From the Medical Research Service, Veterans Affairs
Puget Sound Health Care System, Seattle, Washington 98108, the
¶ Division of Molecular Medicine, Fred Hutchinson Cancer Research
Center, Seattle, Washington 98109, and the § Divisions
of Hematology and Oncology, Department of Medicine, University of
Washington School of Medicine, Seattle, Washington 98195
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The tel gene, recently shown to be translocated in a spectrum of acute and chronic human leukemias, belongs to the ets family of sequence-specific transcription factors. To determine the role of Tel in normal hematopoietic development, we used the tel gene as the bait in the yeast two-hybrid system to screen a hematopoietic stem cell library. Two partners were identified: Tel binds to itself, and Tel binds to the ets family member Fli-1. In vitro and in vivo assays confirmed these interactions. In transient transfection assays, Fli-1 transactivates megakaryocytic specific promoters, and Tel inhibits this effect of Fli-1. Transactivation studies using deletion mutants of Tel, and the Tel-AML-1 fusion protein, indicate that the helix-loop-helix domain of Tel only partially inhibits transactivation and that complete inhibition requires the full-length Tel molecule, including the DNA binding domain. The Tel and Fli-1 proteins are expressed early in hematopoiesis, and the inability of Tel fusion proteins such as Tel-AML-1 to counteract Fli-1 mediated transactivation may contribute to the malignant phenotype in human leukemias where this fusion protein is present.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acute and chronic human leukemias are frequently associated with chromosomal translocations which result in the formation of chimeric proteins. Considerable evidence indicates that these chimeric proteins play a role in transformation (1). At least one member of the chimeric protein is frequently a transcription factor, implicating disordered regulation of target genes as a mechanism of transformation. In most cases the role of these transcription factors in normal hematopoiesis has not been determined.
Recently a member of the ets family of transcription
factors, Tel, was identified at the site of chromosomal breakage in
chronic myelomonocytic leukemia (CMML) where it forms a chimeric
protein with the transmembrane and tyrosine kinase domains of the
platelet-derived growth factor receptor chain (PDGFR
) (2). Tel
has now been shown to be involved in a number chromosomal
translocations in human leukemias. The Tel-AML-1 fusion is present in
approximately 40% of cases of childhood pre-B cell acute lymphoblastic
leukemia (ALL),1 making it
the most common molecular abnormality in childhood cancer (3-5). In a
number of cases involving tel translocations, the remaining
tel allele is deleted, suggesting that loss of functional Tel may contribute to leukemic transformation.
Tel contains the highly conserved ETS DNA-binding domain at the carboxyl-terminal region as well as a distinct 5' region with weak homology to the well described helix-loop-helix (HLH) domain (also referred to as the pointed domain) (2). The predicted HLH secondary structure in the amino-terminal region suggests that this region may be involved in protein-protein interactions (6).
To identify potential protein partners of Tel, we screened a hematopoietic stem cell library using Tel in the bait plasmid (7). We identified two partners of Tel, Tel itself, and the ets protein Fli-1.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two-hybrid Library Screening--
Two-hybrid screens and parent
vectors pBTM116 and pVP16 were as described by (8). Plasmid
pLexA-Tel-(1-371) was constructed by insertion of the Tel polymerase
chain reaction (PCR) product encoding amino acids from 1 to 371 into
EcoRI-BamHI sites of pBTM116, resulting in an
open reading frame encoding a LexA-Tel fusion protein. Tel was
constructed by PCR with the following primers: 5'-CGGAATTCCGGATGTCTGAGACTCCTGCTCAGT-3' (coding strand) and
5'-CGGGATCCCGTGAGGTGGACTGTTGGTTCCTTC-3' (noncoding strand). The
PCR product was cut with EcoRI and BamHI, and the
1125-bp fragment was cloned. The EML-1 cell line cDNA library was
amplified once in DH5 and transformed into yeast containing
pLexA-Tel-(1-371) (7). Plasmids pVP-Tel-(19-163), pVP-Fli-(32-334),
and pVP-Fli-(14-230) were identified as clones that activated lacZ
transcription and conferred histidine prototrophy in the presence of
pLexA-Tel-(1-371). Binding specificity was confirmed in the mating
test with specific bait pLexA-Tel-(1-371) versus
nonspecific bait pLexA-Lamin (pBTM116 expressing LexA-Lamin fusion
protein). Plasmids were sequenced by automatic fluorescent sequencing
(Applied Biosystems).
