From the New England Baptist Bone and Joint Institute, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts 02115
Received for publication, November 6, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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We report here the isolation of
Tel-2, a novel member of the Ets transcription
factor family, with high homology to Tel/ETV-6. Tel-2 is
the second mammalian member of the Tel Ets family subclass whose
prototype Tel is involved in various chromosomal
translocations in human cancers. Six differentially expressed
alternative splice products of Tel-2 were characterized
encoding different Tel-2 isoforms which either contain or lack the
amino-terminal Pointed domain and also vary at the carboxyl terminus.
In contrast to Tel, which is highly expressed in several
different cell types and tissues, Tel-2 is only weakly
expressed in a variety of tissues and cell types, including placenta,
prostate, spleen, liver, and lung. Tel-2 binds to functionally relevant
Ets-binding sites of several genes and only the Tel-2 isoform
containing the Pointed domain and the DNA-binding domain acts as a
strong repressor of transcription. The retinoic acid
receptor Deregulated gene expression due to aberrant transcription factor
expression is a critical determinant in tumorigenesis. Many oncogenes
and tumor suppressor genes encode transcription factors (1, 2).
Chromosomal translocations, mutations, gene amplifications, and
deletions involving transcription factor genes appear to be frequent in
various human malignancies and are specific for a particular tumor
type, thereby emphasizing the importance of a stringent control of
these factors in normal physiological conditions (1). One particular
transcription factor family, the Ets family, has recently gotten a lot
of attention with regard to human cancer. The Ets transcription
factor/oncogene family contains almost 30 different mammalian members
(3, 4) that function as transcription factors under normal
physiological conditions (5-8). All Ets factors share a highly
conserved DNA-binding domain, the Ets domain which is sufficient to
interact specifically with DNA sequences (5-8).
Various members of the Ets transcription factor family have been shown
to cause cellular transformation, when aberrantly expressed. The
relevance of Ets transcription factors in human cancer has recently
been highlighted by the discovery of several distinct and very specific
chromosomal translocations involving various members of the Ets family
in different types of human cancer (5-8). The Ets gene
Tel/ETV6 on chromosome 12 is involved in many translocations leading to fusion of Tel to different genes including
various tyrosine kinases such as abl, JAK2, NTRK3, and
platelet-derived growth factor receptor as well as other
transcription factors such as AML1, MN1, CDX2, MDS/EVI1, and
STL, the fatty acyl-CoA synthetase 2 gene,
ACS2, and a gene of unknown function, BTL
(9-21). Similarly to Tel, fusion of the Ets factor
erg to the fus gene occurs in AML (22). Another
type of human cancer, Ewing's sarcoma, is characterized by
translocations of a number of different Ets factors, erg, ETV1,
E1-AF, FEV, or fli-1, to the EWS gene
(23-27).
Under normal physiological conditions, Ets factors play a critical role
in the regulation of genes involved in tissue-development, differentiation, angiogenesis, cell cycle control, and cell
proliferation as both transcriptional enhancers or repressors (5-8).
The relevance of Ets factors for cell differentiation has been
substantiated in knockout mice lacking an Ets factor gene
and Tel knockout mice are embryonal lethal due to a yolk sac
angiogenic defect (28-30).
Tel is the prototype and sole member of a distinct subclass of Ets
factors with the closest homologue being the Drosophila Ets
factor Yan. A recent deposition of the full-length sequence of a human
genomic DNA PAC clone derived from chromosome 6p21.3 within the MHC
cluster region indicated the potential existence of a novel Tel-related
Ets factor. We now report the cloning and characterization of
full-length cDNAs for this new member of the Tel subclass of Ets
factors, Tel-2, which is present in at least six alternative
splice forms.
Cell Culture--
Human foreskin keratinocytes, CV-1 (green
monkey kidney), LNCaP (human prostate), HEK293 (human fetal epithelial
kidney), C-33A (human cervical carcinoma), A431 (human vulvar
carcinoma), HeLa (human cervical carcinoma), H157 (human large cell
lung carcinoma), H249 (human small cell lung carcinoma), HUVEC (human
endothelial), U-87 Mg, and U-138 Mg (human glioma cells), U-937 (human
monocytes), MG-63 (human osteosarcoma), human synovial fibroblasts, and
human chondrocytes were grown as described (31-33).
Isolation and Analysis of cDNA Clones Encoding a Novel
Ets-related Protein--
To search for novel members of the Ets family
human public DNA data bases were searched for sequences homologous to
known Ets members as described (32). The human DNA sequence from PAC 50J22 on chromosome 6p21.3 contained sequences predicting homology to
the Ets-related protein Tel. These sequence homologies were spread over
several putative exons indicating that it does not encode a pseudogene.
PCR1 primers across two exons
spanning the Ets domain were synthesized and used to determine whether
we could detect any transcripts for this Ets related factor in
different tissues. RT-PCR using mRNA derived from human pancreas
and prostate as described (34) yielded the expected amplification
product of 229 bp. Sequence analysis confirmed that this fragment
encoded the new Tel-related cDNA Tel-2.
5' and 3' RACE Primer Extension--
The 229-bp cDNA
amplified from human prostate RNA contained an open reading frame
throughout this clone suggesting that part of the 5' and 3' end were
missing. To determine the 5' and 3' end of Tel-2 we
performed the RACE method using human adult prostate cDNA ready for
5'-RACE (CLONTECH) and nested primers specific for
the 5' and 3' ends of the partial Tel-2 cDNA as
described (35). Amplified DNA fragments were subcloned and sequenced as described (35). The sequences of the Tel-2 cDNAs were
confirmed by repeating amplification using primers specific for both
ends of the longest RACE products obtained in the first two rounds of
PCR amplification. The 5' and 3' end sequences of the Tel-2 cDNAs were confirmed by repeating 5'- and 3'-RACE PCR amplification using primers specific for the 5' and 3' ends of the longest RACE products obtained in the first two rounds of PCR amplification. Six
alternative splice variants of Tel-2 were isolated and
confirmed by full-length cloning and sequencing.
DNA Sequencing and Computer Analyses--
Nucleotide sequences
were determined at the Beth Israel Deaconess Medical Center DNA
sequencing facility using an Applied Biosystems Prism Automatic DNA
Sequencer Model 377 using the Taq DyeDeoxy Terminator Cycle
Sequencing kit (Applied Biosystems). Sequence analysis utilized DNA
Strider, Lasergene (DNASTAR) and BLAST, BEAUTY, and Clustal W searches
(NCBI). All oligonucleotides were purchased from Life Technologies, Inc.
RT-PCR Analysis--
cDNAs were generated from 1 µg of
mRNA isolated from different cells or tissues using
oligo(dT)12-18 priming (Life Technologies, Inc.) and
Moloney murine leukemia reverse transcriptase (Life Technologies, Inc.)
in deoxyribonuclease I (Life Technologies, Inc.)-treated samples.
cDNAs derived from different fetal and adult human tissues were
obtained from CLONTECH. Each PCR reaction used
equivalent amounts of 0.1 ng of cDNA, 4 ng/µl of each primer, 0.25 units of Taq polymerase (Promega, Madison, WI), 150 µM of each dNTP, 3 mM of MgCl2,
reaction buffer, and water to a final volume of 25 µl and were
covered with mineral oil. The sequences of the ubiquitous
Tel-2 specific primers were: sense,
5'-CCTACGAGAAGATGTCTCGTG-3' and antisense, 5'-CCAGGTGGCTGTGCTTGTCC-3'
with an expected amplification product of 134 bp. The sequences of the
Tel-2 specific primers for the 3' exon 8 were: sense,
5'-ACGTGTATCAGCTGCTCCTTG-3' and antisense, 5'-CCAGGTGGCTGTGCTTGTCC-3'
and crossed two introns between exons 6 and 8 with an expected
amplification product of 273 bp. The sequences of the
Tel-2e/Tel-2f specific primers were: sense,
5'-GTCACTGTGCAGAGCTCGGC-3' and antisense, 5'-CAGGAAGTGTGGGTCCTCCAA-3' and crossed three introns between exons 5 and 9 with an expected amplification product of 438 bp. The sequences of the
Tel-2 5' end specific primers were: sense,
5'-GCGCTCAAGACAGAAAGCCGG-3' and antisense, 5'-CTGGAGCAGCTCATACAGGAC-3'
and crossed three introns between exons 1 and 4 with expected
amplification products of 418 bp for Tel-2a, 377 bp for
Tel-2b, 241 bp for Tel-2c, and 212 bp for
Tel-2d. The sequences of the primers for GAPDH were: sense, 5'-CAAAGTTGTCATGGATGACC-3' and antisense, 5'-CCATGGAGAAGGCTGGGG-3' with
an expected amplification product of 200 bp. The sequences of the
primers for retinoic acid receptor
RT-PCR amplifications were carried out using a PerkinElmer Life
Sciences thermal cycler 480 as follows: 20-35 cycles of 1 min at
94 °C, 1 min at 58 °C, and 1 min at 72 °C followed by 10 min
at 72 °C. 10 µl of the amplification product was analyzed on a 2%
agarose gel.
Expression Vector and Luciferase Reporter Gene
Constructs--
The full-length Tel-2 cDNAs were
inserted into the EcoRI site of the pCI (Promega) eukaryotic
expression vector downstream of the T7 and cytomegalovirus promoters.
