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Published as doi: 10.1096/fj.09-129619.
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(The FASEB Journal. 2009;23:2587-2594.)
© 2009 FASEB

Developmental studies of Xenopus shelterin complexes: the message to reset telomere length is already present in the egg

Dzeneta Vizlin-Hodzic, Jessica Ryme, Stina Simonsson and Tomas Simonsson1

Department of Biomedicine, Medical Biochemistry and Cell Biology Division, University of Gothenburg, Gothenburg, Sweden

1 Correspondence: University of Gothenburg, Department of Biomedicine, Medical Biochemistry and Cell Biology Division, P.O. Box 440, SE 405 30 Gothenburg, Sweden. E-mail: tomas.simonsson{at}gu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The 6-protein complex shelterin protects the telomeres of human chromosomes. The recent discovery that telomeres are important for epigenetic gene regulation and vertebrate embryonic development calls for the establishment of model organisms to study shelterin and telomere function under normal developmental conditions. Here, we report the sequences of the shelterin-encoding genes in Xenopus laevis and its close relation Xenopus tropicalis. In vitro expression and biochemical characterization of the Xenopus shelterin proteins TRF1, TRF2, POT1, TIN2, RAP1, TPP1, and the shelterin accessory factor PINX1 indicate that all main functions of their human orthologs are conserved in Xenopus. The XlTRF1 and XtTRF1 proteins bind double-stranded telomeric DNA sequence specifically and interact with XlTIN2 and XtTIN2, respectively. Similarly, the XlTRF2 and XtTRF2 proteins bind double-stranded telomeric DNA and interact with XlRAP1 and XtRAP1, respectively, whereas the XlPOT1 and XtPOT1 proteins bind single-stranded telomeric DNA. Real-time PCR further reveals the gene expression profiles for telomerase and the shelterin genes during embryogenesis. Notably, the composition of shelterin and the formation of its subcomplexes appear to be temporally regulated during embryonic development. Moreover, unexpectedly high telomerase and shelterin gene expression during early embryogenesis may reflect a telomere length-resetting mechanism, similar to that reported for induced pluripotent stem cells and for animals cloned through somatic nuclear transfer.—Vizlin-Hodzic, D., Ryme, J., Simonsson, S., Simonsson, T. Developmental studies of Xenopus shelterin complexes: the message to reset telomere length is already present in the egg.


Key Words: telosome • differentiation • ES cell • iPS cell • quadruplex • cellular senescence


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TELOMERES ARE THE DYNAMIC DNA-protein complexes that make up the natural ends of eukaryotic chromosomes. Early cytological and genetic studies revealed that telomeres are necessary for chromosome stability and genome maintenance (1 , 2) . It was demonstrated that broken chromosomes fuse end-to-end and become dicentric or ring-formed, or adopt other unstable forms that cause genomic instability. The instability caused by broken chromosome ends contrasted the stability of natural chromosome ends and suggested that telomeres are essential structures that make the natural ends of eukaryotic chromosomes unique. Without telomeres, eukaryotic chromosomes are unstable and suffer chromosomal rearrangements. In addition to their now established roles in cellular aging, stem cell biology, and cancer (3 4 5 6) , telomeres have more recently been found to have specific functions in epigenetic gene regulation and vertebrate embryonic development (7 8 9) .