In Vitro Transcription Translation and Protein Binding
Assay--
In vitro transcription-translation was performed
in TNT rabbit reticulocyte lysate (Promega, Madison, WI) in the
presence of L-[35S]methionine (specific
activity 37.0 TBq/mmol) (Amersham Pharmacia Biotech). All clones were
transcribed under the control of T7 promoter. In each case, 2 µl of a
25-µl translation reaction was analyzed by SDS-PAGE (see Fig.
2A.). Radiolabeled Fli-1 protein (5 µl of translation
reaction) was mixed with one of each radiolabeled proteins, PU.1, CD18,
CD11a, RAR, Luciferase, or Tel-(1-371) (5 µl of translation
reaction in each case) and immunoprecipitated with mouse monoclonal
anti human Fli-1 antibody (PharMingen, San Diego, CA) and assayed by
SDS-PAGE followed by autoradiography.
GST Fusion Protein Binding Assay--
Plasmids pGST-Fli and
pGST-Tel-(1-371) were cloned by linking the full-length coding
sequence of Fli-1 protein (amino acids 1-452) and the coding sequence
for amino acids from 1 to 371 of Tel into
BamHI/XhoI and EcoRI/NotI
sites of pGEX4T-1 vector (Amersham Pharmacia Biotech), respectively.
This resulted in vectors expressing GST-Fli-1 and GST-Tel fusion
proteins. Both fusion proteins were expressed in DH5 cells according
to directions from the GST gene fusion system (Amersham Pharmacia
Biotech). Cell lysates were obtained by sonication 4-6 times for
15 s on ice. GST fusion proteins were bound to the
glutathione-Sepharose by incubating 500 µl of bacterial cell lysate
with 20 µl of Sepharose beads for 1 h at 4 °C and assayed by
SDS-PAGE followed by autoradiography.
In Vivo Tel-Tel Protein Binding Assay--
For the in
vivo protein binding assay, coding sequence for full-length Tel
protein (amino acids 1-452) was cloned into the XhoI/XbaI site in pCS2+MT vector expressing six
Myc domains (9). This cloning resulted in a vector expressing the
fusion Myc-Tel proteins with the Myc domain located at the amino
terminus of Tel protein. Wild type protein was expressed from pSG5-Tel
vector (full-length Tel coding sequence for amino acids 1-452 in
EcoRI/BglII site of pSG5 vector). 293 cells were
cultured on 100-mm tissue culture plates in Dulbecco's modified
Eagle's medium plus 10% fetal bovine serum. Transfections were
performed with 10 µg of each plasmid with the calcium phosphate
method (10). Forty eight h after transfection, the cells were washed
with PBS and lysed by incubation for 10 min at 4 °C in cell lysis
buffer (150 mM NaCl, 20 mM Tris (pH 8.0), 5 mM EDTA, 1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 0.1 mM aprotinin, 1 mM leupeptin). 20-100 µl of 293 cell extracts were
immunoprecipitated with mouse monoclonal anti-Myc antibody (myc
9E10, Sigma) or nonspecific mouse IgG (Sigma). Immunoprecipitated
proteins were resolved by electrophoresis in 7.5% SDS gels. Protein
immunoblots were probed with Tel antiserum produced by five sequential
immunizations of a rabbit with synthesized peptide
H-TNHRPSPDPEQRPLRC-OH (Research Genetics, Huntsville, AL) emulsified
with Freund's Adjuvant. This antiserum detected all forms of human Tel
protein used in this study on Western blots and showed no
cross-reactivity with any other human protein.