Hemagglutinin (HA) epitope-tagged Tel-2 isoforms were generated as
follows. A 111-bp trimer of HA tags was inserted into the XhoI and NotI sites of the pCI vector as a
COOH-terminal epitope generating pCI/HA. Full-length Tel-2a,
Tel-2b, and Tel-2c cDNAs with flanking
SalI sites were amplified from pCI/Tel-2a, pCI/Tel-2b, and
pCI/Tel-2c using a common pair of primers:
5'-TCATGTCGACTCCTCACCTCCACCTGTA-3' and
5'-ATCTAGTCGACCGGAGAGATTTCTGGCCT-3'. The PCR products were subcloned
into the SalI site of pCI/HA in-frame with the COOH-terminal HA epitope tag.
Synthetic wild type E74 Ets site oligonucleotides containing
SalI and XhoI ends were inserted into the
SalI site of the DNA Transfection Assays--
Co-transfections of 3 × 105 CV-1 cells were carried out with 3.5 µg of
E74/
To compare relative expression levels of Tel-2 isoforms after
transfection 3 µg of pCI/HA Tel-2a, Tel-2b, and Tel-2c expression vectors were transfected into COS cells using 12.5 µl of
LipofectAMINE. Whole cell extracts were generated 16 h later using
a lysis buffer containing 1 × phosphate-buffered saline, 5 mM EDTA, 0.5% Triton X-100, 0.1 mM
phenylmethylsulfonyl fluoride, 10 µM pepstatin A, 10 µM leupeptin, 25 µg/ml aprotinin.
In Vitro Transcription-translation--
Coupled in
vitro transcription-translation reactions with the different
untagged and HA-tagged Tel-2 splice variants inserted downstream of the T7 promoter into the pCI vector were performed in TNT
rabbit reticulocyte lysate (Promega, Madison, WI) in the presence of
[35S]methionine (PerKinElmer Life Sciences) as described
(37).
Electrophoretic Mobility Shift Assays (EMSA)--
EMSAs and
supershift assays were performed as described (33, 37) using 2 µl of
in vitro translation product or 3 µl of whole cell extract
and 0.1-0.2 ng of 32P-labeled double stranded
oligonucleotide probes (5,000-20,000 cpm) in the presence or absence
of competitor oligonucleotides (1, 5, 10, 25, and 50 ng) or 1 µl of
anti-HA tag or anti-mouse IgG antibody and run on 4% polyacrylamide
gels, containing as buffer 0.5 × TBE as described (32). The
anti-influenza HA tag monoclonal antibody was from Roche Molecular
Biochemicals and anti-mouse IgG was from Pharmingen.
Oligonucleotides used as probes and for competition studies are
as follows. 1) Drosophila E74 WT oligonucleotide:
5'-TCGAGTAACCGGAAGTAACTCAG-3' and 3'-CATTGGCCTTCATTGAGTCAGCT-5'; 2)
Drosophila E74 MUT oligonucleotide: 5'-TCGAGTAACCGGTTGTAACTCAG-3' and 3'-CATTGGCCAACATTGAGTCAGCT-5'; 3)
murine blk promoter WT oligonucleotide:
5'-TCGAGTCTCCAGGAAGTATTTTTCAGAC-3' and
3'-CAGAGGTCCTTCATAAAAAGTCTGTCGA-5'; 4) IgH enhancer RNA Isolation and Northern Blot
Analysis--
Poly(A)+ mRNAs were isolated as
described by Libermann et al. (31). Total cellular RNA was
isolated using guanidine isothiocyanate nucleic acid extraction and
cesium chloride gradient ultracentrifugation (38). Northern
blots and dot blots containing poly(A)+-selected mRNA
derived from different human tissues (CLONTECH) were hybridized with random prime-labeled Tel-2b full-length
cDNA in QuickHyb solution (Stratagene) as described (32) and washed at 50 °C with 0.2 × SSC, 0.2% SDS.
cDNA Microarrays--
106 human osteosarcoma
MG-63 cells were transiently transfected in duplicates with 8 µg of
pCI/Tel-2b or pCI plasmid using 30 µl of Superfect (Life
Technologies, Inc.). Total RNA was isolated separately from each plate
18 and 20 h after transfection using the RNeasy Kit (Qiagen Inc.,
Valencia, CA) according to the manufacturer's instructions. Three
types of Atlas human cDNA microarray nylon membranes from
CLONTECH Laboratories, Inc. (Palo Alto, CA), each containing 1136 different genes membrane, were hybridized with [32P]dATP-labeled cDNAs derived from 4 µg of total
RNA according to the manufacturer at 68 °C in ExpressHyb solution
(CLONTECH Laboratories, Inc.) using a Model 2000 Micro Hybridization Incubator (Robbins Scientific). The filters were
washed according to the user's instruction and exposed to Bio-Max MS
film (Fisher) for different exposure times. Hybridizations were
performed with duplicate experiments. The spot intensities reflecting
gene expression levels on the Atlas human cDNA array were
quantified using the Atlas Image 1.5 Software
(CLONTECH). The Tel-2b gene expression
profiles were compared with the gene expression profile with the
parental pCI vector normalizing spot intensities based on the average
of the intensities of all spots. Genes up-regulated or down-regulated by Tel-2b were validated by RT-PCR.
Isolation and Characterization of Six Alternative Splice Products
of the Human Ets-related cDNA, Tel-2--
To search for novel
members of the Ets family the GenBankTM data base was
searched for sequences homologous to the Ets domain. A human genomic
DNA sequence from PAC 50J22 (Z84484) on chromosome 6p21.3 contained
sequences related to the Ets factor Tel and was used for further
analysis. Since the Tel-related sequences encoding the DNA-binding
domain were spread over several putative exons separated by apparent
introns, we assumed that this Ets-related gene does not encode a
pseudogene. To test whether this putative Ets-related gene is indeed
transcribed into mRNA, we designed PCR primers spanning several
putative exons and performed RT-PCR using mRNA from various human
tissues as well as human genomic DNA. Genomic DNA gave the expected
amplification product. Human pancreas and prostate-derived cDNA
resulted in a smaller amplification product which upon sequencing
confirmed the existence of a correctly spliced mRNA expressed in
those tissues. We describe here the isolation and characterization of
full-length cDNA clones for this new member of the Ets family, the
second member of the Tel subclass, which we have named
Tel-2. To clone the full-length cDNA encoding
Tel-2 we performed the 5' and 3' RACE method as described
(35) using RACE ready human prostate Marathon cDNA.
Both strands of the full-length Tel-2 cDNAs were
sequenced entirely by double-stranded dideoxy sequencing using T7 and
T3 polymerase sequencing primers, and Tel-2-specific primers
based on partial DNA sequencing (Fig. 1).
Four alternative splice products of Tel-2, Tel-2a, Tel-2b,
Tel-2c, and Tel-2d, which differed in their coding
regions, were identified. The 5' end sequences of the Tel-2
cDNAs were confirmed by repeating 5'-RACE PCR amplification using
primers specific for the 5' end of the longest 5'-RACE products obtained in the first two rounds of PCR amplification. The 3' end of
these four Tel-2 splice variants contains a long poly(A) tract which is preceded by a classical polyadenylation site (Fig. 1) at
an appropriate distance. All four Tel-2 splice products contain the same 3' and 5' ends and are alternative splice forms as was
confirmed by PCR amplification and sequencing of full-length cDNAs
encoding all four splice products. The length of the Tel-2a full-length cDNA is 1592 bp, of Tel-2b 1551 bp, of
Tel-2c 1415 bp, and that of Tel-2d is 1385 bp
(Fig. 1). Inspection of the public EST data base identified four ESTs
encoding an apparent fifth alternative splice product
(Tel-2e and Tel-2f) that diverges at the 3' end
of the cDNA in comparison to the other four Tel-2 splice
variants and contains alternative polyadenylation sequences. To clone
this putative fifth splice product we used Tel-2e/f-specific primers at the 3' end of Tel-2e/f and primers from the 5'
end of Tel-2 and performed RT-PCR using the human prostate
Marathon cDNA. Two divergent full-length amplification products of
the expected sizes containing the 5' ends of Tel-2b and
Tel-2d, respectively, were obtained and fully sequenced
confirming the existence of a Tel-2e and Tel-2f
splice product (Fig. 1c). Interestingly, the 3' end of
Tel-2e and Tel-2f contains three apparent
polyadenylation sites and an L1 LINE repetitive element (Fig.
1c) (39).
Genomic Organization of the Tel-2 Gene--
Since the full-length
sequence of the human genomic PAC clone had been deposited in
GenBankTM, we were able to align the cDNA sequences of
the six Tel-2 alternative splice products to the genomic
sequence. A perfect match with the deposited genomic sequence was
obtained and enabled us to determine the intron/exon structure of the
Tel-2 gene. Sequence analysis revealed that the total length
of the transcribed portion of the Tel-2 gene covers more
than 33 kilobases and contains 9 exons (Fig.
2a). The first exon, common to
all splice products, contains the 5'-untranslated region and the first
two amino acids (Figs. 1 and 2). All intron/exon splice junction
borders conform with the splice site consensus (G/G)T ...