Telomeric DNA consists of extended arrays of tandem repeats with common endings. One strand is rich in guanines and thymines and forms a single-stranded 3' overhang (10) . Consequently, the complementary strand is rich in cytosines and adenosines and has a recessed 5' end. Telomeric repeat sequences are well conserved through evolution. All vertebrates, including Xenopus laevis and Xenopus tropicalis, share an identical TTAGGG hexanucleotide telomeric repeat sequence (11) . Because conventional replication of linear chromosomes fails to make a complete copy of the lagging strand due to discontinuous DNA synthesis and the requirement for RNA priming (12) , telomeric DNA gradually shortens in differentiated cells (10) . It has been suggested that this provides a molecular clock that tells cellular age and that telomere shortening serves as a tumor suppressor mechanism that prevents accumulation of mutations (13 , 14) . When its shortest telomere eventually becomes critically short (15) , a differentiated cell enters an irreversible state of arrested growth known as replicative senescence (16) . In contrast, the ribonucleoprotein complex telomerase makes up for loss of telomeric DNA in stem cells and germ line cells. Telomerase is a unique homodimeric reverse transcriptase that adds back telomeric DNA repeats by iterated reverse transcription of its internal RNA template (17 18 19 20) . Albeit telomere extension by telomerase depends on the conformation of the telomeric DNA (21 , 22) , which is modulated by shelterin (23) , a 6-protein complex consisting of telomeric DNA-binding proteins and their interacting partners: The structurally related telomeric repeat-binding factors 1 (TRF1) (24) and 2 (TRF2) (25 , 26) bind double-stranded telomeric DNA and interact with TRF1 interacting nuclear factor 2 (TIN2) (27 , 28) and repressor activator protein 1 (RAP1) (29) , respectively, whereas protection of telomeres 1 (POT1) (30 31 32) binds telomeric single-stranded 3' overhangs and interacts with POT1 interacting protein 1 (TPP1; a.k.a. PTOP, PIP1, TINT1, ACD) to control synthesis of telomeric DNA by telomerase (33 34 35 36 37) .

The African clawed frog X. laevis and its close relation X. tropicalis are frequently employed model organisms to explore vertebrate embryonic development and cellular fate. However, studies aiming to investigate roles of telomeres in epigenetic gene regulation and vertebrate embryonic development have been hampered by lack of knowledge about the Xenopus shelterin complex. Only TRF1 and TRF2 orthologs have been identified in X. laevis to date (38 39 40) . Here, we identify and characterize the remaining four shelterin components in X. laevis, as well as the entire X. tropicalis shelterin complex. Moreover, shelterin gene expression has never been previously studied during the development of a multicellular organism. Here, we determine the shelterin gene expression profiles during embryogenesis in Xenopus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sequence analysis
Possible orthologs of human shelterin components were identified from X. laevis and X. tropicalis cDNA libraries. Full-length cDNA sequences were amplified by standard PCR using Vent polymerase (New England Biolabs, Beverly, MA, USA) and were subjected to DNA sequencing (Geneservice). Each full-length shelterin cDNA was subject to a minimum of three complete independent sequence reads, and ClustalW sequence alignments were made using the BLOSUM62 substitution scoring matrix with "open gap" and "extend gap" penalties of 10 and 0.5, respectively.

Xenopus eggs and embryos
X. laevis eggs were obtained by injecting adult females with human chorionic gonadotropin (Sigma, St. Louis, MO, USA). Embryos were obtained by in vitro fertilization and manually staged under the microscope according to Nieuwkoop and Faber (41) .

Cloning
PCR products containing full-length cDNA sequences were cloned into a modified Pet30a vector (Novagen) fitted with an N-terminal 6xHis-tag/S-tag followed by a tobacco etch virus (TEV) protease cleavage site.

Protein expression and purification
Constructed plasmids were transformed into Escherichia coli Rosetta cells (Novagen, Madison, WI, USA), and protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM when the cell cultures reached an optical density ({lambda}=600 nm) of ~1. Cells were harvested by centrifugation (5000 g for 15 min), and pellets were resuspended in lysis buffer (50 mM Tris/HCl, pH 8.0;, 500 mM KCl; 1% Triton X-100; 2 mM DTT) containing protease inhibitors (Complete EDTA free protease inhibitor cocktail; Roche Diagnostics, Mannheim, Germany). Cells were disrupted by sonication, and following centrifugation (25,000 g for 30 min), the supernatant was incubated with lysis buffer-equilibrated Ni-NTA resin (Qiagen, Valencia, CA, USA) for 60 min. After centrifugation (200 g for 3 min), the Ni-NTA resin was washed with wash buffer (50 mM Tris/HCl, 500 mM KCl, 10 mM imidazole, 2 mM DTT, 10% glycerol), and the protein was eluted with wash buffer containing 200 mM imidazole and dialyzed into digestion buffer (50 mM Tris/HCl, pH 8.0; 500 mM KCl; 2 mM DTT; 10% glycerol). TEV proteolytic digestion was performed overnight with TEV protease at ~1:100 to eluted protein. The TEV protease was itself 6xHis-tagged, which allowed the cleaved protein to be purified from TEV protease and cleaved N-terminal 6xHis-tag/S-tag by binding to Ni-NTA resin equilibrated with digestion buffer. The proteins were purified to homogeneity by gel filtration on a Superdex 200 column equilibrated with digestion buffer, and dialyzed into binding buffer (50 mM Tris/HCl, pH 8.0; 125 mM KCl; 5 mM DTT; 10% glycerol).