In Vivo Tel-Fli-1 Protein Binding Assay-- Human K562 erythroleukemia cells were cultured in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. For electroporations, 300 µl of ~8 × 106 cells/ml, washed and resuspended in RPMI 1640, was mixed with 10 µg of pL(Fli-1)SN plasmid (wild type human Fli-1 coding sequence ligated into HpaI/XhoI site of pLXSN vector) and 10 µg of pSG5-Tel-(1-452) in a 2-mm gap electroporation cuvette (BTX, San Diego, CA). Cells were electroporated with 275 V, 600 microfarads, and 13 ohm using Elecro Cell Manipulator 600 (BTX). After 48 h of incubation in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum, cells were harvested and lysates were prepared as described previously. 100-200 µl of lysate was immunoprecipitated with mouse monoclonal anti-human Fli-1 antibody (PharMingen). Rabbit polyclonal antiserum directed against the human Tel protein was used for detection.
Western Blot Detection of Tel and Fli-1 in CD34+ Cells-- CD34+ cells were purified and cultured as described (11). For differentiation, growth factors interleukin-3 (IL-3), IL-6, G-CSF, and GM-CSF and stem cell factor (SCF) were added to a final concentration of 50 ng/ml. Lysates were created from identical numbers of cells at designated time periods. Anti-Tel and anti-Fli-1 antibodies were used to detect their respective proteins.
Transactivation Luciferase Assays-- For transactivation assays, 293 cells were transiently transfected by the calcium-phosphate method with 5 µg of each of the indicated plasmids, and cell lysates were prepared after 48 h of incubation. For internal control of transfection efficiency, all samples were co-transfected with the vector pCMV-GH (Nichols Institute, San Juan Capistrano, CA) expressing human growth hormone. Twenty µl of cell lysate was assayed for luciferase activity by using Promega luciferase assay kit according to the instructions of the manufacturer. The activity was assayed by using a Turner two-dimensional 20e luminometer. The growth hormone assay was performed by the use of HGH-TGES 100T kit (Nichols Institute) according to the instructions of the manufacturer.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
To identify potential partners of Tel in normal hematopoietic cell development, we used a LexA-Tel fusion protein consisting of the first 371 amino acids of Tel to screen a yeast two-hybrid library constructed from a murine pluripotent hematopoietic cell line (EML-1) (7). The fragment of Tel used in the bait plasmid contains the HLH domain. Five positive clones were identified from 2 × 106 transformants screened. Two ets family members were identified as partners of Tel, Tel itself and Fli-1 (Fig. 1). The two 435-bp Tel cDNA clones that were isolated include the entire HLH domain, and the three positive clones coding for Fli-1 also include the HLH domain (Fig. 1) (12). These results suggest that the amino-terminal HLH domain in each protein is involved in the interaction.
|
To confirm the specificity of the Tel/Tel and Tel/Fli-1 interaction, we conducted both in vitro and in vivo experiments. Initially, we expressed several proteins known to be involved in protein-protein interactions using rabbit reticulocyte lysates (Fig. 2A). These proteins were then used in a co-immunoprecipitation assay (Fig. 2B). Monoclonal antibodies directed against Fli-1 immunoprecipitated the human Tel protein (amino acids 1-371) but none of the control proteins (Fig. 2B, lane Fli-1 + Tel). Additional in vitro experiments addressed the specificity of protein-protein interactions. In in vitro studies, GST-Fli-1 bound to 35S-Met-Tel protein (amino acids 1-371) but not to control 35S-Met-Luciferase (Fig. 2C). Similarly, GST-Tel associated with S-Met-Tel (1-371 amino acid fragment), but not with 35S-Met-Luciferase (Fig. 2C).
|
In vivo experiments confirmed the interaction of Tel and Fli-1. When the Tel protein containing the Myc domain at its amino terminus and wild type Tel protein were expressed in 293 cells and immunoprecipitated with anti-Myc antibody, the anti-Myc antibody immunoprecipitated wild type Tel protein as well as the Myc-Tel protein (Fig. 2D). Neither mouse nonspecific IgG (Fig. 2D) nor anti-Myc antibody (data not shown) were able to immunoprecipitate the wild type Tel protein alone. The Tel-Fli-1 interaction in vivo was tested in K562 cells electroporated with expression vectors harboring wild type Fli-1 and wild type Tel. Cell lysates immunoprecipitated with an anti-Fli-1 antibody revealed the presence of the accompanying Tel protein on Western blots using an anti-Tel antibody (Fig. 2E). The Tel protein was not present in lysates immunoprecipitated with nonspecific mouse IgG and probed with the anti-Tel antibody (Fig. 2E).