CA(G/)rule (40). Several introns are relatively large with the largest
being ~11.5 kilobases between exon 8 and 9 and ~9.5 kilobases
between exon 2 and 3 (Fig. 2a). Four of the six cloned
Tel-2 isoforms are due to alternative splicing of exons 2 and 3. Both, Tel-2a and Tel-2b are made up of the
first 8 exons; however, Tel-2a contains in addition an alternative splice form of exon 3, which extends exon 3 from 166 bp by
an additional 41-bp exon 3a. Both alternative splice forms of exon 3 are flanked by consensus splice sites (40). Tel-2c contains
exon 1 and exons 3 to 8, but is missing exon 2, whereas Tel-2d contains exons 1 and 2 and exons 4 to 8 and lacks
exon 3. The fifth and sixth alternative splice forms Tel-2e
and Tel-2f lack exon 8 which has been replaced by a further
downstream exon 9. Tel-2e contains the Tel-2b 5'
exons, whereas Tel-2f lacks exon 3 and, thus, is identical
to Tel-2d at the 5' end.
Based on 5'-RACE RT-PCR the major transcription start site for the
human Tel-2 gene can be fairly accurately assigned to the nucleotide G as indicated in Fig. 3.
However, we cannot rule out the possibility of multiple start sites
with shorter 5'-untranslated sequences. A typical TATA box is not found
upstream of the apparent transcription start site at the expected
distance; instead, a classical initiator Inr element CCAGTT is found 48 bp upstream of the transcription start site (Fig. 2c) (41).
Upstream of the transcription start site is a putative E2F-binding
site. A potential Ets-binding site partially overlaps the Inr element. Several additional Ets sites as well as NF- Predicted Amino Acid Sequence of Tel-2--
Sequence analysis of
the Tel-2 isoforms revealed a 1023-nucleotide open reading
frame encoding a 341-amino acid protein with a predicted molecular mass
of 39 kDa for Tel-2b, starting with an ATG at position 76 and
terminating with a TGA at position 1101 (Figs. 1 and 2b).
Similar to the highly related Ets factor Tel, the Tel-2b protein can be
subdivided into several structural and functional domains. The
amino-terminal Pointed domain which is potentially involved in
protein-protein interactions and is conserved in a subclass of Ets
factors is encoded by exons 2 to 4 (Figs. 1 and 2). The COOH-terminal
Ets domain which is the DNA-binding domain conserved among all
members of the Ets family is divided into three exons (exons 6 to 8)
(Figs. 1 and 2). The open reading frame of Tel-2b is
interrupted in Tel-2a due to the insertion of the 41-bp long
alternative exon 3a. As a result Tel-2a is potentially a
bicistronic mRNA which can encode a polypeptide of 111 amino acids
containing the amino terminus of Tel-2b including most of the Pointed
domain as well as a polypeptide of 282 amino acids starting with an
internal ATG in exon 3 created due to the frameshift by the alternative
exon 3a containing a new amino terminus, the middle part of Tel-2b and
the COOH-terminal Ets domain (Figs. 1 and 2). Sequence analysis of the
Tel-2c cDNA which lacks exon 2 revealed a 780-nucleotide
open reading frame encoding a 260-amino acid protein with a predicted
molecular mass of 30 kDa, starting with an internal ATG in exon 3 and
resulting in a protein that lacks the amino-terminal half of the
Pointed domain and the amino terminus of Tel-2b (Figs. 1 and 2).
Sequence analysis of the Tel-2d cDNA revealed a
858-nucleotide open reading frame encoding a 286-amino acid protein
with a predicted molecular mass of 32.6 kDa, starting with the same ATG
in exon 1 as Tel-2b and continuing in-frame until the carboxyl terminus
(Figs. 1 and 2). However, due to the in-frame deletion of exon 3 the
Tel-2d polypeptide lacks most of the Pointed domain, but contains the
amino terminus of Tel-2b and the Ets domain. Analysis of the
Tel-2e and Tel-2f isoforms revealed a
951-nucleotide open reading frame encoding a 317-amino acid protein
with a predicted molecular mass of 36.2-kDa for Tel-2e and a
786-nucleotide open reading frame encoding a 262-amino acid protein
with a predicted molecular mass of 29.8 kDa for Tel-2f, starting with
the same ATG in exon 1 as Tel-2b (Figs. 1 and 2). However, due to the
replacement of exon 8 by a further downstream exon 9 the last amino
acid of the Ets domain and all amino acids immediately downstream of
the Ets domain are eliminated and replaced by an alternative carboxyl
terminus encoded by exon 9. The carboxyl terminus of Tel-2e and Tel-2f
encoding 13 amino acids exhibits no homology to any known protein, but
is leucine/isoleucine-rich. The Tel-2e and Tel-2f isoforms may
express proteins with distinct characteristics from the other Tel-2 isoforms.
The ATG initiator codons only partially conform to the consensus
eukaryotic translation initiation sequence (42). There are several
reasons to believe that the Tel-2b ATG is the translation initiation codon. No additional ATG is found in-frame and an in-frame termination codon is found 70 bp upstream of the ATG. The same ATG is
being used in Tel-2a and Tel-2d. However, in
Tel-2a it is possible that an internal ATG generated by the
frameshift inserted by exon 3a also leads to translation into a protein
encoding the COOH-terminal half of Tel-2b. In vitro
translation and EMSA analysis indeed indicate that an internal ATG may
be used to generate a fragment containing the DNA-binding domain.
Tel-2c also apparently uses an internal ATG to generate a
protein encoding the COOH-terminal half of Tel-2b as demonstrated by
in vitro translation and EMSA. However, we cannot exclude
the possibility that translation of some of the Tel-2
isoforms starts either at another more preferrable ATG or at a codon
different from ATG.
The deduced amino acid sequence of Tel-2b predicts a protein rich in
leucine (11%), glutamic acid (7%), glycine (7%), proline (9%), and
arginine (8%). A putative PEST sequence with a PEST score of 9.4 is
located between amino acids 138 and 155 in Tel-2b. PEST sequences have
been implicated in protein degradation and may suggest a low stability
of the Tel-2b protein (43). Several potential phosphorylation sites for
Akt, p90rsk, protein kinase C, casein kinase II, cdc2 kinase,
and tyrosine kinase are present in Tel-2 (44-48). The predicted Tel-2b
protein sequence reveals, furthermore, seven potential MAP kinase
phosphorylation sites ((S/T)P) for ERK, jnk, p38, one of them
containing the optimal PX(S/T)P sequence (49, 50).
Interestingly three MAP kinase phosphorylation sites and a tyrosine
kinase phosphorylation site are conserved in the highly related Tel as
well. The importance of MAP kinase phosphorylation sites for the
biological function of Ets-related factors has recently been
demonstrated suggesting that at least some of these putative sites
might be functionally relevant for Tel-2 as well (51-53).
Sequence Comparison of Tel-2 to Other Members of the Ets
Family--
Comparison of the deduced amino acid sequence of Tel-2
with other members of the Ets family revealed the highest homology to
Tel and to a lesser extent to the Drosophila Ets factor
Yan (Fig. 3). Overall, the amino acid homology between Tel-2b
and Tel is ~50%. Homologies are clustered in several primary
regions, a putative protein-protein interaction domain A (Pointed
domain) at the amino terminus, the putative DNA-binding domain B (ETS domain) which extends over ~85 amino acids at the carboxyl terminus of the gene, a smaller region just upstream of the Pointed domain which
includes a conserved optimal Map kinase phosphorylation site, as well
as a short acidic domain (C domain, Fig. 2) downstream of the Ets
domain (Fig. 3a). Similarly, Yan shows high homology to
Tel-2 in the Pointed and Ets domain as well as some lower homology regions throughout the protein (data not shown). Interestingly, the
Tel-2e/Tel-2f isoforms have an alternative carboxyl terminus that eliminates the acidic C domain, a region of Tel-2 with high homology to Tel. This conserved C-terminal region of Tel and Tel-2 may
have functional significance for these factors such as transactivation or protein-protein interaction.
Alignment of the ETS domain of Tel-2 with that of other members of the
Ets family reveals highest homology to Tel (88%) and to a lesser
extent to Yan (48%) (Fig. 3b). Sequence identity to all other members of the Ets family is 34-44%. Besides the highly conserved ETS DNA-binding domain, the amino terminus of Tel-2 contains
a region with significant homology to the Pointed domain present in Tel
and a subset of Ets factors (Fig. 3c). Again homology is
highest to Tel (63%). Based on the sequence comparisons it is clear
that Tel-2 represents the second mammalian member of the Tel subfamily
of Ets factors.
Expression Pattern of Tel-2 in Human Tissues--
To determine the
expression pattern of Tel-2 we performed PCR with cDNA
reverse transcribed from RNA derived from different human fetal and
adult tissues, since Northern blot and dot blot hybridizations with
full-length Tel-2b cDNA probes did not result in any
specific signal (data not shown) indicating very low abundance of
Tel-2 mRNAs in most tissues. A multiple tissue cDNA
panel set of normalized (using several housekeeping genes),
first-strand cDNA ready for use in PCR analysis and generated using
poly(A)+ RNA from different human tissues was obtained from
CLONTECH. To control for RNA quality and quantity,
we performed PCR with primers specific for GAPDH as well
(Fig. 4a). Whereas only 20 PCR
cycles were applied for GAPDH, 35 cycles were required for Tel-2, further confirming the low abundance of
Tel-2 transcripts. Due to the high number of PCR cycles
required to obtain a significant amplification product for
Tel-2 and the very low abundance of Tel-2
mRNA in most tissues, a relatively high degree of error in the
relative quantities of Tel-2 transcripts can be expected. Nevertheless, the PCR reactions were repeated two times with very similar results. Since Tel-2 is expressed in the form of at
least six splice variants, we used PCR primers that are either specific for the 5' end alternative splice variants Tel-2a, Tel-2b,
Tel-2c, and Tel-2d resulting in differently sized
amplification products, primers specific for the 3' end of
Tel-2e/Tel-2f including exon 9 or primers
specific for the 3' end of Tel-2 containing exon 8. The
results indicate that the Tel-2 gene is expressed, although with very low abundance in a variety of fetal and adult tissues tested
(Fig. 4a). Strikingly, each Tel-2 splice product
displays a distinct expression pattern. In adult tissues, placenta
expressed the highest levels of Tel-2, followed by liver,
prostate, lung, ovary, spleen, thymus, and peripheral blood lymphocytes
(Fig. 4a). Lower levels of Tel-2 mRNA were
expressed in most other tissues. In human fetal tissues, lung and
kidney expressed the highest amounts of Tel-2 (Fig.