Electrophoretic mobility shift assays (EMSAs)
A synthetic telomeric single-stranded undecamer (5'-GTTAGGGTTAG-3'; MWG Biotech, Ebersberg, Germany) was purified by denaturing polyacrylamide gel electrophoresis and used for POT1 EMSAs. The pTH3 plasmid was digested by HindIII and KpnI (New England Biolabs) to produce a double-stranded fragment containing 3.5 telomeric TTAGGG repeats, which was purified by standard native agarose gel electrophoresis and used for TRF1 and TRF2 EMSAs. Telomeric DNA probes were radiolabeled using 5'-[{gamma}-32P]-triphosphate (Amersham) and T4 polynucleotide kinase (New England Biolabs). Unincorporated 5'-[{gamma}-32P]-triphosphate was removed on Bio-Spin P10 columns (Bio-Rad). Radiolabeled telomeric probes were incubated with protein in binding buffer (50 mM Tris/HCl, pH 8.0; 125 mM KCl; 5 mM DTT; 10% glycerol) containing 100 µg/ml bovine serum albumin (New England Biolabs) and 10 µg/ml sheared E. coli DNA (Sigma) for 30 min at 4°C. Reaction mixtures were analyzed with native agarose (BioGene HiPure Low EEO agarose; BioGene, Huntingdon, UK) gel electrophoresis (0.5% agarose, 0.25xTB) at 7.5 V/cm for 60 min at +4°C. Gels were dried onto DE81 anion exchange chromatography paper (Whatman, Clifton, NJ, USA) and scanned using a Typhoon 8600 imaging system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Gene expression analysis
Total RNA samples were extracted from X. laevis developmental stages 8, 8–9, 9+, 9, 10, 10.25, 10.75, 11.25, and >11.25 (5 embryos/stage) using the RNeasy kit (Qiagen), including digestion of contaminating genomic DNA by RNase-Free DNase kit (Qiagen). cDNA was synthesized using random primers and Superscript III RNase H- reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Endogenous mRNA levels were measured by real-time PCR (RT-PCR) analysis based on SYBR Green detection. Briefly, the real-time PCR mixture contained 1 µl of cDNA in total volume of 20 µl, containing 1x SYBR Green mix reagent (Applied Biosystems, Foster City, CA, USA), 50 nM forward primer and 50 nM reverse primer using the primers listed in Table 1 . Each primer pair gave a single product, as confirmed by dissociation curve analysis and electrophoresis. Standard curves were generated for each gene by serial dilution of cDNA. Unknown quantities were calculated by comparison to the standard curve for each gene. Experiments were repeated a minimum of 3 times on different embryo batches to ensure reproducibility.


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  		Table 1. Primers used for real-time PCR


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TRF1 sequence analysis
The 420-aa X. laevis TRF1 (xlTRF1), with a calculated molecular mass of 49.3 kDa, binds double-stranded telomeric DNA and was the first shelterin component to be identified in a multicellular organism (38) . Just like the xlTRF1, the 421-aa X. tropicalis TRF1 (xtTRF1), with an almost identical calculated molecular mass of 49.3 kDa, includes most known features of the xlTRF1 and the 439-aa human TRF1 (hTRF1). The TRF1 family of proteins shares a common architecture with two separate structural domains. An N-terminal domain is necessary and sufficient for the formation of homodimers, and a C-terminal MYB/homeodomain mediates sequence-specific recognition of double-stranded telomeric DNA. A clustalW alignment of hTRF1 and its Xenopus orthologs reveals that the amino acid sequences of hTRF1 and xtTRF1 are 81% similar, with 32% identity (Supplemental Fig. 1). In comparison, the amino acid sequences of hTRF1 and xlTRF1 are 85% similar, with 34% identity. The xlTRF1 and xtTRF1 are 82.5% identical. The acidic hTRF1 N terminus, which is lacking in xlTRF1 (39) , is missing also in xtTRF1 (Supplemental Fig. 1).

