(Received for publication, March 14, 1996, and in revised form, October 17, 1996)
From Physiological Chemistry I, Theodor Boveri Institute for Biosciences (Biocenter), University of Würzburg, Am Hubland, 97074 Würzburg, Federal Republic of Germany
Melanoma formation in Xiphophorus is caused by overexpression of the Xmrk gene. The promoter region of the Xmrk oncogene differs strikingly from the corresponding proto-oncogenic sequences and was acquired in the course of a nonhomologous recombination with another gene locus, D. In order to identify regulatory elements leading to the strong transcriptional activation of Xmrk in melanoma tissue and to contribute to an understanding of the role the regulatory locus R might play in suppressing the tumor phenotype in wild-type Xiphophorus, we performed functional analysis of the Xmrk oncogene promoter. Transient transfections in melanoma and nonmelanoma cells revealed the existence of a potent positive regulatory element positioned close to the transcriptional start site. Contained within this promoter segment is a GC-rich sequence identical to the binding site described for human Sp1. In vitro binding studies and biochemical characterizations demonstrated the existence of GC-binding proteins in fish that share immunological properties with members of the human Sp family of transcription factors and appear to be involved in the high transcriptional activation of the Xmrk oncogene. Since the identified cis element is functional in both melanoma and nonmelanoma cells, additional silencer elements suppressing Xmrk expression in nonpigment cells must exist, thereby suggesting a negative regulatory function for the genetically defined R locus.
Melanoma formation in the teleost fish Xiphophorus provides an in vivo model for studies on the genetic basis of tumor induction and the role of receptor tyrosine kinases in neoplastic transformation (1, 2). In these animals tumors are caused by a single, dominantly acting gene locus, Tu. According to the model developed to explain melanoma formation in Xiphophorus (3), the tumor-inducing potential of the Tu locus is suppressed in wild-type fish by an unlinked locus, R, which is proposed to function as a tumor suppressor and is progressively eliminated upon crossing with a parental fish containing neither of the two loci. The stepwise displacement of R genes in Xiphophorus hybrids is thought to allow expression of the Tu phenotype, thus leading to benign or malignant melanoma depending on the copy number of R still present in the hybrid genome. The crossing data could, however, be similarly interpreted by assuming that instead of elimination of suppressor genes the introduction of intensifier genes leads to Tu-dependent melanoma formation. So far no genetic evidence could be obtained to decide between both possibilities.
The Xmrk1 oncogene, which is the critical constituent of the Tu locus, encodes a receptor tyrosine kinase with high similarity to the human epidermal growth factor receptor and is responsible for the hereditary malignant melanoma in Xiphophorus (4). Besides the oncogenic version of Xmrk, a second copy exists in the Xiphophorus genome. This proto-oncogenic version of Xmrk is present in all individuals of the genus examined so far (5) and is expressed at low levels in a variety of tissues.2 Expression of the Xmrk proto-oncogene is not associated with the tumor phenotype; however, the oncogene transcript is highly overexpressed in melanoma, whereas no expression is detectable in normal tissues in Northern blots (4, 7), and only very low level expression is seen in reverse transcriptase-PCRs (8). The amount of oncogene transcript found in the tumors is positively correlated with their malignancy (4, 7). It thus appears that it is the overexpression of the Xmrk oncogene which is primarily responsible for melanoma formation in Xiphophorus hybrids (9, 10).
Molecular genetic analyses revealed that both versions of
Xmrk, proto-oncogene and oncogene, are highly identical in
their coding region but differ significantly in their promoter regions (11). This situation is explained by a nonhomologous recombination event between the Xmrk proto-oncogene and another gene locus
(designated D) (12), giving rise to the oncogene as an
additional copy of Xmrk with altered 5 sequences. This
upstream region contains TATA- and CAAT-like sequences and could thus
represent a "non-housekeeping gene" promoter (13) in contrast to
the GC-rich sequences driving transcription of closely related receptor
tyrosine kinases like the human epidermal growth factor receptor (14)
or the rat HER2/neu gene (15). It is suggestive that these
newly acquired upstream sequences account for the observed
Xmrk overexpression in Xiphophorus hybrids and
that the R locus might be involved in their transcriptional regulation. Analysis of the Xmrk oncogene promoter and its
transcriptional control elements is therefore important to obtain
insight into the mechanisms leading to the tissue-specific
overexpression of the Xmrk oncogene. These analyses might
further help to decide whether a silencing or an enhancing mode of
action has to be proposed for the regulatory gene(s) encoded by the
R locus.