4a). Among the different 5' splice forms of Tel-2
Tel-2d, lacking the Pointed domain, was the most frequently expressed transcript which was expressed in the majority of human tissues to some extent. Highest levels of Tel-2d were
observed in liver, prostate, placenta, and lung. Tel-2b,
encoding the full-length protein including the Pointed domain, was also
highly expressed in placenta, prostate, liver, and lung, and to a lower
extent in several other tissues (Fig. 4a). But a significant
number of tissues such as testis, brain, and several fetal tissues
expressed Tel-2d without any evidence for Tel-2b
expression indicating that their expression is regulated in a
tissue-specific manner. Only very low levels of Tel-2a and
Tel-2c were detected and only in a few tissues suggesting
that these isoforms may play a more limited role. Strikingly
differential expression was also observed for the alternative 3' exons
8 and 9. The Tel-2 isoforms containing exon 8 and, thus, the
complete Ets domain were expressed in most of the tissues that express
Tel-2 except ovary and heart (Fig. 4a). Highest
expression was seen in placenta, lung, liver, pancreas, peripheral
blood leukocytes, and fetal lung. Tel-2e/Tel-2f,
which contain exon 9, miss the last amino acid of the Ets domain and replace a sequence conserved in Tel-2 and Tel
with an alternative carboxyl terminus were highly expressed in a subset
of tissues, with highest expression in placenta followed by spleen,
prostate, thymus, and ovary (Fig. 4a). Low levels of
Tel-2e/Tel-2f were also observed in the heart,
whereas none of the other tissues expressed any detectable levels of
Tel-2e/Tel-2f. Tel-2e/Tel-2f expression is much more restricted than expression of the exon 8 containing Tel-2 transcripts. Interestingly,
Tel-2e/Tel-2f expression in ovary, prostate,
heart, spleen, and thymus appears to be higher than the exon 8 Tel-2 splice forms demonstrating that the unusual Tel-2e/Tel-2f transcripts are likely to be
functionally relevant. These results indicate that several of the
Tel-2 isoforms are expressed at significant levels in
various tissues, although at strikingly different ratios. Unexpectedly,
the Tel-2 isoform closest related to Tel, Tel-2b,
which contains the Pointed domain and the Ets domain is not the most
frequently expressed transcript.
To determine in more detail expression of Tel-2 in different
cell types we performed RT-PCR with RNA derived from different cell
types using both primary cells and cancer-derived cell lines and
primers for a common region of Tel-2 that would amplify all splice variants of Tel-2. Like for the fetal and adult
tissues most cell lines expressed some Tel-2 mRNA,
although in most cell lines expression was very low as well (Fig.
4b). However, some cell lines including A431 squamous
carcinoma cells, U-937 monocytic cells, and primary human chondrocytes
expressed highly elevated levels of Tel-2. This was
particularly striking for A431 cells which expressed by far the highest
level of Tel-2 from all RNAs tested. Thus, although
Tel-2 is weakly expressed in many tissues and cell types,
Tel-2 expression might be enhanced in a subset of cell types
or cancer-derived cells.
Tel-2 Binds Specifically to Functionally Important Ets-related
Binding Sites in a Variety of Genes--
To determine whether Tel-2
can bind in a sequence-specific manner to DNA, Tel-2a, Tel-2b, and
Tel-2c were in vitro transcribed and translated into protein
in a coupled reticulocyte lysate system. Based on the cDNA
sequences of the different Tel-2 isoforms we expected to obtain a
full-length Tel-2 protein for Tel-2b and truncated forms for Tel-2a and
Tel-2c lacking either the amino terminus or the carboxyl terminus of
Tel-2b. SDS-polyacrylamide gel electrophoresis analysis of the
[35S]methionine-labeled in vitro translation
reactions revealed as the major products for Tel-2b two equal intensity
proteins with the molecular weight close to the expected molecular
weight (Fig. 5a). The lower
molecular weight Tel-2b product might be a result of an internal
translation start site 17 amino acids downstream of the first ATG. A
similar alternative translation start site has been observed for the
highly related Tel as well resulting in the translation of two
alternative Tel proteins. Tel-2c in vitro translation
resulted in the synthesis of a protein with the expected molecular
weight, but also in a strong lower molecular weight product which may
either encode a degradation product, early termination, or an internal
start site. We believe that this lower molecular weight product
represents an internal start site, since the Tel-2c ATG which would be
used to generate the full-length Tel-2c protein is an internal ATG in
Tel-2b and does not conform with the optimal Kozak sequence very well.
However, nucleotides surrounding an ATG codon just upstream of the Ets domain are much closer to the Kozak sequence. Tel-2a as described above
possibly encodes a bicistronic RNA which could encode either a protein
that starts at the original ATG and results in a protein containing the
amino terminus of Tel-2b including the Pointed domain or a protein
starting from an internal ATG in a different reading frame leading to a
protein encoding the COOH-terminal Ets domain of Tel-2b. In
vitro translation of Tel-2a revealed indeed two products, although
the amino-terminal shorter product was far more abundant than the
protein presumably encoding the DNA-binding domain (Fig.
5a). Smaller amounts of additional faster migrating proteins
were visible as well in some of the reactions due to partial
proteolysis, premature translational termination, or alternative
internal initiation codons.
An EMSA was performed using equivalent amounts of in
vitro translated full-length Tel-2b as well as the Tel-2a and
Tel-2c isoforms to determine their relative ability to bind to an
oligonucleotide encoding the Drosophila E74 Ets-binding
site, which has previously been shown to bind to several members of the
Ets family (54). The E74 oligonucleotide formed several higher
molecular weight complexes with both the control reticulocyte lysate
(Fig. 5b, lane 1) and reticulocyte lysates expressing Tel-2
proteins (lanes 2-4). A specific protein-DNA complex was
formed by the full-length Tel-2b reticulocyte lysate that was not
present in either the control reticulocyte lysate or in the Tel-2a or
Tel-2c in vitro translation (Fig. 5b). Two
additional faster migrating protein-DNA complexes were also
specifically formed, which were absent from control lysate, but present
in the Tel-2a, Tel-2b, and Tel-2c lysate (Fig. 5b). The
exact nature of these two complexes is not clear, since they comigrate
in all three Tel-2 isoform lysates, even though each of the isoforms
generates proteins with slightly different molecular weights. The
specificity of the protein-DNA complexes was confirmed by competition
analysis. Increasing amounts of wild type, but not mutant E74
oligonucleotide competed specifically with all three complexes formed
by Tel-2b (Fig. 5b). That indeed all three protein-DNA
complexes are due to binding of Tel-2-derived polypeptides was
confirmed in a supershift assay using HA-tagged in vitro
translated Tel-2 isoforms (Fig. 5c). The HA tags were fused
downstream of the COOH-terminal Ets DNA-binding domain. The anti-HA
antibody, but not an anti-mouse IgG antibody supershifted all three
Tel-2-related complexes, but not several nonspecific complexes
demonstrating the specificity of these complexes (Fig. 5c).
This result demonstrates that full-length Tel-2b protein binds
efficiently to DNA and that Tel-2a and Tel-2c apparently also generate
proteins that can interact with DNA.
To analyze the DNA sequence requirements for the binding of Tel-2 we
designed oligonucleotides encoding a whole spectrum of different,
functionally relevant binding sites for Ets-related factors (32, 35).
The relative binding affinity and specificity of Tel-2b for these sites
was compared with its affinity for the E74 site in competition
experiments. Equivalent amounts of wild-type oligonucleotides were used
as competitors in EMSA with equal amounts of Tel-2b in vitro
translated protein and the E74 oligonucleotide as a probe. The
wild-type E74 oligonucleotide competed effectively with complexes
formed by Tel-2b, whereas the mutant E74 oligonucleotide was unable to
inhibit binding of Tel-2b even at high concentrations (Fig.
