(Received for publication, March 9, 1995; and in revised form, July 26, 1995)
From the
The T-cell-specific protooncogene lck, a src-related tyrosine kinase, is under the control of two promoters that give rise to transcripts differing only in their 5`-untranslated regions. The distal promoter is transcriptionally active in both peripheral and thymic T-cells, whereas expression of the proximal promoter is highest in developing thymocytes. The proximal promoter has also been shown to be selectively activated in a number of colon carcinoma cell lines. Approximately 570 base pairs of proximal promoter sequence is required for expression in both T-cells and colon carcinoma cell lines. Protein binding studies were initiated with an oligonucleotide homologous to a region that, when deleted, causes an increase in promoter activity in transgenic animals. Two proteins with approximate molecular masses of 35 and 75 kDa were found to bind to this region as determined by UV cross-linking studies. Absence of specific protein binding is correlated with a high level of proximal promoter expression. Competitive gel retardation analysis identified a 9-base pair binding site within the proximal lck promoter that is necessary for repression of transcription in cells that contain specific binding activity. Mutants of this binding site do not repress transcription. Repression does not occur in a cell line that expresses lck and lacks this activity. These data support the hypothesis that activation of lck transcription in colon carcinoma is due, at least in part, to the loss of a transcriptional repressor.
In T-cells, expression of lck, a src-related tyrosine kinase, is driven by two promoters located approximately 35 kilobases apart that give rise to transcripts differing only in their 5`-untranslated regions(1) . Transgenes containing lck proximal promoter sequences are actively transcribed in thymocytes of transgenic animals, whereas in similar studies the distal promoter can support transcription in both thymocytes and peripheral T-cells(2, 3) . Therefore, these two apparently independently acting promoters may lead to both the developmental and cell type specific regulation of lck.
In T-cells lck is physically associated with the CD4 and CD8 components of the
T-cell receptor complex (4, 5) and has been reported
to be associated with the -chain of the interleukin-2
receptor(6) . Although the exact functions of lck in
T-cells are not known, it has been shown to be an essential component
of the T-cell receptor signaling pathway (7, 8) and
may also be involved in T-cell maturation in the thymus(9) . lck has been classified as a protooncogene because of its
homology to src and its activation by promoter insertion in
certain retroviral induced murine lymphomas (1, 3) and
by translocation in human T-cell leukemias(10) . Also,
transfection of NIH3T3 cells with an lck mutant that encodes
an activated protein kinase results in malignant transformation (11) . However, the functional significance of overexpression
of wild type lck to the transformed phenotype has yet to be
determined.
In addition to its expression in T-cells and T-cell lymphomas, lck is expressed in a subset of colon carcinoma cell lines derived from metastatic lesions as well as in some small cell lung carcinomas and B-cell leukemias(12, 13, 14) . In solid tumor cell lines, lck expression results from activation of the proximal promoter, and no obvious rearrangements or amplifications of the gene have been detected in these cell lines(12) .
The proximal lck promoter, like that of other src family
members(15, 16) , is TATA-less and contains multiple
start sites for initiation of transcription. Previously, deletion
analysis has determined minimal promoter sequences required for both in vitro(17) and in vivo(2) expression of this gene. These studies identified a
potentially important region between positions -565 and
-433, which, when deleted, caused an increase in expression of
both a reporter gene and of lck driven by the proximal
promoter in transgenic animals(2) . In order to investigate the
transcriptional regulatory mechanisms involved in the expression of lck in colon cancer we studied this region of the proximal
promoter. We now report that a novel sequence is located at positions
-474 to -466 and specifically binds two proteins of
approximate molecular mass 35 and 75 kDa. This binding activity is
present in a number of cell lines that do not express lck as
well as in a cell line that expresses only small amounts of lck. Specific binding was not detected using extracts from two
cell lines expressing high levels of lck. The 9-bp ()sequence acts to repress a heterologous minimal promoter
in cell lines that contain specific binding activity. Repression does
not occur in a colon carcinoma cell line that expresses high levels of lck and lacks this binding activity. Mutants of the sequence
that do not bind the proteins do not repress transcription. We propose
that activity of the proximal lck promoter is regulated by a
repressor that binds to this sequence and that expression of lck in colon cancer cell lines is, in part, due to loss of this
repression.