Here we report on the functional characterization of Xmrk promoter sequences leading to the identification of a potent cis element and its corresponding transacting factors enhancing transcription of the Xmrk oncogene. A GC box positioned close to the transcriptional start site was found to play a major role for Xmrk promoter activity in both melanoma and nonmelanoma cells. Analyses of the corresponding transcription factors point toward the involvement of fish homologues of human Sp proteins in the transcriptional control of Xmrk. Since transcriptional activation is not restricted to melanoma cells, the existence of additional potent silencer elements down-regulating Xmrk expression in nonpigment cells has to be postulated, thus supporting the possibility that the R locus might exert a negative regulatory function on the Xmrk oncogene.
To obtain sequences of the Xmrk promoter
upstream of the previously isolated region (11), a PCR was performed on
genomic DNA from Xiphophorus maculatus of the genotype
SdDr/SrAr using a downstream primer from the
Xmrk breakpoint region (DA 11) and an upstream primer (JA 8)
derived from the D locus (12). PCR amplification was
performed in a total volume of 50 µl with 200 ng of genomic DNA as
template and 2.5 units of Taq polymerase. The buffer
conditions were 100 mM Tris-Cl, pH 9.0, 50 mM
KCl, 1.5 mM MgCl2, 0.1% gelatin, 1% Triton
X-100. After initial denaturation for 4 min at 92 °C, amplification
was performed for 35 cycles in a two-step PCR with 70 °C as
annealing/extension temperature. The primers used had the following
sequences. DA 11, 5-CCTTTCTGTCCGGGTCTGTGCTGCAGCAG-3
; JA 8, 5
-CTCGGATCCCTCAAGGCAGACTGG-3
.
The resulting 0.8-kilobase amplification product was specific for the
Xmrk oncogene Sd allele.3 To
minimize the risk of cloning DNA stretches containing mutated nucleotides as a result of polymerase errors only a
BamHI/EcoRI fragment upstream of the previously
identified promoter region was inserted into Bluescript II KS(+) to
create pBSXmrk675/
272, and four independent plasmid clones were
sequenced.
XmrkCAT277/+34 contains a
HindIII/NcoI fragment spanning the
Xmrk promoter (11) and parts of the CAT coding region in the backbone of pBLCAT6 (16). The 397-bp insert of pBSXmrk
675/
272 was
cloned into XmrkCAT
277/+34 to create XmrkCAT-675/+34. A 5
deletion
series was constructed using the following restriction sites:
StuI (
194), Sau3A (
166), TaqI
(
125), and EcoO109I (
49).
Insertion of a BamHI/HindIII Xmrk
promoter fragment containing residues 277 to +34 into
pBLCAT5/BamHI/HindIII resulted in XmrktkCAT
277/+34 rev. All other XmrktkCAT fusions were
constructed by inserting various Xmrk promoter fragments
into the blunted SalI site of pBLCAT5.
The plasmid ptkTATA CATII was constructed by replacing the
BamHI/BglII fragment from pBLCAT5 (16) containing
the tk promoter region from 105 to +51 with the corresponding
fragment from a linker scanning mutant ending at
32 (17).
To obtain XmrkTATACAT 67/
20, an 840-bp
BsaHI/BamHI fragment from XmrktkCAT
67/
20 was
inserted into ptkTATACATII opened with the same enzymes.
Double-stranded oligonucleotides Xmrk
67/
32, Xmrk
67/
32 mut,
and Sp1 (for sequences see Fig. 5A) were inserted into the
XbaI site of ptkTATACATII to yield XmrkTATACAT
67/
32 wt
and mut and Sp1TATACAT, respectively. All plasmid contructs were
verified by sequence analysis.
DNA Sequencing and Sequence Analysis
Double-stranded DNA
sequencing was performed using the SequenaseTM-kit (USB) and
-35S-dATP according to the manufacturer's
recommendations. Sequence assembly and comparison was done using the
UWGCG program package.4
The embryonic epithelial cell line A2 (18) and the melanoma cell line PSM (19), both from Xiphophorus, were cultured under the conditions described (20).