5d). 10 ng of wild-type competitor completely abolished binding of Tel-2b to the E74 probe. Oligonucleotides encoding the
Ets-binding sites found in the regulatory regions of the blk, lyn, Endo A, and SPRR2a competed efficiently with
Tel-2b although none of them demonstrated the same affinity as E74
(Fig. 5d). HIV-2 LTR, and HTLV-1 LTR
also partially competed with Tel-2b, whereas other sites including the
IgH enhancer Tel-2b, but Not Tel-2a or Tel-2c Acts as a Strong Repressor of
Transcription--
Since the alternative splice products of Tel-2
encode different protein isoforms which either lack or contain the
Pointed domain or the Ets DNA-binding domain, we were interested to
know whether the different splice products of Tel-2 would express any differences in their functions as transcriptional regulators and whether Tel-2 would act as a transcriptional enhancer or repressor. Furthermore, since the highly related Tel had previously been shown to
be a repressor of transcription, we compared the activity of Tel-2 to
Tel. Tel-2a, Tel-2b, Tel-2c, and Tel were
inserted into the eukaryotic expression vector pCI and co-transfected
into CV-1 cells together with a pGL3 reporter gene construct containing the luciferase gene in which two copies of the E74
Ets-binding site were inserted upstream of the minimal c-fos
promoter
A dose curve with differing amounts of Tel-2 expression vectors and
constant amounts of the E74 luciferase construct demonstrated that
increasing amounts of Tel-2b led to enhanced repression (Fig. 6c). In contrast, increased levels of Tel-2c did not affect
E74 transactivation at all. Interestingly, Tel-2a again reproducibly enhanced E74 activity in a dose-dependent manner up to
2.5-fold (Fig. 6c).
To determine whether Tel-2 can repress enhancers or promoters of
mammalian genes that contain functionally important Ets-binding sites,
we co-transfected CV-1 cells with either the lyn promoter luciferase construct (lyn/pGL3) or the IgH enhancer
luciferase construct (IgH- The RAR The Ets factor family has evolved as a family of transcription
factor genes that play intricate roles in many facets of development and cell differentiation (5-8). Disturbances in Ets factor functions lead invariably to developmental defects and in extreme cases to tumor
formation. Indeed the first members of the Ets factor family were
derived as transforming oncogenes from retroviruses as well as
integration sites of retroviruses raising the notion that defects in
Ets factor genes may be involved in human cancer as well (5-8). This
hypothesis was confirmed by the findings that at least six different
members of the Ets family are involved in various chromosomal
translocations in different types of human cancer (9-21). Particularly
striking is the involvement of the Ets factor Tel in the
majority of 12p13 chromosomal abnormalities present in various human
hematological malignancies as well as in certain solid tumors such as
fibrosarcomas (9-21, 25-27). Interestingly, Tel
translocation partners are derived from various types of proteins including both membrane-bound and cytoplasmic protein-tyrosine kinases
as well as nuclear transcription factors. Similarly striking is the
variability in the domains of Tel that are involved in these
chromosomal translocations and the consequent different mechanisms of
Tel mediated transformation. The Tel protein can be divided into two
primary functional domains, the amino-terminal Pointed dimerization
domain and the COOH-terminal Ets DNA-binding domain. Under normal
circumstances Tel appears to act as a transcriptional repressor (57,
58). Chromosomal translocations of Tel to tyrosine kinases
such as abl, platelet-derived growth factor-R, JAK2, and NTRK3 invariably involve the fusion of the amino terminus of
Tel including the Pointed domain to the carboxyl terminus of the
tyrosine kinase including the tyrosine kinase catalytic domain. This
fusion leads to constitutive activation of the fused tyrosine kinase due to forced dimerization via the Tel Pointed domain (59). In
contrast, fusion of the Tel Pointed domain to the DNA-binding and
transactivation domains of the transcription factor AML-1 leads to a
switch of AML-1 function from transcriptional enhancement to repression
(18, 60). Additional Tel translocations lead to fusion of the
COOH-terminal Tel DNA-binding domain to putative transactivation
domains of apparent transcription factors such as MN1 and STL which may
have the opposite effect of converting the transcriptional repressor
Tel into a transcriptional enhancer (15, 21).
Tel has up to now been the sole mammalian member of its subclass in the
Ets family. Tel-2 is a second member of the Tel subclass which may play
similar roles as Tel in human cancer. Tel-2 shows high homology to Tel
throughout the coding sequence with an overall homology of 50%. Like
Tel, Tel-2 contains an amino-terminal Pointed domain and a
COOH-terminal Ets domain which are highly conserved between Tel and
Tel-2. In addition, both Tel and Tel-2 contain an optimal
phosphorylation site for members of the MAP kinase pathway,
PXSP, immediately upstream of the Pointed domain. No function for this site has been demonstrated in Tel up to now, but it
is intriguing that this site is highly conserved in both genes at the
same position. Furthermore, the repressor activity of the Ets factor
closest related to Tel and Tel-2, the Drosophila Yan, is
tightly regulated by the MAP kinase pathway (61). Indeed, it has been
shown that Tel is phosphorylated in vivo (62). Another similarity between Tel and Tel-2 is the translational initiation of Tel
mRNA at two different in-frame ATGs at positions 1 and 43 (62). Our
in vitro translation studies with Tel-2b show two equal
strength translation products which most likely encode proteins starting at positions 1 and 17. Interestingly, both for Tel and Tel-2,
the alternative in-frame starting points eliminate the putative MAP
kinase phosphorylation sites. The existence of both translation
products for Tel has been demonstrated in vivo using Tel-specific antibodies (62).
Like Tel the homologous Tel-2 isoform Tel-2b acts as a strong
transcriptional repressor indicating that Tel and Tel-2b might have
related functions. The transcriptional repressor domain of Tel-2 is
most likely located at the amino terminus of Tel-2, since the two
isoforms of Tel-2, Tel-2a and Tel-2c, which encode proteins lacking
either the amino terminus or the carboxyl terminus do not repress
transcription. These results also indicate that both the amino terminus
including the Pointed domain and the carboxyl terminus including the
DNA-binding domain are necessary to generate an active repressor
protein. Interestingly, the Tel-2a isoform appears to increase rather
than repress transcription slightly in a dose-dependent
manner. This is somewhat surprising, since Tel-2a may make two
different proteins encoding either the amino terminus only including
the Pointed domain or the carboxyl terminus by itself including the Ets
domain. A possible scenario may be that one of these proteins binds to
a corepressor and, thus, limits the availability of the corepressor for
other transcription factors. The repressor domain in Tel has been
located to the Pointed domain and the central domain between the
Pointed domain and the Ets domain, and it is striking that the
Drosophila Ets factor Yan, the closest relative of Tel, also
acts as a transcriptional repressor (57, 58, 61). Comparison of the
amino acid sequences of Tel-2, Tel, and Yan reveals primarily
homologies in the Pointed domain and the Ets domain that are common to
all three factors. Nevertheless, additional short stretches of homology
between these three repressor Ets factors are seen upstream and
downstream of the Pointed domain that may be involved in repressor
function. Within the Pointed domain there are several stretches of high homology between these three Ets factors that are less conserved in
other Pointed domain containing Ets factors. One sequence
G(R/K)AL(C/L)(I/L)LT is of particular interest, because a similar
sequence is also found in the SAM domain of another repressor factor,
the polycomb protein SCM (63). Recent data suggest that Tel acts as a
repressor due to recruitment of the corepressor Sin3a which interacts
with the amino-terminal 127 amino acids of Tel presumably via the
Pointed domain (57, 58). We are now evaluating whether Tel-2 also interacts with Sin3a.
Our transcriptional profiling data also support the notion that Tel-2b
acts as a transcriptional repressor in vivo, since the
majority of genes affected by Tel-2b in MG-63 osteosarcoma cells were
down-regulated. These experiments also give the first clues about the
potential biological function of Tel-2. Two of the genes repressed by
Tel-2b, BMP-6 and RAR In contrast to Tel, which does not bind efficiently to DNA as a
full-length protein, the equivalent Tel-2 isoform Tel-2b binds to a
variety of functionally important Ets-binding sites as demonstrated by
EMSA. Two faster migrating complexes are also formed that comigrate with complexes formed by the other two isoforms Tel-2a and Tel-2c. In vitro translation reveals the synthesis of a ~22-kDa
protein common to all isoforms in addition to their expected proteins. This protein may be the result of an internal translation start site
using a methionine upstream of the Ets domain with a preferred Kozak
consensus sequence. Whether this protein is being synthesized in
vivo is not known, but whole cell extracts from cells transfected with expression vectors for the Tel-2 isoforms reveal the same size
proteins as in vitro translated Tel-2. The DNA binding
specificity does not seem to be significantly different from most other
Ets factors, although binding is restricted to sites containing a GGAA
core rather than GGAT and Tel-2 strongly prefers a T in the +2 position
(Table I).
Tel has been shown to dimerize via the Pointed domain (59). The Pointed
domain is present in several other members of the Ets family and in
addition to Tel Fli-1, Ets-1, and Ets-2 are apparently able to form
homo- and hetero-dimers via the Pointed domain (77). The Pointed domain
of Tel-2 is the one closest related to Tel suggesting that Tel-2 may
dimerize as well. The Pointed domain is a subclass of the highly
conserved SAM domain which is found in various types of proteins from
yeast to human including among others the EPH receptors and polycomb
proteins (78). The SAM domain has been implicated in both homo- and
hetero-dimerization. Since most Ets factors that contain the Pointed
domain do not dimerize with each other, it is tempting to speculate
that these Ets factors interact with other SAM domain containing
proteins such as the polycomb proteins.