To identify the stage of hematopoietic cell development at which Tel and Fli-1 are expressed, human CD34+ cells were differentiated with IL-3, IL-6, SCF, G-CSF, and GM-CSF, and the appearance of Tel and Fli-1 proteins was detected by Western blotting. Fli-1 protein was expressed at day 0, peaked at day 2 of differentiation, and decreased to undetectable levels by day 4 (Fig. 3A). Interestingly, the Tel protein displayed a similar pattern of appearance with a peak at 2 days of differentiation and a decrease to undetectable levels by day 4 (Fig. 3B).
|
To determine whether Tel and Fli-1 displayed a functional interaction,
the transactivation abilities of Tel and Fli-1 proteins were tested
individually and in concert on promoters containing ETS binding sites
(Fig. 4A). Recombinant Fli-1
has been shown to bind to DNA in a sequence-specific manner and to
transcriptionally activate the platelet glycoprotein IIb promoter (12,
13). In this study, we used the promoters from the related
megakaryocytic genes GPIb (14) and GPIX (15) which contain ETS
sequences. The GPIb
promoter region extended to
567 bp upstream
from the transcriptional start site, and the GPIX promoter region
covered 203 bp proximal to the transcriptional start site. Transfection of Fli-1 resulted in a 3.5-fold increase of activity of the GPIb
promoter and a 7-fold increase in the activity of the GPIX promoter (Fig. 4, B and C). Expression of Tel alone did
not activate either promoter; however, co-transfection of Tel with
Fli-1 together resulted in complete inhibition of Fli-1-mediated
transactivation (Fig. 4, B and C). Transfection
of Fli-1, Tel, or the two ets factors together did not
transactivate a GPIX promoter construct in which the ETS site was
deleted (pGPIX-EETS-Luc) (Fig. 4D). Thus, Tel inhibits
Fli-1-mediated transactivation of megakaryocytic promoters as GPIb
and GPIX (Fig. 4) and as GPIIb (data not shown), and the ETS binding
site in the promoter sequence is required for transactivation.
|
To analyze the mechanism whereby Tel inhibits Fli-1-mediated
transactivation in more detail, we generated deletion mutants of Tel.
The Tel H protein consists of the HLH domain of Tel, and Tel D consists
of the DNA binding domain of Tel (Fig.
5A). To assess whether Tel
fusion proteins identified in human leukemias are capable of inhibiting
the transactivation mediated by Fli-1, we also expressed the Tel-AML-1
fusion protein present in childhood acute lymphoblastic leukemia. These
constructs were tested in transactivation assays with Fli-1 on the GPIX
promoter. Transfection of the Tel and Fli-1 constructs were adjusted to
achieve expression of equivalent amounts of respective proteins (Fig.
5C). In these experiments, wild type Tel was able to
completely abrogate transactivation (Fig. 5B). The Tel H,
Tel D, and Tel-AML-1 constructs partially inhibited Fli-1-mediated
transactivation (Fig. 5B). Similar data have been obtained
using the GPIb promoter (data not shown). These data indicate that
full-length Tel protein is necessary to completely repress
Fli-1-mediated transactivation. The Tel H construct, lacking the DNA
binding domain, inhibits Fli-1-mediated transactivation by
approximately 50%, indicating that protein-protein interaction between
Tel and Fli-1 most likely accounts for this effect. The mechanism
whereby the Tel D construct inhibits transactivation has not been
established. We and others have not been able to demonstrate
DNA-binding activity by Tel (data not shown). It is possible that the
Tel DNA-binding domain may recruit a generalized transcriptional
repressor to the promoter.