TransfectionsCells were collected and resuspended to a density of 1 × 107 cells/ml (PSM) or 2 × 107 cells/ml (A2) in F-12 containing 5% fetal calf serum and placed on ice. 350 µl of this cell suspension were mixed with 50 µl of DNA (0.6 mg/ml in TE) directly in a 4-mm electroporation cuvette and subjected to electroporation at 250 V/1200 µF using an Easyject Plus electroporator (EUROGENTEC, Belgium) in single pulse mode. After the pulse, the cells were placed on ice for 5-15 min before they were seeded onto 6-cm Primaria culture dishes (Falcon) in 5 ml of regular growth medium and cultured for 2 days before harvesting.
CAT AssaysTransfected cells were washed once with PBS, harvested in 1 ml of TEN (40 mM Tris-Cl, pH 7.5, 1 mM EDTA, 150 mM NaCl) using a rubber policeman, transferred to 1.5-ml Eppendorf tubes, and spun down for 5 min at 5000 rpm. Cells were resuspended in 100-150 µl of 0.25 M Tris-Cl, pH 7.8, and lysed by three cycles of freezing/thawing. Cellular debris was removed by centrifugation for 15 min at 14,000 rpm, and the supernatants were used for all following assays. Protein concentrations of the lysates were determined according to Bradford (21), and equal amounts of protein were used to measure CAT enzyme activity.
CAT assays were performed according to Seed and Sheen (22) with modifications described by Crabb and Dixon (23). Lysates were complemented up to 88 µl with 0.25 M Tris-Cl, pH 7.8, and mixed with 10 µl of butyryl-CoA (5 mg/ml), 1 µl 0.5 M EDTA, and 1 µl of 2 mM [ring-3,5-3H]chloramphenicol (0.25 µCi/µl in EtOH). The reaction was allowed to proceed for 2-16 h at 37 °C and stopped by addition of 200 µl of mixed xylenes. After vortexing and centrifugation, the organic phase was reextracted twice with 100 µl of TE. Conversion rates were calculated after counting the butyrylated chloramphenicol in the organic phase and the nonreacted chloramphenicol in the aqueous phase.
Microinjection into Early Fish EmbryosMedaka embryos were injected cytoplasmically with approximately 500 pl (equivalent to 25 pg) of supercoiled plasmid DNA into one cell of a two-cell stage embryo and assayed as described previously (24).
Nuclear ExtractsNuclear extracts of PSM cells were
prepared as described by Dignam et al. (25) with minor
modifications. Cells were grown to near confluency, washed, and
collected in PBS and pelleted by centrifugation for 5 min at 1000 × g. All the following steps were performed at 4 °C. The
cells were resuspended in 5 packed cell volumes of 10 mM
HEPES-KOH, pH 7.8, 0.1 mM EDTA, 0.1 mM EGTA, 0.75 mM spermidine, 0.15 mM spermine, 1 mM dithiothreitol, 1 mM benzamidine, and 0.5 mM PMSF. After 10 min incubation on ice, the cells were
lysed by homogenization in a Dounce homogenizer with 12 strokes using a
"B" pestle. After addition of (null)/1;10 volume of 10 × PBS
to stabilize the nuclei, the lysate was centrifuged in a SS-34 rotor
for 8 min at 4000 rpm. The nuclear pellet was resuspended in 3 packed
cell volumes 50 mM Tris-Cl, pH 7.5, 10% sucrose, 420 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 1 mM benzamidine, and 0.5 mM PMSF. The lysate was
gently stirred for 1 h at 4 °C and then centrifuged for 1 h at 40,000 rpm. The supernatant was passed over a PD-10 desalting column (Pharmacia Biotech Inc.), equilibrated with 20 mM
HEPES-KOH, pH 7.8, 12.5 mM MgCl2, 1 mM EDTA, 100 mM KCl, 0.1% Nonidet P-40, 20%
glycerol, 1 mM dithiothreitol, 1 mM
benzamidine, and 0.5 mM PMSF, and eluted in the same
buffer. The extract was aliquoted, frozen in liquid nitrogen, and
stored at 80 °C.