The evolutionary relationship between Tel-2 and Tel is also conserved
in the genomic organization with both the Tel-2 and Tel genes being encoded by eight exons and exon/intron
borders being highly conserved. Whether Tel has the
alternative 3' exon 9 is not known. The Pointed domain of both Tel and
Tel-2 is encoded by exons 2, 3, and 4 and the Ets domain by exons 6, 7, and 8 with identical exon/intron borders. The Tel-2 gene has
an alternative out-of-frame exon 3a of 41 nucleotides which has not
been seen for Tel up to now. We actually have isolated five
alternative splice products of Tel-2 which lead to the
expression of various Tel-2 isoforms. Only Tel-2b encodes the
full-length protein homologous to Tel. The other three isoforms encode
proteins in which either the Pointed domain alone or together with the
rest of the amino terminus is deleted or proteins which contain only
the amino terminus of Tel-2 including the Pointed domain, but lack the
Ets DNA-binding domain. The fifth and sixth splice variants Tel-2e and
Tel-2f delete the COOH-terminal 38 amino acids immediately downstream of the Ets domain including the last amino acid of the Ets domain. Whether conversion of the last amino acid of the Ets domain from phenylalanine to asparagine affects DNA binding or DNA binding specificity is not known. Nevertheless, this amino acid is part of the
In contrast to Tel which is abundantly and ubiquitously
expressed, Tel-2 expression appears to be very low in all
fetal and adult tissues tested. We were so far unable to detect any
Tel-2 mRNA by Northern blot or dot blot analysis of
poly(A+) mRNA in a whole set of human fetal and adult
tissues. However, using RT-PCR we detected low levels of
Tel-2 mRNA expression in many fetal and adult tissues as
well as in most human cell lines. No specific pattern of expression was
observed which would indicate the role of Tel-2 in a
specific organ or cell type. However, the differential expression of
the different splice variants of Tel-2 was striking.
Surprisingly, the most common splice product was Tel-2d
which lacks the Pointed domain, but contains the amino terminus
in-frame with the carboxyl terminus. Indeed in several tissues
Tel-2d was the sole splice form expressed indicating that Tel-2d may play as important a function as Tel-2b.
Tel-2b was also expressed at relatively high levels in several
tissues, whereas Tel-2a and Tel-2c expression was
very low. Highest levels of Tel-2b mRNA were detected in
prostate, liver, lung, and placenta, the same tissues that express the
highest levels of Tel-2d. The majority of these splice forms
are combined with the Tel-2 isoform containing exon 8 which
results in a full-length Ets domain. However, the Tel-2e/Tel-2f isoforms splicing exon 9 rather
than exon 8 to the carboxyl terminus are also significantly expressed
in a restricted set of tissues. Again placenta and prostate express
significant amounts of this splice form. But some tissues such as ovary
and heart, and to a lesser extent prostate, spleen, and thymus, almost exclusively express the Tel-2e/Tel-2f isoforms.
By far the highest levels of Tel-2 mRNA were detected in
the A431 squamous carcinoma cell line suggesting that Tel-2
may be overexpressed in some cancer cells. This relatively low
abundance of Tel-2 is also reflected in the
GenBankTM EST data base where only 14 Tel-2-derived ESTs have been deposited. These ESTs are
derived from two colon adenocarcinomas, two lung carcinomas, an adrenal
adenoma, a chronic B lymphocytic leukemia, a germ cell tumor, two
ovarian cancers, an endometrial tumor, and germinal center B cells
possibly indicating enhanced expression in human cancers.
Interestingly, the Tel-2e/Tel-2f ESTs containing exon 9 were all derived from ovarian and endometrial cancers
correlating with our expression analysis, and exon 8 was not present in
any entry from these tissues indicating the tissue-specific splicing. In addition, SAGE analysis suggest enhanced expression of
Tel-2 in the brain or brain tumors.
Tel-2 is located on human chromosome 6p21.3 in the MHC
cluster region. This region has been implicated in a variety of
different cancers such as chondroid hamartomas, thyroid adenomas,
ductal carcinoma in situ of the breast, B-cell
non-Hodgkin's lymphoma, cervical cancer, astrocytoma, nonsmall cell
lung carcinomas, and ovarian carcinomas (82-91). Since chromosomal
translocations of Tel are frequent events in various
leukemias and fibrosarcoma, it is likely that Tel-2 plays
similar roles.
After submission of this manuscript two independent groups reported
similar findings (92, 93) on the cloning and characterization of
Tel-2 confirming our data about the repressor activity and DNA binding capacity. Both reports, in addition, demonstrate that Tel-2
can homo-dimerize as well as hetero-dimerize with Tel via the Pointed
domain (92, 93). Elevated levels of Tel-2 were detected in
several hematopoietic tissues including fetal liver and bone marrow
(92). Our results add additional information about the various
alternative splice products of Tel-2 and possible target
genes regulated by Tel-2.
and bone morphogenetic protein-6B
(BMP-6) genes are specifically repressed by Tel-2
indicating a function for Tel-2 as an inhibitor of differentiation. Due
to the important involvement of Tel in human cancer and the
location of Tel-2 within the MHC cluster region,
Tel-2 might be involved in chromosomal translocations in
human cancer as well.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(RAR
)
were: sense, 5'-GTCCGACAAGGTCATCCTGT-3' and antisense,
5'-GTGTCACCGGGACAAGAACT-3' with an expected amplification product of
360 bp. The sequences of the primers for BMP-6 were: sense,
5'-CTCGGGGTTCATAAGGTGAA-3' and antisense, 5'-ACAGCATAACATGGGGCTTC-3'
with an expected amplification product of 412 bp.
56-c-fos-pGL3 plasmid as
described (32). The lyn promoter and IgH enhancer pGL3 luciferase constructs are as described (32).
56-pGL3, lyn/pGL3 or IgH/
56-pGL3 reporter gene construct DNA
and 0.5, 1, 1.5, or 2 µg of Tel-2 expression vector DNA using 12.5 µl of LipofectAMINE (Life Technologies, Inc.) as described (32). The
cells were assayed 16 h after transfection. Transfections for
every construct were performed independently in duplicates and repeated
3 to 4 times with two different plasmid preparations with similar
results. Equal amounts of cell extract protein were used to normalize
the luciferase assay. Co-transfection of a second plasmid for
determination of transfection efficiency was omitted because potential
artifacts with this technique have been reported (36) and because many
commonly used viral promoters contain potential binding sites for Ets factors.
WT
site oligonucleotide: 5'-TCGACTGGCAGGAAGCAGGTCATGC-3' and
3'-GACCGTCCTTCGTCCAGTACGAGCT-5'; 5) polyoma virus PEA3 WT
oligonucleotide: 5'-TCGAGCAGGAAGTGACG-3' and 3'-CGTCCTTCACTGCAGCT-5';
6) HIV-2 LTR WT oligonucleotide: 5'-TCGAGTTAAAGACAGGAACAGCTATG-3' and
3'-CAATTTCTGTCCTTGTCGATACAGCT-5'; 7) HTLV-1 LTR WT
oligonucleotide: 5'-TCGAGGGGAGGAAATGGGTG-3' and 3'-CCCCTCCTTTACCCACAGCT-5'; 8) T-cell receptor
enhancer
T
2 WT oligonucleotide: 5'-TCCCGCAGAAGCCACATCCTCTG-3' and
3'-AGGGCGTCTTCGGTGTAGGAGAC-5'; 9) Fos SRE WT
oligonucleotide: 5'-TCGA- GCTTACACAGGATGTCCATATTAGGACATCTG-3' and
3'-CGAATGTGTCCTACAGGTATAATCCTGTAGACAGCT-5'; 10) MHC class II
promoter WT oligonucleotide: 5'-TCGAGAGTGAGGAACCAATCAG-3' and 3'-CTCACTCCTTGGTTAGTCAGCT-5'; 11) murine IgH µB WT site
oligonucleotide: 5'-TCGAGCTATTTGGGGAAGGGAAAATAAA- AC-3' and
3'-CGATAAACCCCTTCCCTTTTATTTTGTCGA-5'; 12) human lyn promoter
WT oligonucleotide: 5'-TCGAGCACCAGGAAGTAGCTGGGAC-3' and
3'-CGTGGTCCTTCATCGACCCTGTCGA-5'; 13) mouse EndoA enhancer WT
oligonucleotide: 5'-TCGACCAGACTGGACAGGAAGTAGGAACAGAC-3' and 3'-GGTCTGACCTGTCCTTCATCCTTGTCTGAGCT-5'; 14) human SPRR2A
promoter WT oligonucleotide: 5'-TCGAGCAGCAGGAAGTGAAACTACCCG-3' and
3'-CGTCGTCCTTCACTTTGATGGGCAGCT-5'.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Complete nucleotide sequence and predicted
amino acid sequence of Tel-2. The nucleotide
sequences of human Tel-2a, Tel-2b, Tel-2c, Tel-2d,
Tel-2e, and Tel-2f, with the deduced amino acid
sequences (one-letter code) of the major open reading frames are shown.
Nucleotides and amino acids are numbered. The full-length
sequence of Tel-2b is shown (a). The
amino-terminal alternatively spliced exons of Tel-2a,
Tel-2c, and Tel-2d (b) and the COOH-terminal
alternatively spliced exons of Tel-2e/Tel-2f
(c) are shown below. The Tel-2a alternative exon
3a is boxed and shaded and marked on the
right side. The two alternative overlapping out-of-frame
open reading frames for Tel-2a are indicated
below the nucleotide sequence. Alternative internal
translation start methionines in Tel-2a are
underlined. The arrows indicate the splice sites
and deletions of exons are indicated above the sequence. The
Pointed domain, the Ets domain, and the C domain are boxed
and shaded and marked on the right side. The
termination codons in-frame with the reading frame upstream and
downstream are indicated by asterisks. The putative
polyadenylation sequences, ATAAA, close to the polyadenylated 3' end of
the mRNAs are double underlined.
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Fig. 2.