TRF2 sequence analysis
The 500-aa human TRF2 (hTRF2), with a calculated molecular mass of 55.6 kDa, binds double-stranded DNA and was the second shelterin component to be identified in humans (25 , 26) . An X. laevis ortholog to hTRF2 was recently reported (40) . The 468-aa X. laevis TRF2 (xlTRF2) and the 472-aa X. tropicalis TRF2 (xtTRF2) identified here, with calculated molecular masses of 54.2 and 54.7 kDa, respectively, share several known features of the hTRF2. The TRF2 family of proteins has the same overall structure as the TRF1 family of proteins. An N-terminal homodimerization domain consists of 9 {alpha}-helices and a 3-helix bundle makes up the C-terminal DNA-binding MYB/homeodomain. A clustalW alignment of hTRF2 and its Xenopus orthologs (Supplemental Fig. 2) reveals that the amino acid sequence of hTRF2 shares 90% similarity and 36% identity with xlTRF2, and 88% similarity and 36% identity with xtTRF2. The xlTRF2 and xtTRF2 are 80.5% identical. Both Xenopus TRF2 orthologs lacks the basic hTRF2 N terminus.

POT1 sequence analysis
The 634-aa human POT1 (hPOT1), with a calculated molecular mass of 71.4 kDa was the third DNA-binding shelterin component to be identified in humans (30) . The hPOT1 is made up of three oligosaccharide/oligonucleotide binding (OB) folds (21) . The structure of the two N-terminal hPOT1 OB-folds, which mediate sequence specific binding of single-stranded telomeric 3' overhangs, was recently reported (35) . The C-terminal hPOT1 OB-fold is likely responsible for protein-protein interactions. Xenopus POT1 orthologs have not been reported until now. The two 621-aa X. laevis POT1 (xlPOT1) and X. tropicalis POT1 (xtPOT1) have calculated molecular masses of 70.5 and 70.9 kDa, respectively. A clustalW alignment of hPOT1 and the Xenopus POT1 orthologs reveals that the amino acid sequence of hPOT1 shares 67% similarity and 50% identity with xlPOT1, as well as xtPOT1 (Supplemental Fig. 3). The xlPOT1 and xtPOT1 are 84% identical. The only noticeable difference between the hPOT1 and its Xenopus orthologs is that the peptide linker, which connects the two N-terminal DNA-binding OB-folds to a putative C-terminal protein-binding OB-fold, is slightly longer in hPOT1.

TIN2 sequence analysis
The 451-aa human TIN2 (hTIN2), with a calculated molecular mass of 50.0 kDa, is a shelterin component that does not interact directly with telomeric DNA. It was initially discovered as a hTRF1 partner (28) and later found to also interact with TRF2 (42 , 43) and TPP1 (34) . The crystal structure of the hTRF1 binding domain of hTIN2 (aa. 256–276) in complex with the TRF1 homodimerization domain was recently reported (27) . It reveals how amino acids 257–268 of hTIN2 forms an extensive network of intermolecular hydrogen bonding, ion pairing, and hydrophobic interactions primarily with {alpha}-helices 2 and 3 of the TRF1 homodimerization domain. Xenopus TIN2 orthologs have not been reported so far. The 364-aa X. laevis TIN2 (xlTIN2) has a calculated molecular mass of 41.2 kDa, and the 362-aa X. tropicalis TIN2 (xtTIN2) has a calculated molecular mass of 41.0 kDa. A clustalW alignment of hTIN2 and the identified Xenopus orthologs shows that the amino acid sequence of hTIN2 shares 66% similarity and 26% identity with xlTIN2, and 65% similarity and 25% identity with xtTIN2 (Supplemental Fig. 4). The xlTIN2 and xtTIN2 are 96% identical. Just like hTIN2 the Xenopus orthologs lack conserved domains, but notably, the hTRF1 (aa 256–276) and hTRF2 (aa 1–220) binding domains of hTIN2 correspond to highly conserved regions in Xenopus TIN2.