|
To determine whether overexpression of the Tel truncation constructs abrogated Fli-1-mediated transactivation of the GPIX promoter, titration experiments were performed. Transactivation experiments were conducted using increasing molar ratios of Tel expression constructs (Tel, Tel H, Tel D, and Tel-AML-1) to Fli-1 expressing plasmid DNA (Fig. 6). Three sets of transactivations were performed with Tel constructs to Fli-1 DNA molar ratios of 1:1, 2.5:1, and 5:1, respectively. Increasing amounts of Tel H or Tel D protein resulted in only a small effect on the ability of these proteins to inhibit Fli-1 transactivation of the GPIX promoter (Fig. 6). The addition of increasing amounts of Tel-AML-1 resulted in a modest increase in the ability of the fusion protein to repress Fli-1-mediated transactivation. These results confirm our previous observation that the full-length Tel protein is necessary to completely inhibit Fli-1 transactivation of the GPIX promoter, and they indicate that the effect of the Tel mutant proteins appears to reach a plateau at a 1:1 molar ratio.
|
With Tel-AML-1, the expression of the chimeric protein in these studies may not represent the physiological level of the protein in cells from ALL patients because, in at least one ALL cell line expressing Tel-AML-1 protein (the REH cell line), the level of expression of Fli-1 was more than 10-fold higher then the expression of Tel-AML-1 (data not shown). The wild type Tel protein is not expressed by REH cells. In this cell line, it is possible that the low molar ratio of Tel-AML-1 protein to Fli-1 protein expression may not be sufficient to inhibit the activation of Fli-1 target genes in these ALL cells.
Ets family members have been identified at the site of chromosomal translocations in primitive stem cell tumors including leukemia (16). The identification of Fli-1 as a partner of Tel is of interest for several reasons. First, the interaction of Tel with Fli-1 was unexpected since ets family members have previously not been demonstrated to form heterodimers with others ets family members (17). Second, Fli-1 is involved in malignancies in both mouse and man. Mouse fli-1 (Friend leukemia integration site 1) gene was identified originally as a proto-oncogene insertionally activated in 75% of erythroleukemias derived from newborn mice infected with F-MuLV (Friend murine leukemia virus (18)). Erythroleukemia cell lines induced by F-MuLV expressed high levels of Fli-1 transcript (18). The human homologue of fli-1 is rearranged in Ewing's sarcoma and neuroepithelioma as a result of a reciprocal chromosomal translocation t(11;22) that fuses the DNA binding domain of Fli-1 to a novel amino-terminal sequence of the Ews gene (19). Thus, not only is Fli-1 involved in human and mouse malignancies, but its DNA-binding domain is preserved in both instances.
There are reasons to suspect that Tel and Fli-1 may have opposing roles in development. Whereas Fli-1 translocations preserve the DNA binding domain of Fli-1, the translocations involving Tel almost invariably result in the loss of the Tel DNA binding domain and preserve the helix-loop-helix domain of Tel in the fusion protein. This suggests that Fli-1 retains its ability to recognize its target genes as a fusion protein and Tel does not.
In Tel translocations, the chimeric Tel fusion protein may also dimerize with the normal Tel protein from the other allele and result in a loss of normal, wild-type Tel function (3-5). The observation that the remaining Tel allele is deleted in up to one-third of patients with the Tel/AML-1 translocation is consistent with the loss-of-function hypothesis of Tel as a result of the generation of the chimeric protein (3-5). Loss of Tel would also result in the loss of its ability to dimerize with Fli-1.
Future studies will address the effect of Tel and Fli-1, and their respective fusion proteins, on the process of malignant transformation.
![]() |
FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed: Medical Research
(151), Veterans Affairs Puget Sound Health Care System, 1660 S. Columbian Way, Seattle, WA 98108. Tel.: 206-764-2705; Fax
206-764-2827.
1 The abbreviations used are: ALL, acute lymphoblastic leukemia; HLH, helix-loop-helix; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; SCF, stem cell factor; PVDF, polyvinylidene difluoride; PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|