Genomic organization of the human
Tel-2 gene. a, the human
Tel-2 genomic structure is shown on the top.
Exons 1 to 9 are represented by the filled boxes and the
introns in between by lines. The lengths of exons (bp) and
introns (kilobases) are shown below the exons and
above the introns. Functional domains are indicated. The
exon configuration of the six alternative Tel-2 splice
products is shown below the genomic structure. b, schematic
description of the expected proteins encoded by the six alternative
Tel-2 isoforms. Functional domains and the location of
potential MAP kinase phosphorylation sites (MAPK) are
indicated above the figure. c, nucleotide
sequence of the immediate 5'-flanking sequence of the human
Tel-2 gene. The apparent major transcription start site is
indicated by the arrow and 1. Potential
regulatory elements in the promoter region are boxed.
B-, AP1-, Ikaros-, and
HLH-binding sites are present in the immediate upstream region.
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Fig. 3.
Sequence comparison of Tel-2.
a, comparison of the amino acid sequence of Tel-2b with that
of Tel. The predicted amino acid sequence of full-length human Tel-2b
and human and mouse Tel was compared with each other. Amino acids that
are identical in at least two proteins are shaded. Gaps are
introduced to optimize the alignment. The two major homology regions,
the Pointed and Ets domain, are boxed. b,
comparison of the Ets domain of Tel-2 with those of all known
members of the Ets gene family. Percent identity of each Ets
domain with Tel-2 is indicated on the right side.
Shaded amino acids denote amino acid identity with Tel-2.
Gaps are introduced to optimize alignment. The Ets proteins
examined are indicated on the left side. Only human and
Drosophila ETS factors are included for simplicity.
GenBankTM accession numbers: ERG, M21536; ERG-B/FLI-1,
Y17293; ETS-3, M88473; FEV, Y08976; ETS-6, M88475; ERF, U15655;
PE-1/ETV3, L16464; GABP- /E4TF1-60, Q06546; ELG, M88471; ETS-1,
X14798; ETS-2, AF017257; POINTED, S33167; ER71/ETV2, AC002115; ERP/NET,
Z36715; SAP-1, P28323; ELK1, P19419; ER81/ETV1, U17163; ERM, X96381;
PEA3/E1AF/ETV4, U18018; ETS4, M88474; PDEF, AF071538; MEF, U32645;
NERF, U43188; ELF-1, P32519; E74, A53225; TEL/ETV6, U11732; TEL-2,
AF116509; YAN, Q01842; ESE-1, U73844; ESE-2, AF115402; ESE-3, AF124439;
PU.1, X52056; SpiB, X66079; SpiC, AF098863. Tentative
-helices and
-sheets as predicted by NMR and x-ray crystallography for some of
the Ets domains are indicated above the sequence by
boxes, respectively, arrows. c,
comparison of the Pointed domain of Tel-2 with all known members of the
Ets gene family and selected members of the SAM domain superfamily.
Shown are the homologies between the Tel-2 Pointed domain and the
corresponding regions present in a subset of the Ets family. Only human
and Drosophila Ets factors are included for simplicity. The
consensus line indicates which residues are highly conserved.
GenBankTM accession numbers: see legend to b.
The sequences are compared with the SAM domain containing proteins PH
(Drosophila Polyhomeotic, P39769), SCM
(Drosophila Sex comb on Midleg, U49793) and EphB2 receptor
tyrosine kinase (L25890).
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Fig. 4.
Differential expression of Tel-2
and the different isoforms in various human fetal and adult
tissues and cell types. a, expression of Tel-2 in
different human fetal and adult tissues. PCR analysis of cDNA
reverse transcribed from poly(A)+ mRNA from human fetal
and adult tissues as indicated above the figure using primers specific
for the amino-terminal splice variants of Tel-2 (upper
panel), the COOH-terminal exon 9 Tel-2e/Tel-2f splice variants (second
panel), the COOH-terminal exon 8 Tel-2 variants
(third panel), or GAPDH (lower panel).
Peripheral blood leukocytes (PBL). The different
Tel-2 splice variants are indicated on the right
side. Below the figure is a schematic diagram of the intron/exon
structure of the Tel-2 gene. Arrows below the
exons indicate the location of the different PCR primers (see also
"Materials and Methods," "Results," and Fig. 2). b,
expression of Tel-2 in various epithelial and nonepithelial
cell types. RT-PCR analysis of reverse transcribed poly(A)+
mRNA from human foreskin epithelium, LNCaP (human prostate), HEK293
(human epithelial kidney), C-33A (human cervical carcinoma), A431
(human vulvar carcinoma), HeLa (human cervical carcinoma), H157 human
(large cell lung carcinoma), H249 (human small cell lung carcinoma),
HUVEC (human endothelial cells), U-87 Mg and U-138 Mg (human glioma
cells), fetal brain, U-937 (human monocytes), human synovial
fibroblasts and human chondrocytes using Tel-2 (upper
panel), or GAPDH (lower panel) specific
primers as described under "Materials and Methods."
View larger version (68K):
[in a new window]
Fig. 5.
Interaction of Tel-2 with functionally
relevant Ets sites. a, in vitro translation
products of unprogrammed reticulocyte lysate ( ), full-length Tel-2a,
Tel-2b, and Tel-2c separated by SDS-polyacrylamide gel electrophoresis
and visualized by autoradiography. b, DNA binding of
full-length Tel-2a, Tel-2b, and Tel-2c in an EMSA using synthetic
oligonucleotides coding for the E74 Ets site. EMSAs of in
vitro translated Tel-2a, Tel-2b, and Tel-2c incubated with the
labeled E74 oligonucleotide probe were carried out with either no
competitor (left panel), 1, 5, 25, and 50 ng of unlabeled
wild type E74 oligonucleotide or mutant E74 oligonucleotide
(right panel). The arrows indicate the specific
DNA-protein complexes. c, EMSA supershift assay
demonstrating DNA binding specificity of full-length HA-tagged Tel-2a,
Tel-2b, and Tel-2c. EMSAs of in vitro translated HA-tagged
Tel-2a, Tel-2b, and Tel-2c or the empty pCI/HA vector incubated with
the labeled E74 oligonucleotide probe were carried out with either no
antibody, anti-HA tag antibody, or as a control anti-mouse IgG
antibody. The arrows indicate the specific DNA-protein
complexes. d, interaction of Tel-2b with functionally
relevant regulatory sites in different genes. Relative DNA binding
activities of Tel-2b for different Ets-binding sites in an EMSA using
synthetic oligonucleotides coding for the E74 site. Tel-2b was
incubated with the labeled E74 oligonucleotide probe in the presence or
absence of unlabeled competitor oligonucleotides containing
functionally relevant Ets sites from transcriptional regulatory regions
of different genes. Sequences present in the regulatory regions of
various genes that have been shown to bind Ets-related factors
including blk, lyn, E74, polyoma PEA3,
IgH enhancer
and µB sites (33, 94), HIV-2
LTR CD3R, HTLV-1 LTR, c-fos SRE,
TCR
enhancer T
2, SPRR2A, Endo A, and MHC class II
promoter are shown.
site, IgH enhancer µB site, c-fos
SRE, MHC II, TCR
enhancer, and
polyoma virus PEA3 did not compete (Fig. 5d).
These results demonstrate that Tel-2 binds specifically to a subset of
functionally relevant Ets-binding sites in a variety of genes. Table
I summarizes the results obtained in this
competition analysis indicating the relative binding affinity of the
different sites for Tel-2b and the DNA sequence of the binding core.
Based on this experiment we have compiled a putative high affinity
consensus binding site for Tel-2b (Table I, at the bottom) which is
very similar to the consensus recognition sequences for many other Ets
factors.
The relative DNA binding affinities of full-length Tel-2b towards each
site as determined by EMSA (see Fig. 5c) are shown on the right. A
potential consensus high affinity binding site for Tel-2b based on
comparing the sequences of the high and medium affinity Tel-2-binding
sites is summarized at the bottom. Capital letters designate
nucleotides present in high affinity binding sites, whereas small case
letters indicate nucleotides in lower affinity binding sites.
56 (55).
56-pGL3 containing only the minimal
c-fos promoter expressed very little luciferase activity
above the background of the parental promoterless pGL3 vector (Fig.
6a). Two copies of the wild
type E74 promoter Ets site enhanced transcription more than 50-fold as
compared with the minimal c-fos promoter due to the action of endogenous Ets factors. Co-transfection with the Tel expression vector resulted in a ~3-fold transcriptional repression of the E74
site (56, 57). Similarly to Tel, the Tel-2b expression vector strongly
repressed E74 transcriptional activity by ~6-fold indicating that the
full-length Tel-2 protein is an at least as or more potent repressor
than Tel (Fig. 6a). Tel-2a and Tel-2c, in contrast, did not
significantly affect E74 transcriptional activity, but Tel-2a actually
slightly and reproducibly enhanced transcription suggesting that the
Tel-2 Pointed domain alone or the Tel-2 Ets DNA-binding domain alone
are insufficient to repress transcription. These results also support
the notion that Tel-2 repression of transcription is an active process
presumably involving corepressors as shown for Tel (57, 58) and both
the DNA-binding domain and the Pointed domain and is not just a result
of competition for DNA binding. Alternatively, proteins generated by
Tel-2a or Tel-2c may not be stable or may not be translocated to the
nucleus. To evaluate whether all Tel-2 isoforms generate stable
proteins at similar levels after transfection into cells, the HA-tagged Tel-2 isoform expressing vectors were transfected into COS cells. Whole
cell extracts of transfected cells were analyzed by EMSA and compared
with in vitro-translated Tel-2 demonstrating that Tel-2 b
and Tel-2c express large quantities of proteins able to bind to DNA
which comigrated with the in vitro translated Tel-2 isoforms, whereas Tel-2a generated significantly lower levels of
DNA-binding proteins (Fig. 6b). Indeed although Tel-2c was unable to
repress luciferase activity, the level of Tel-2c protein after
transfection appeared to be even higher than Tel-2b. Specificity of the
protein-DNA complexes was confirmed using the anti-HA tag antibody
(Fig. 6b). Since this assay measured the ability of
transfected Tel-2 to bind to DNA, these results further support the
notion that DNA binding alone (Tel-2c) is not sufficient to act as a repressor of transcription.