RAP1 sequence analysis
The 399-aa human RAP1 (hRAP1), with a calculated molecular mass of 44.3 kDa, is another shelterin component that does not directly interact with telomeric DNA, at least not on its own. The hRAP1 was originally discovered as a hTRF2 partner (29) , and the solution structure of the hRAP1 MYB motif was reported shortly thereafter (44) . Xenopus hRAP1 orthologs have not been characterized to date. The 513-aa X. laevis RAP1 (xlRAP1) has a calculated molecular mass of 57.1 kDa, and the 509-aa X. tropicalis RAP1 (xtRAP1) has a calculated molecular mass of 56.4 kDa. A clustalW alignment of hRAP1 and the identified Xenopus orthologs show that the amino acid sequence of hRAP1 shares 77% similarity and 32% identity with xlRAP1, and 79% similarity and 33% identity with xtRAP1 (Supplemental Fig. 5). The xlRAP1 and xtRAP1 are 72% identical. Similarly to hRAP1, the Xenopus orthologs contain a putative MYB domain (aa 189–243 in X. laevis and aa 189–241 in X. tropicalis), as well as a conserved C-terminal TRF2 interacting domain (aa 402–513 in X. laevis and aa 398–509 in X. tropicalis).

TPP1 sequence analysis
The 544-aa human TPP1 (hTPP1), with a calculated molecular mass of 57.7 kDa, was the sixth and last core component of shelterin to be identified (34 , 37) and requires hPOT1 to interact with single-stranded telomeric 3' overhangs (35 , 36) through its OB-fold (aa 98–242). The 705-aa X. laevis TPP1 (xlTPP1) has a calculated molecular mass of 78.6 kDa and the 708-aa X. tropicalis TPP1 (xtTPP1) has a calculated molecular mass of 78.9 kDa. A clustalW alignment of hTPP1 and the identified Xenopus orthologs shows that the amino acid sequence of hTPP1 shares 50% similarity and 19% identity with xlTPP1, and 51% similarity and 20% identity with xtTPP1 (Supplemental Fig. 6). The xlTPP1 and xtTPP1 are 68% identical. Notably, the 87-aa N terminus of hTPP1, which appears to be functionally dispensable in human cells, is missing in the identified Xenopus orthologs, as well as in other species (34 , 37 , 45) . Moreover, the OB-fold and the POT interacting region (aa 244–337 in hTPP1) are both highly conserved in xlTPP1 and xtTPP1 (Supplemental Fig. 6).

PINX1 sequence analysis
The 328-aa shelterin accessory factor human PINX1 (hPINX1), with a calculated molecular mass of 37.0 kDa, was initially reported to be a potent inhibitor of telomerase and a TRF1 interaction partner (46) . The 353-aa X. laevis PINX1 (xlPINX1) has a calculated molecular mass of 40.3 kDa, and the 337-aa X. tropicalis PINX1 (xtPINX1) has a calculated molecular mass of 38.6 kDa. A clustalW alignment of hPINX1 and the identified Xenopus orthologs show that the amino acid sequence of hPINX1 shares 78.4% similarity and 48.5% identity with xlPINX1, and 79.0% similarity and 50.3% identity with xtPINX1 (Supplemental Fig. 7). The xlPINX1 and xtPINX1 are 77.4% identical.

EMSAs
EMSAs under native conditions confirm that full-length Xenopus TRF1 (Fig. 1 ), just like hTRF1, binds double-stranded telomeric DNA with high affinity and specificity in vitro. In addition, they reveal that Xenopus TRF1 remains bound to telomeric DNA, while simultaneously forming a complex with Xenopus TIN2 (Fig. 1) . Xenopus TIN2 exhibits no detectable affinity for telomeric DNA on its own (Supplemental Fig. 8).


Figure 1
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  		Figure 1. X. laevis TRF1 protein binds double-stranded telomeric DNA sequence specifically on its own (right lane). A stepwise titration of telomeric DNA bound by X. laevis TRF1 with X. laevis TIN2 in 2-fold dilution steps results in a slower-migrating band corresponding to a TRF1/TIN2 complex bound to telomeric DNA.