Gel retardation analyses were performed with a
double-stranded oligonucleotide, synthesized by the SKI Microchemistry
Core Facility, representing sequences -560 to -420 of
upstream lck sequence:
AACAGGCACACATTTATCACTTTACTCCTATGGAGTTCTGCTTGATTCATCAGACAAA (19) . Other oligonucleotides used in this study were derived
from this sequence and are described in detail in Fig. 2A. The AP1 consensus oligonucleotide was
purchased from Stratagene, and the nuclear factor of activated T-cells
(NFAT) oligonucleotide was a gift from S. Nimer (Memorial
Sloan-Kettering Cancer Center). Double-stranded oligonucleotides were
prepared by slow annealing and purified on an 18% polyacrylamide gel,
and 25 ng was P-end-labeled using T4 DNA kinase (Life
Technologies, Inc.) by standard methods(20) . Gel shifts were
done as described(21) . Gel shift buffer contained 20
mM Hepes, pH7.9, 75 mM NaCl, 0.5 mM EDTA, 1
mM dithiothreitol, 10 mM MgCl
. Each
reaction contained approximately 0.05 ng of oligonucleotide (20,000
cpm), 6 mg of BSA, 4 µg of poly(dI-dC), and 60 µg of crude
extract. These reactions were incubated at 30 °C for 30 min,
electrophoresed on a nondenaturing 6% acrylamide gel, dried, and
subjected to autoradiography.
Figure 2: Localization of the binding site by competition in a gel retardation assay. Panel A shows the -520 to -460 oligonucleotide used as a probe in the gel shift assay as well as the competitor oligonucleotides used to map the binding site. Oligonucleotides 1-7 show the mutations, either C to T or G to A conversions used as competitors. The deduced binding site is shown within the -520 to -460 region in boldface letters. Panel B is the gel shift analysis using HT29 nuclear extract and no competitor (0) or various competitors at different levels of molar excess, 1000-fold molar excess of a nonspecific competitor (N), the -520 to -460 oligonucleotide in 10, 100, and 1000-fold molar excess, and the 5`, middle, 3`, and mutant 1-3 competitor oligonucleotides also in 1000-fold molar excess. Panel C is identical to panel B except that the last four lanes are competitions with 1000-fold molar excess of mutant oligos 4-7.
A search for known transcription factors that bind the mapped sequence was performed using the Transcription Factor Data Base from the National Center For Biotechnology, National Library of Medicine.
Concatamerization of both the binding site and a corresponding mutant was performed by cloning double-stranded oligonucleotides with flanking BglII and BamHI sites into a pCAT vector constructed by H.L. Grimes. By utilizing an EcoRI site within the pCAT sequence and sequential BamHI/EcoRI and BglII/EcoRI ligations, the binding site was duplicated and cloned into a TKCAT vector, also provided by H. L. Grimes. All plasmids were sequenced using Sequenase in a procedure described by the manufacturer (U.S. Biochemical Corp.).
Proper initiation of transcription of the TKCAT templates was determined by RNase protection analysis (12) of total RNA isolated from transfected cells. The Riboprobe was comprised of sequences between -109 and +52 of the thymidine kinase (TK) promoter.
Figure 1:
Gel retardation analysis. Panel A shows the result of a gel shift assay using oligonucleotide
complementary to -520 to -460 of the proximal lck promoter and 60 µg of crude nuclear extracts from HT29. The
following competitors were used: 0, no competitor; N,
a nonspecific oligonucleotide in 1000-fold molar excess and 10, 100,
and 1000-fold molar excess of the -520 to -460
oligonucleotide. In panel B, the analysis in panel A was repeated using COLO205 nuclear extracts. On the right of panel B a consensus AP1 oligonucleotide was used in a
gel retardation using COLO205 extracts and 1000-fold molar excess of a
nonspecific oligonucleotide (N) and the AP1 oligonucleotide (1000 ).