View larger version (31K):
[in a new window]
Fig. 6.
Tel-2b, but not Tel-2a or Tel-2c acts as a
potent transcriptional repressor. a, Tel-2b is a strong
repressor of transcription. CV-1 cells were co-transfected with the
indicated Tel-2 and Tel expression vector constructs or the parental
pCI expression vector and a pGL3/c-fos promoter luciferase construct
containing the E74 Ets site oligonucleotide or the empty pGL3/c-fos
promoter plasmid. Luciferase activity in the lysates was determined as
described. Data shown are means of triplicate measurements from one
representative transfection. The experiment was repeated four times
with different plasmid preparations with comparable results.
b, EMSA and supershift of full-length HA-tagged Tel-2a,
Tel-2b, and Tel-2c after transfection into COS cells. EMSAs of in
vitro translated (in vitro) and transfected (in
vivo) HA-tagged Tel-2a, Tel-2b, and Tel-2c or the empty pCI/HA
vector incubated with the labeled E74 oligonucleotide probe were
carried out with either no antibody or anti-HA tag antibody. The
arrows indicate the specific DNA-protein complexes.
c, CV-1 cells were co-transfected with the indicated amounts
of Tel-2a, Tel-2b, or Tel-2c pCI expression vector constructs or the
parental pCI expression vector and luciferase constructs containing
either the E74 Ets site, the lyn promoter, or the
IgH enhancer upstream of the minimal c-fos
promoter. Luciferase activity in the lysates was determined as
described. Data shown are means of triplicate measurements from one
representative transfection.
56/pGL3) together with increasing amounts
of the different Tel-2 expression vectors. Increasing amounts of Tel-2b led to a dose-dependent repression of the lyn
promoter similar like for E74 correlating with the ability of Tel-2 to
bind with high affinity to the lyn promoter Ets site (Fig.
6c). In contrast, Tel-2b did not significantly repress the
IgH enhancer which contains two relevant Ets sites that do
not interact with Tel-2 with high affinity (Fig. 6c). Again
Tel-2a slightly enhanced lyn promoter activity at higher doses.
and BMP-6 Genes Are Target Genes for Tel-2b in MG-63
Osteosarcoma Cells--
To understand more about the biological role
of Tel-2 and to determine the physiological targets for Tel-2 we used
transcriptional profiling methods. Human MG-63 osteosarcoma cells which
can be transfected to 80-90% efficiency were transiently transfected with the Tel-2b expression vector (pCI/Tel-2b) or the parental expression vector (pCI). Total RNA was isolated 18 or 20 h after transfection and reverse transcribed into cDNA.
32P-Labeled cDNA probes derived from two independent
experiments each were hybridized to the Human 1.2, Human 1.2 II, and
Human Cancer 1.2 Atlas Microarray membranes that contain each more than 1100 genes. Autoradiographs were analyzed using the
CLONTECH Atlas Image software. The majority of
hybridizing genes gave similar relative intensities of hybridization
indicating the reproducibility and that expression of most genes did
not change significantly upon expression of Tel-2b. Only a small number
of genes (RAR
, BMP-6, HIV-EP2, and
thymosin
-10) were affected by Tel-2b, the majority of them being repressed by Tel-2b as could be expected from
our luciferase experiments. A small section of two autoradiographs of
the Human 1.2 II Atlas array is shown in Fig.
7a highlighting the similar
relative expression pattern of most genes and the differential
expression of RAR
. Thymosin
-10
was the only gene up-regulated 2.5-3.2-fold in the two experiments,
whereas RAR
was repressed 4-5-fold, BMP-6
5-10-fold, and interleukin-6 2-fold. To validate the
microarray data with an independent method we performed RT-PCR for the
RAR
and BMP-6 genes with the RNAs derived from
the transfected MG-63 cells (Fig. 7b). Whereas the
amplification product for the control GAPDH gene did not
differ in intensity among the different samples, a drastic reduction in
amplification was observed for both RAR
and
BMP-6 in Tel-2b-transfected cells when compared with the
control cells. These results confirm that Tel-2b represses expression
of the RAR
and BMP-6 genes in MG-63 cells.
Since both RAR
and BMP-6 are inducers of cell
differentiation, their repression by Tel-2b implies a role for Tel-2b
in blocking cell differentiation.
View larger version (37K):
[in a new window]
Fig. 7.
Tel-2b represses RAR
and BMP-6 gene expression in MG-63 osteosarcoma
cells. a, transcriptional profiling of MG-63 cells
transfected with pCI/Tel-2b or pCI. CLONTECH Atlas
cDNA microarrays were hybridized to 32P-labeled
cDNA probes generated from total RNA isolated from transfected
MG-63 cells. Shown are the autoradiographs of a small section of the
Atlas Human 1.2 II microarrays from pCI and pCI/Tel-2b transfected
cells that include the hybridization signals for the RAR
gene (arrow). b, expression of RAR
and BMP-6 in MG-63 cells after transfection with pCI/Tel-2b
or pCI. RT-PCR analysis of reverse transcribed total RNA from MG-63
cells after transfection with pCI/Tel-2b or pCI for 18 h and
20 h using BMP-6 (upper panel),
RAR
(middle panel), or GAPDH
(lower panel) specific primers as described under
"Materials and Methods."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, play important roles in
development and cell differentiation and, in particular, regulate bone
remodeling and osteoblast differentiation (64-72). Indeed some of the
effects of BMP-6 on osteoblast differentiation are similar to the
effects of retinoic acid on osteoblasts (66). But whereas BMP-6 induces
osteoblast differentiation in all models studied, retinoic acid effects
on osteoblast cultures and cell lines have been less straightforward,
ranging from the induction to inhibition of differentiation (65, 68,
71-76). Because Tel-2b inhibits BMP-6 and RAR
expression in MG-63 cells, Tel-2b is expected to play a critical role
as an inhibitor of cell differentiation.
4
-sheet based on three-dimensional structure analyses of other
Ets factors, a domain critical for DNA binding (79, 80). Tel-2e/Tel-2f
also eliminate an acidic domain COOH downstream of the Ets domain which
is conserved in both Tel-2 and Tel. No function has been attributed to
domain C yet, but its acidic nature may suggest a role in
transactivation. Whether similar isoforms exist for Tel is not clear.
The function of the different Tel-2 isoforms is not yet clear, since
Tel-2b itself appears to act as a repressor. A protein encoding
exclusively the Pointed domain of Tel may be able to interact with
proteins that normally interact with Tel-2b and, thus, might limit or
modulate the function of Tel-2b. However, this protein may also lack a
nuclear localization signal and may, thus, not translocate to the
nucleus. A Tel-2 protein lacking the Pointed domain should still be
able to bind to the Tel-2 DNA-binding sites, and may not act as an
active repressor, but as a weaker repressor due to competition with
other Ets factors or as a transcriptional enhancer. Our co-transfection
experiments indeed demonstrate that only Tel-2b is a strong repressor.
Interestingly, the Tel-2a isoform appears to enhance transcription
slightly, possibly due to competition for a corepressor or elimination
of a repressor domain in the truncated protein. Whether Tel-2 contains also a transactivation domain whose activity may be blocked by the
repression domain, is not yet clear. It is possible that
phosphorylation of Tel-2 may switch Tel-2 from a repressor into an
enhancer as has been shown for the ets factor ERP/NET (37, 81).
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge fruitful discussions with Dr. Phil Auron and Koen Kas.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants RO1 CA76323 and CA72009 (to T. A. L.) and National Institutes of Health Grant KO8/CA 71429 (to P. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF116508 (Tel-2a), AF116509 (Tel-2b), AF116510 (Tel-2c), AF218235 (Tel-2d), AF218365 (Tel-2e), and AF218366 (Tel-2f).
Contributed equally to the results of this work.
§ To whom correspondence should be addressed: New England Baptist Bone & Joint Institute, Dept. of Medicine, Beth Israel Deaconess Medical Center, 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-667-3393; Fax: 617-975-5299; E-mail: tliberma@caregroup.harvard.edu.
Published, JBC Papers in Press, December 6, 2000, DOI 10.1074/jbc.M010070200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PCR, polymerase
chain reaction;
RT, reverse transcriptase;
RACE, rapid amplification of
cDNA ends;
HA, hemagglutinin;
EMSA, electrophoretic mobility shift
assay;
RAR, retinoic acid receptor
;
BMP, bone morphogenetic
protein;
PBL, peripheral blood leukocytes;
bp, base pair;
MAP, mitogen-activated protein;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
HIV, human immunodeficiency virus;
LTR, long terminal
repeat;
MHC, myosin heavy chain.
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