Full-length Xenopus TRF2 binds double-stranded telomeric DNA as a high molecular weight aggregate in vitro (Fig. 2A ). Similar observations have been reported for hTRF2. The high molecular weight complex formed by TRF2 in the presence of telomeric DNA (Supplemental Fig. 8) is disrupted on addition of Xenopus RAP1 (Fig. 2A ). Together TRF2 and RAP1 form a distinct complex, which binds double-stranded telomeric DNA sequence specifically (Fig. 2A ). Moreover, the Xenopus RAP1 protein exhibits no detectable affinity for telomeric DNA on its own, despite the presence of a conserved MYB-domain (Fig. 2B ). Notably, the human RAP1 protein also does not bind telomeric DNA on its own (29) . This observation has been attributed to a lack of positive surface charge typical of DNA-binding domains (44) .


Figure 2
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  		Figure 2. A) X. laevis TRF2-RAP1 complex binds double-stranded telomeric DNA sequence specifically. A stepwise titration of telomeric DNA bound by X. laevis TRF2 with X. laevis RAP1 in 2-fold dilution steps results in a band corresponding to a TRF2-RAP1 complex bound to double-stranded telomeric DNA. Note that telomeric DNA bound by X. laevis TRF2 alone forms higher-order aggregates, which do not enter the gel, and that these higher-order aggregates are dissolved with increasing RAP1 concentrations. Addition of RAP1 is necessary to form a complex of defined, albeit unknown, stoichiometry. The same phenomenon has been reported for the human TRF2-RAP1 complex (29) . B) Just like human RAP1 (29) , X. laevis RAP1 does not bind telomeric DNA on its own. A very weak band, likely representing unspecific binding, can be detected close to the well only at the highest 100 nM RAP1 concentration.

Finally, EMSAs under native conditions reveal that recombinant Xenopus POT1, just like recombinant human POT1 (30 31 32) , binds single-stranded telomeric DNA with high affinity and specificity in vitro (Fig. 3 ). Taken together, the DNA binding studies indicate that the X. laevis and X. tropicalis shelterin complexes interact with telomeric DNA in the same manner as the human shelterin complex interacts with telomeric DNA.


Figure 3
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  		Figure 3. X. laevis POT1 binds single-stranded telomeric DNA sequence specifically. The undecamer GTTAGGGTTAG corresponds to the exact sequence permutation generated by telomerase if the entire RNA template region is used for synthesis of telomeric DNA and may form 4-stranded G-quadruplex DNA structures based on the guanine tetrad base-pairing scheme (58) .

Gene expression analysis
To determine the shelterin gene expression profiles during Xenopus embryogenesis, we quantified mRNA levels for developmental stages 8, 8.5, 9+, 10, 10.25, 10.75, 11.25, and > 11.25 by RT-PCR (Fig. 4 ). All shelterin components, as well as the shelterin accessory factor PINX1 and telomerase, undergo major gene expression changes during embryogenesis. The expression of all shelterin genes decreases, but there is no apparent overall covariance between their expression profiles. The expression profile of telomerase is different from the expression profiles of the shelterin genes (Fig. 4A ). However, subgroups of shelterin genes exhibit pronounced covariances. The pot1, tpp1, and tin2 genes constitute a shelterin subgroup whose expression profiles are very similar (Fig. 4B ) and also resemble that of telomerase. The expression profile of trf1 is very similar to that of pinx1 (Fig. 4C ), whereas the trf2 and rap1 genes constitute another shelterin subgroup whose expression profiles are very similar (Fig. 4D ). Xenopus reference genes like odc, L8, and apod yield distinctly different expression profiles (Supplemental Fig. 9). This agrees with the recent report that normalization of RT-PCR expression measurements is highly unsuitable for development studies in Xenopus (47) .