Figure 3:
Molecular characterization of proteins
binding the -520 to -460 oligonucleotide. Panel A shows a 10% SDS-polyacrylamide gel electrophoresis gel indicating
the relative migration of the protein-DNA complexes from either HT29 or
SW620 nuclear extracts that bind to a doubly labeled probe
corresponding to -520 to -460 of the proximal promoter
after UV cross-linking. The molecular weight marker is listed at the left. The lane marked 0 is no competitor added; 100
and 1000
indicate where either 100- or 1000-fold molar
excess of specific competitor was used. Panel B shows the
migration patterns of both the upper and lower protein-DNA complexes
excised from gel retardations using either HT29 or SW620 nuclear
extracts.
Figure 4: The effect of a site-directed mutation in the mapped binding site on the proximal lck promoter. Comparison in HT29 and SW620 of promoter activity of the wild type and mutant measured by CAT activity, quantitated by phosphor imaging and expressed as relative to the luciferase activity obtained by cotransfection of Rous sarcoma virus-luciferase. Transfections were done in triplicate in three independent experiments. A representative experiment is shown.
Since loss of transcriptional repression at this site may not be sufficient to fully activate this promoter in HT29, the effect of the site on a minimal promoter was investigated. An oligonucleotide containing the binding site was cloned in one or two copies just upstream of the herpes simplex virus TK minimal promoter in a CAT-containing expression vector (Fig. 5A) and these constructs were used in transient transfection assays. The results of a representative experiment for HT29 and SW620 are shown in Fig. 5, B and C, respectively. Comparison of CAT activity driven either by the TK promoter alone or with one or two sites just 5` to the TK promoter reveals the ability of this element to completely abolish transcription from this minimal promoter element in HT29 (Fig. 5A), and identical results were obtained in HeLa (data not shown). These experiments were repeated in SW620, and although complete repression was not documented, this site caused an 8-fold reduction in promoter activity in this cell line. As a control, a mutant binding site, GGGCATACT, was also cloned upstream of the TK promoter, and the transient assays were repeated. For both HT29 (Fig. 5B) and SW620 (Fig. 5C) a mutant binding site that did not compete for protein binding in gel retardation analysis had no effect on the transcriptional activity of the TK promoter in either of these cell lines. To insure that the CAT activity measured was due to accurate transcription initiation in the TK promoter, RNase protection analysis was performed using a probe containing the TK promoter. Proper initiation of transcription of the TKCAT construct was observed (data not shown).
Figure 5: The binding site functions to repress transcription of the TKCAT construct. Graphs of transient expression of TKCAT as compared with expression obtained with either one or two wild type binding sites as well as mutant binding site (mut) cloned 5` of the TK promoter. All transfections were done in at least triplicate in three different experiments. Diagrams of the constructs used in this analysis are shown in panel A. The quantitation of one experiment is shown in panel B and panel C using either HT29 or SW620, respectively. CAT assays were quantitated by phosphor imaging and expressed as relative to luciferase activity measured by luminometry (RLU).
Since this sequence acts to repress transcription in cells containing specific binding activity, transfection studies were performed in a cell line lacking this activity. As shown in Fig. 6, the sequence does not suppress the TK promoter in COLO205, a cell line that expresses high levels of the proximal lck transcript. Proper transcript initiation was also observed in this cell line (data not shown). Thus, repression of promoter activity seems to require both the intact binding sequence and the binding activity.
Figure 6: Promoter activity is not affected by the binding site in COLO205. Transfections were performed as in Fig. 5using COLO205 as the recipient cell line. CAT assays were repeated three times in at least triplicate, quantitated by phosphor imaging, and expressed relative to luciferase activity. A representative experiment is shown.
In this study we have defined a sequence in the human lck proximal promoter located at positions -474 to -466, which acts as a strong repressor of transcription. The approximate molecular masses of proteins that specifically bind this sequence are 35 and 75 kDa. The binding activity was detected in four human tumor cell lines; three of these do not express lck (HT29, T84, and HeLa), and the other expresses nominal amounts (SW620). Specific binding was not detected in two cell lines that express high levels of proximal lck promoter transcripts. This sequence acted to repress transcription of a heterologous promoter in cell lines that contained the binding activity but had no effect on the promoter in a cell line lacking this activity. These data suggest that activation of the proximal lck promoter is at least partly due to a loss of transcriptional repression mediated by the loss of specific protein binding to this 9-bp sequence.
