The FASEB Journal • Research Communication
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
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 singlestranded 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 lengthresetting 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.
FASEB J. 23, 2587–2594 (2009)
ABSTRACT
Key Words: telosome 䡠 differentiation 䡠 ES cell 䡠 iPS cell 䡠 quadruplex 䡠 cellular senescence
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
0892-6638/09/0023-2587 © FASEB
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– 6), telomeres have more
recently been found to have specific functions in
epigenetic gene regulation and vertebrate embryonic
development (7–9).
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
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:
[email protected]
doi: 10.1096/fj.09-129619
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reverse transcription of its internal RNA template (17–
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 –32) binds telomeric singlestranded 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 (33–37).
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 – 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
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 6⫻His-tag/S-tag followed by a tobacco etch
virus (TEV) protease cleavage site.
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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-␤-d-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM when
the cell cultures reached an optical density (␭⫽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 6⫻His-tagged, which
allowed the cleaved protein to be purified from TEV protease
and cleaved N-terminal 6⫻His-tag/S-tag by binding to NiNTA 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⬘-[␥-32P]-triphosphate (Amersham) and T4 polynucleotide kinase (New England Biolabs). Unincorporated 5⬘-[␥-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 ␮g/ml bovine serum albumin
(New England Biolabs) and 10 ␮g/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, 0.25⫻TB) 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
The FASEB Journal
VIZLIN-HODZIC ET AL.
were measured by real-time PCR (RT-PCR) analysis based on
SYBR Green detection. Briefly, the real-time PCR mixture
contained 1 ␮l of cDNA in total volume of 20 ␮l, containing
1⫻ 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.
RESULTS
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 sequencespecific 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
TABLE 1.
Primers used for real-time PCR
Primer
TERT fwd
TERT rev
TRF1 fwd
TRF1 rev
TRF2 fwd
TRF2 rev
POTl fwd
POT1 rev
TIN2 fwd
TIN2 rev
RAP1 fwd
RAPT rev
TPP1 fwd
TPP1 rev
PINX1 fwd
PINX1 rev
APOD fwd
APOD rev
L8 fwd
L8 rev
ODCfwd
ODC rev
Sequence
5⬘-GAGGGAGACTCTGAGAAGTTC-3⬘
5⬘-CAACTAAGCCATAACCAAAAGC-3⬘
5⬘-AGAGAAGATCGAACGGAGG-3⬘
5⬘-GCGGTTTCTAAAGGTGTAAGG-3⬘
5⬘-TCCGTCTTACTTCTTCGTCC-3⬘
5⬘-ATCAAGAGGCATTTCTTCCTG-3⬘
5⬘-GAAGCAAAGGAACCGATTATTG-3⬘
5⬘-AGCCACCACTATTAATTCCTTG-3⬘
5⬘-AGAGAAGAGAGACACAGCAC-3⬘
5⬘-GGGGGAAAATGGAGATTTAGC-3⬘
5⬘-AAGGGGCTCAGTATGTATCC-3⬘
5⬘-GGCCTTCACCAACCTTAAC-3⬘
5⬘-CCGAGGCATCATCACAAAG-3⬘
5⬘-TGTTTCCATCGGCAATATACTG-3⬘
5⬘-TCACCAGGATAACTGGCTC-3⬘
5⬘-TGAGAGGTCCTTCCCTTTG-3⬘
5⬘-CCCTGGAAGACCAAGATCTATG-3⬘
5⬘-GCTTCCCACTGGTTCTTATTC-3⬘
5⬘-TCCGTGGTGTGGCTATGAATCC-3⬘
5⬘-GACGACCAGTACGACGAGCAG-3⬘
5⬘-GCCATTGTGAAGACTCTCTCCATT-3⬘
5⬘-TTCGGGTGATTCCTTGCCAC-3⬘
XENOPUS SHELTERIN COMPLEXES
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 ␣-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 proteinprotein 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
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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 ␣-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).
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 doublestranded 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).
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
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
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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.
The FASEB Journal
VIZLIN-HODZIC ET AL.
Figure 2. A) X. laevis TRF2-RAP1 complex binds doublestranded 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.
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).
Finally, EMSAs under native conditions reveal that
recombinant Xenopus POT1, just like recombinant human POT1 (30 –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.
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).
DISCUSSION
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
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
XENOPUS SHELTERIN COMPLEXES
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 Gquadruplex DNA structures based on the guanine tetrad
base-pairing scheme (58).
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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.
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 doublestranded 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
2592
Vol. 23
August 2009
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 OBfolds 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 second-
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 DNAbinding 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.
The FASEB Journal
VIZLIN-HODZIC ET AL.
ary 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.
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 TPP1POT1 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 RAP1bound TRF2 homodimer are specific for doublestranded 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 (53–55). 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
XENOPUS SHELTERIN COMPLEXES
egg and provides a molecular framework to explore how
telomeres acquire embryonic stem cell characteristics in
induced pluripotent stem cells (57).
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.
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Received for publication January 15, 2009.
Accepted for publication February 26, 2009.
VIZLIN-HODZIC ET AL.