Figure 4
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  		Figure 4. Real-time PCR reveals covariances in shelterin gene expression during X. laevis embryogenesis. A) Expression profile of telomerase is unique, and telomerase expression decreases for every developmental stage. B) The pot1, tpp1, and tin2 genes constitute a shelterin subgroup whose expression profiles are very similar, with a peak at developmental stage 8.5. C) Expression profile of trf1 is very similar to that of pinx1. The trf1 and pinx1 mRNA levels are almost constant between developmental stages 8 and 10 and then decrease between 10 and 10.25. D) The trf2 and rap1 genes constitute another shelterin subgroup whose expression profiles are very similar. The trf2 and rap1 mRNA levels peak at developmental stage 9+ and have a minor peak at developmental stage 10.75.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have identified the shelterin-encoding genes and determined their expression profiles during embryogenesis in X. laevis and X. tropicalis, two model organisms that are frequently employed to explore vertebrate embryonic development and cellular fate. Although the encoded Xenopus shelterin orthologs appear to fulfill all major functions of the human shelterin components, there are still some features worth considering. It has already been reported that X. laevis TRF1 lacks the acidic N terminus of hTRF1 (39) , which is responsible for interactions with the shelterin accessory factor Tankyrase in humans (48) . Here, we also confirm that the X. tropicalis TRF1 lacks an acidic N terminus. Because Tankyrase interacts with the acidic hTRF1 N terminus and regulates its binding to telomeric DNA in humans (48) , the missing acidic N terminus in Xenopus TRF1 likely has implications for TRF1 regulation by Tankyrase in Xenopus. Sequence analysis further indicates that Xenopus TRF1 forms homodimers that interact with telomeric DNA via conserved MYB/homeodomains and also that the TRF1 region, which is responsible for recruiting TIN2 to telomeres, has remained virtually intact in Xenopus (Supplemental Fig. 1). Reciprocally, the TRF1 interacting region of TIN2 is very well conserved in the Xenopus TIN2 orthologs (Supplemental Fig. 4). EMSAs demonstrate that Xenopus TRF1 simultaneously interacts with double-stranded telomeric DNA and Xenopus TIN2 (Fig. 1) in the presence of high concentrations of competitor DNA and protein.

An X. laevis ortholog of hTRF2 was recently reported (40) . The identification of an X. tropicalis TRF2 ortholog now verifies that the major difference between human and Xenopus TRF2 is that the basic N terminus of hTRF2 is missing in Xenopus (Fig. 2) . However, the roles of this TRF2 region remain to be clarified. So far it has not been implicated in interactions with other shelterin components, neither with shelterin accessory factors nor other proteins. Moreover, even though the MYB/homeodomains of TRF1 and TRF2 have similar affinities for telomeric DNA (49) , TRF2 has been shown to associate with nontelomeric chromatin (40) . In contrast, together with RAP1, the TRF2 forms a distinct complex, which is highly specific for telomeric DNA and associates with telomeric chromatin (23 , 29) . The region of TRF2 that is responsible for interactions with RAP1 (Supplemental Fig. 2), as well as the TRF2 interacting region of RAP1 (Supplemental Fig. 5), are highly conserved in Xenopus. EMSAs using the Xenopus orthologs support the view that the RAP1-TRF2 complex has significantly higher specificity for double-stranded telomeric DNA than TRF2 on its own (Fig. 2) . In addition, the hTIN2 binding region of hTRF2 (aa 352–365) (Supplemental Fig. 2), as well as the hTRF2 interacting region of hTIN2 (aa 1–220) (Supplemental Fig. 4), are highly conserved in the Xenopus orthologs.

The identification of Xenopus POT1 orthologs confirms the notion that POT1 proteins constitute the most conserved shelterin component (23 , 30) . The only noticeable difference between the human and Xenopus POT1 orthologs is that the central linker peptide, which connects the two N-terminal DNA-binding OB-folds to the C-terminal protein-interacting OB-fold, is slightly longer in hPOT1 (Supplemental Fig. 3). This peptide linker is believed to function as a molecular ball-and-socket joint, which allows the DNA binding module of POT1 to move relative to its protein-interacting module, so that shelterin can modulate the structure and accessibility of the single-stranded telomeric G-overhang (Fig. 5 ). TPP1 proteins have proven notoriously difficult to study in vitro, and difficulties in producing X. laevis and X. tropicalis TPP1 have so far prevented their biochemical characterization. A sequence analysis nonetheless reveals that fixed secondary structure elements of TPP1, as well as regions responsible for interactions with POT1, are highly conserved between human and Xenopus TPP1. Moreover, it has been reported that mice contain two unique POT1 paralogs, POT1a and POT1b (50) , and that human POT1 is subject to alternative splicing (51) . All five X. laevis POT1 cDNA clones analyzed here had an identical sequence, and also the three X. tropicalis POT1 cDNA clones were of an identical sequence. Even though these eight sequences may not represent a statistically significant Xenopus POT1 mRNA population, evidence for Xenopus POT1 paralogs, as well as alternative splicing of Xenopus POT1, remains to be established.


