacking the C-terminal domain bound the telomere as efficiently as the full-length CEH-37 (lanes 4 and 5). As shown in B, the N-term domain competes with the full-length CEH-37 for telomere binding. Lane 1, without the N-terminal competitor and the full-length CEH-37; lane 2, N-terminal domain alone; lane 3, the full-length CEH-37 alone; and lanes 4–6, 1: 1, 1: 4, and 1: 8 ratio of full-length to N-terminal domain. For lanes 4–6, the templates for both the full-length and the N-terminal domain were co-translated in single reactions. Even at 1:1 competition, the N-terminal domain almost completely blocked the CEH-37 binding. We ensured that the translation the full-length CEH-37 was not affected by adding DNA encoding competitors by S35-labeled translation reaction (data not shown).

 

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TABLE I
Yeast one-hybrid assay with derivatives of CEH-37 for telomere binding in vivo

 

CEH-37 Bends DNA by Binding to Telomeric DNA in Vitro—A 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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FIG. 4.
CEH-37 can bend DNA. A, schematics showing the probes used in the experiment. In B, the left panel shows a result of a gel shift assay using the full-length CEH-37 and the radiolabeled L, M, or R probes (lanes 4–6, respectively). The mobility of the CEH-37-M complex is slower than the other two DNA-protein complexes. Lanes 1–3 are without the CEH-37 translates. The right panel shows a result of a gel shift assay using the C-term truncated CEH-37 (CEH-37{Delta}C) and the radiolabeled L, M, or R probes (lanes 4–6, respectively). The truncated protein bound the probes (the arrow) but could not bend it. Lanes 1–3 are without the CEH-37 translates.

 

CEH-37 Is Mainly Localized to Telomeres in Vivo—To 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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FIG. 5.
CEH-37 is mainly localized to the telomere in vivo. CEH-37 was visualized as a GFP full-length CEH-37 fusion protein expressed by the pie-1 promoter. This figure shows CEH-37 localization in the nuclei of a two-cell embryo. A, the 4,6-diamidino-2-phenylindole images. B, GFP fluorescence images. C, the merged images. Images of higher magnification are shown in the right panels.

 

ceh-37, Together with mrt-2, Is Required for Chromosome Stability—To 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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TABLE II
Genetic interaction of ceh-37 with mrt-2

The results are from three independent experiments.

 

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).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this report, we identified a novel homeodomain-containing protein, CEH-37, as a telomere-binding protein in the nematode C. elegans. Although the closest mammalian homolog of CEH-37 is CRX, a transcription factor that acts in photoreceptors, our results strongly suggest that CEH-37 has different functions. First, the CEH-37 primary structure diverges extensively outside the conserved homeodomain, indicating that although the homeodomain is a DNA binding domain, the remainder of the protein may have functions distinct from those of CRX proteins. Second, three-dimensional modeling of the CEH-37 homeodomain shows that it is structurally almost identical to that of TRF1, although there is no sequence homology between CEH-37 and TRF1. Third, the consensus recognition sequence for the CRX proteins cannot compete with the nematode telomere sequences in CEH-37 binding. Lastly, CEH-37 is primarily localized to the telomeres in vivo. Although CEH-37 shows no similarity to known telomere-binding proteins in terms of amino acid sequence, it shares several features with other telomere-binding proteins. Functionally, CEH-37 binds telomere DNA in vitro and in vivo. Our results suggest that CEH-37 may bind telomeres as dimers and that the dimerization domain may be in its N-terminal region, as other TRF proteins do. The roles of the domains within CEH-37 were suggested by our analyses: the N-term for dimerization, the homeodomain for DNA binding, and the C-term for DNA bending. Further studies would be necessary to directly prove the suggested roles of each domain, such as two-hybrid assays, co-immunoprecipitation, and the examination of the sufficiency of the C-term domain to bend DNA in other contexts. CEH-37 can bend DNA, as some telomere-binding proteins such as TRF1 and Rap1 do. The biochemical properties of CEH-37 as reported in this study indicate that CEH-37 may play roles at the telomeres in vivo.

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.


    FOOTNOTES
 
* This work was supported in part by the Molecular Aging Research Center grant and by a National Research Laboratory Grant (Ministry of Science and Technology (Korea)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

|| 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. Back

2 S. H. Kim, S. B. Hwang, I. K. Chung, and J. Lee, unpublished observation. Back


    ACKNOWLEDGMENTS
 
We thank Drs. R. Barstead for the cDNA library, A. Coulson for the cosmid, A. Fire, and G. Seydoux for the vectors. We also thank the C. elegans knockout consortium at Oklahoma for the ceh-37 deletion mutant strain, and the C. elegans Genetics Center (Minneapolis, MN) for other strains.



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