These findings are consistent with results from transgenic mice experiments, which demonstrated protein binding to an analogous 50-bp region in the murine proximal promoter with nuclear extracts from spleen, a tissue in which this promoter is inactive, and a lack of binding in cells expressing the transgene(2) . Comparisons between mouse and human have demonstrated a high degree of sequence conservation in the proximal promoter element, indicating that this promoter may be similarly regulated in both species. This conservation also extends to the binding site mapped to -474 to -466 in this study (7/9-bp). These data indicate that the murine system may have a functionally similar transcriptional repressor.
Our data, which show measurable
promoter activity using 570 bp of the human proximal promoter sequence,
varies somewhat from previously reported transient assays in which,
when using identical sequence, deletion to -512 was necessary to
show modest promoter activity(17) . In transgenic animals,
however, 584 bp of the murine proximal promoter sequence was sufficient
for promoter activity(2) . In this report CAT activity was
measured rather than primer extension of transient -globin mRNA,
possibly allowing for the detection of lower amounts of promoter
activity. The binding site located within the -520 to -460
oligonucleotide was mapped to the sequence TTTCATCAG at positions
-474 to -466 by competitive gel retardation assays. A
computer search of the Transcription Factor Data Base revealed no
strong homology to known transcription factor binding sites. However,
we noted some homology to the 3` portion of the composite binding site
for NFAT within the interleukin-2 promoter, i.e. TTTCATACAG,
the portion thought to bind Fos or
Jun(25, 26, 27) . The proteins detected in
this study are unlikely to be identical to those in the T-cell NFAT
complex, as neither an oligonucleotide containing an NFAT motif nor one
containing an AP-1 motif competes in 1000-fold molar excess in a gel
retardation assay (data not shown).
Although the region from -520 to -460 was shown to contain a repressive element (2) and gel retardation experiments correlated a lack of binding to this region with a high level of lck expression, site-directed mutagenesis of the binding site located at -474 to -466 had little effect on the expression of the intact 570-bp promoter in transient assays in SW620. However, the binding site does repress the transcriptional activity of a heterologous promoter in this cell line. Since the proximal lck promoter is activated in SW620 and deletion analysis to -433 of the human promoter had little effect on transcription in SW620(17) , it is possible that the promotor is activated at another site. However, a reproducible 2-fold increase in promoter activity was observed in HT29. It is likely that other factors are required for full activation of this promoter.
To determine the effect of the binding site on a minimal promoter the binding site was cloned upstream of a TK promoter containing CAT vector and used in transient transfection assays. The binding site acted to completely abolish transcriptional activity of this minimal promoter in both HeLa and HT29 and reduced transcriptional activity 8-fold in SW620. A mutated site that abolishes protein binding in gel retardation experiments had no effect on promoter activity, indicating that repression was sequence-specific. Since the binding site had no effect of transcription from this promoter in COLO205 it appears that the specific binding proteins, which were not detected by gel retardation, are either absent in this cell line or at least not able to bind to this site.
This paper describes a novel binding site in the proximal lck promoter that acts as a strong repressor of transcription in cell lines that either do not express proximal lck transcripts (HT29 and HELA) or only express small amounts (SW620). The correlation of repressor activity, protein binding activity, and lck expression suggests that this repressor does regulate lck transcription. Whether expression of the wild type lck transcript in colon carcinoma regulates aspects of the transformed phenotype remains to be established. However, the loss of a transcriptional repressor may have more global regulatory effects in colon cancer. In addition, the proximal lck promoter is most active in developing thymocytes; the transcriptional repressor complex defined in this report may play an important role in the silencing of this promoter in peripheral T-cells. Purification and cloning of the binding proteins will allow further characterization of the function of this repressor.