Figure 5
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  		Figure 5. Schematic of the Xenopus shelterin complex on telomeric DNA. The 6-subunit shelterin complex has the capacity to recognize telomeric DNA with at least 6 DNA-binding domains; 2 MYB/homeodomains in each TRF1 and TRF2 homodimer are specific for double-stranded telomeric DNA, whereas 2 OB-folds in each POT1 monomer are specific for single-stranded telomeric DNA. Although TIN2 itself does not directly interact with telomeric DNA, it is the shelterin linchpin that brings together the subcomplexes containing the 6 DNA-binding domains. The shelterin accessory factor PINX1 interacts with TRF1. Nucleosomes have been omitted.

TIN2 is the shelterin linchpin (Fig. 5) , and despite its moderate size, a TIN2 monomer was just found to have the capacity to simultaneously interact with one TRF1 homodimer and one RAP1 bound TRF2 homodimer (27) . In addition, TIN2 can interact with the TPP1-POT1 heterodimer to bring together the shelterin subcomplexes, which together contain 6 DNA-binding domains; 2 MYB/homeodomains in the TRF1 homodimer and 2 MYB/homeodomains in the RAP1-bound TRF2 homodimer are specific for double-stranded telomeric DNA, whereas 2 OB-folds in each POT1 monomer are specific for single-stranded telomeric DNA. Identification of the X. laevis and X. tropicalis shelterin complexes provides evidence for conserved telomere maintenance machineries among vertebrates.

X. laevis has been favored by biologists to investigate embryonic development for many years. Because both X. laevis and the true diploid X. tropicalis share the human TTAGGG telomeric DNA repeat sequence, we believe they are now poised to provide excellent model systems to investigate the roles of telomeres in vertebrate embryonic development. So far, variations in shelterin gene expression during the development of a multicellular organism have not been reported. Expression profiling of Xenopus shelterin genes during embryogenesis thus offers a first glimpse at telomere maintenance in development. Telomere maintenance has previously been demonstrated to depend on spatial control of telomeric proteins (52) . Our results now reveal that the composition of shelterin, and the formation of its subcomplexes, is also temporally regulated during embryonic development. Questions have been raised as to whether cells or organisms derived from somatic nuclear transfers have shorter telomeres than age-matched controls. There is ample evidence for a telomere resetting mechanism during early embryogenesis in cattle (53 54 55) . A telomere resetting mechanism per se requires efficient net synthesis of telomeric DNA, which, in turn depends on high telomerase activity and high levels of shelterin and shelterin accessory factors. The genes encoding telomerase and the telomeric ssDNA-interacting TIN2-POT1-TPP1 shelterin subcomplex, which together likely constitute the core of a telomere resetting machinery, exhibit expression profiles typical for maternal genes (56) . The data presented here thus support the notion that the message to reset telomere lengths is present already in the egg and provides a molecular framework to explore how telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells (57) .


   ACKNOWLEDGMENTS
 
We thank John Gurdon and Nigel Garett at the Wellcome Trust/Cancer Research UK Gurdon Institute for embryo staging and X. laevis cDNA. This work was supported by the Swedish Research Council, the Swedish Cancer Society, the Assar Gabrielsson cancer research foundation, the Johan Jansson foundation, and the Magn. Bergvall Research Foundation. Nucleotide sequences have been deposited to GenBank under the following accession numbers: xlTRF1 EU422974, xlTRF2 EU422975, xlPOT1 EU422976, xlTIN2 EU422981, xlRAP1 EU422979, xlTPP1 AY673986, xlPINX1 EU520258, xtTRF1 EU422977, xtTRF2 EU422978, xtPOT1 AY535401, xtTIN2 EU422982, xtRAP1 EU422980, xtTPP1 AY673987, and xtPINX1 EU520259.

Received for publication January 15, 2009. Accepted for publication February 26, 2009.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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