The
oligonucleotides used as probes were annealed and labeled at the
5-ends using T4 polynucleotide kinase and [
-32P]ATP
(>6000 Ci/mmol). The double-stranded probes were purified over a 15%
polyacrylamide gel, eluted, precipitated with ethanol, and resuspended
in TE buffer. Binding reactions were performed in a final volume of 20 µl containing PSM nuclear extract, 2 µg of
poly(dI·dC)·poly(dI·dC), 2 µg/µl bovine serum albumin, 90 mM KCl, and specific competitor DNA as indicated. The
binding buffer consisted of 10 mM HEPES-KOH, pH 7.8, 2.5 mM EDTA, 5 mM spermidine, 2% Ficoll 400, 6%
glycerol, 1 mM dithiothreitol, and 0.5 mM PMSF. After a 15-min preincubation step at 4 °C, 20,000 cpm (1-2 fmol) of
labeled oligonucleotide was added, and incubation was continued for 15 min. For supershift assays 1 µl of either preimmune, anti-Sp1, or
anti-Sp3 antiserum (26) was added to the binding reaction 10 min prior
to loading of the gel. DNA-protein complexes were resolved on 4-6%
polyacrylamide gels containing 5% glycerol in 0.25 × Tris borate
buffer (22.5 mM Tris base, 22.5 mM boric acid, 0.5 mM EDTA). The gels were run at 4 °C at a constant
current of 12 mA with buffer recirculation, then dried, and
autoradiographed.
According to
Papavassiliou (27) an upscaled EMSA reaction was performed, using a DNA
fragment as probe labeled exclusively at one of its 5-ends. Following
electrophoresis the gel was immersed in 200 ml of 10 mM
Tris-Cl, pH 8.0. After addition of 20 ml 2 mM
1,10-phenanthroline monohydrate, 0.45 mM CuSO4
the chemical nuclease reaction was initiated by adding 20 ml of 58 mM 3-mercaptopropionic acid. The reaction was quenched
after 7 min at 4 °C by adding 20 ml of 28 mM
2,9-dimethyl-1,10-phenanthroline monohydrate. After 2 min incubation
the gel was rinsed four times in distilled water and subsequently
electroblotted on DE-81 membrane for 5 h at 500 mA in 0.5 × TBE. The membrane was autoradiographed overnight, and bands
corresponding to free and bound probe were cut out. The membranes were
washed twice with 100 µl of LS wash buffer (50 mM
Tris-Cl, pH 8.0, 150 mM NaCl, 10 mM EDTA, pH
8.0) and then eluted twice with 100 µl of HS wash buffer (50 mM Tris-Cl, pH 8.0, 1 M NaCl, 10 mM
EDTA, pH 8.0) for recovery of DNA. Following sequential extractions
with phenol/chloroform (1:1 (v/v)) and chloroform the DNA was
precipitated with ethanol. After resuspension in 5 µl of loading
buffer (50% formamide, 0.05% bromphenol blue, 0.05% xylene cyanol),
equal amounts (as determined by Cherenkov counting) were loaded onto a
12% 1 × TBE, 50% urea polyacrylamide gel together with a
G + A sequencing ladder, which was prepared according to Maxam and
Gilbert (28). The gel was run at 60 watts for 3-4 h, dried on Whatman
3MM paper, and autoradiographed.
For Western analysis equal amounts of nuclear proteins were boiled for 5 min in Laemmli buffer and separated on SDS-polyacrylamide gel electrophoresis (10%). Using a transfer buffer containing 4 mM NaH2PO4 and 57 mM Na2HPO4, the proteins were electroblotted to nitrocellulose membranes (Schleicher & Schüll, Dassel, Germany) by standard procedures. Filters were blocked for 1 h at room temperature with NETG (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.05% Triton X-100, 0.25% gelatin). For protein detection anti-Sp1 and anti-Sp3 (26) as well as anti-Sp1(PEP2) (Santa Cruz Biotechnology, Inc.) antisera and as a control preimmune serum were used as primary antibodies. After incubation with primary antibody overnight at 4 °C and washing three times for 20 min at room temperature, the filters were incubated with horseradish peroxidase-coupled anti-mouse or anti-rabbit secondary antibodies for 1 h at room temperature and washed again as above. Nonradioactive detection (enhanced chemiluminescence, Amersham Corp.) was performed according to the supplier's recommendations. To reprobe Western blots with different antibodies, the membrane was incubated in STRIP buffer (62.5 mM Tris-Cl, pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol) for 1 h at 50 °C. To remove any remaining SDS, filters were washed three times in PBS and then blocked again for 1 h with NETG.
Sequences further upstream of the previously
isolated promoter region of the Xmrk oncogene (11) were
obtained by PCR using primers deduced from the D locus (12)
and the Xmrk coding region. This segment containing 675 bp
upstream of the transcriptional start site was inserted into a CAT
reporter gene vector to yield XmrkCAT 675/+34. Transient transfection
of this promoter construct into two different Xiphophorus
cell lines of melanoma (PSM) and nonmelanoma (A2) origin revealed
similar high activity in both cell types (Fig. 1). The
seemingly different transcriptional activity of Xmrk
675/+34 observed
upon comparison with a HSV tk promoter construct was only due to a
differential transcriptional potential of the viral promoter in the two
cell lines, which was demonstrated using a construct containing only
the tk-derived TATA box as a reference. Thus, the existence of one or
several strong, positive regulatory element(s) within the Xmrk
675/+34 region has to be proposed, driving transcription of the
Xmrk oncogene in a nontissue-specific manner.
Gene transfer of the Xmrk 675/+34 CAT fusion into early embryos of
medaka fish (Oryzias latipes), a genus closely related to
Xiphophorus, revealed that the investigated promoter
fragment was not only active in tissue culture cells but was also
functional in vivo in a whole animal system
(Fig. 2). CAT activity observed in embryos 3 days after
injection with Xmrk
675/+34 CAT was on average about 50% of CAT
expression driven by one of the strongest enhancer-promoter
combinations in this in vivo assay, CMV-tk, indicating the
high transcriptional activity of the Xmrk regulatory sequences. A promoterless CAT construct sharing the plasmid backbone with Xmrk
675/+34 CAT led to expression at background level
comparable with that of noninjected embryos.
To determine the position of the regulatory elements responsible for
this high level activation, the reporter gene activity of a set of 5
deletions spanning the
675/+34 segment of the Xmrk
upstream region was quantitated in the melanoma cell line PSM. Whereas
deletion to
194 led to a reduction in reporter activity to about 60%
of
675/+34 (Fig. 3A), an even larger
decrease in promoter activity to approximately 20% of
675/+34 was
observed in a construct lacking sequences upstream of
49. These
results indicate the presence of probably two positive cis
regulatory elements positioned in the promoter region downstream of
277 and
125, respectively. For a more precise localization of the regulatory elements various Xmrk promoter fragments were
then fused to the tk promoter and tested for their transcriptional activation potential (Fig. 3B). Since fragments
277/+34
rev and
194/
20 did not differ significantly regarding their CAT
expression, obviously no enhancer element was detectable in this assay
within the region between
277 and
194. However, a fragment
containing the region between
67 and
20 proved to be sufficient to
activate a heterologous promoter to a similar level as all longer
Xmrk fragments demonstrating the presence of functional
activating elements within these 48 bp.
DNA-Protein Interactions with Xmrk
To test whether
the in vivo observed positive regulatory activity within
67 and
20 was also reflected by in vitro DNA-protein interactions, EMSA was performed using an 88-bp
HindIII/BamHI fragment of XmrktkCAT
67/
20 as
probe and nuclear extract of PSM cells (Fig.
4A). One major complex was formed with this fragment. To
precisely localize the position of the complex we performed copper-phenanthroline footprint analysis applying the same conditions as in the previous EMSA. The complex produces one clear footprint positioned between
50 and
41 with a hypersensitive site at position
46 (Fig. 4B). Interestingly, the protected region
coincides with a GC-rich region within positions
48 and
41 of the
Xmrk promoter that has the sequence 5
-CCCGCCCC-3
, thus
resembling the previously determined Sp1 consensus binding motif (29).
These data suggest that the complex is formed over the Sp1 consensus
within
67/
20, implying the possible involvement of DNA-binding
proteins related to Sp1 in complex formation.
To test further whether Sp1-like proteins were involved in complex
formation, EMSA was performed using an oligonucleotide as probe, which
was derived from the Xmrk sequence between 67 and
32
(Xmrk
67/
32) (Fig. 5A), in order to avoid
undesired side effects from the endogenous Xmrk TATA box positioned
within residues
34 to
27. High resolution gel analysis of the
DNA-protein complex revealed two bands of slightly different mobility
(C, C
) (Fig. 5B). Both complexes
proved to be specific since unlabeled Xmrk
67/
32 inhibited complex
formation, whereas an oligonucleotide mutated in two positions within
the GC box (Xmrk
67/
32 mut) did not, suggesting the involvement of
Sp1-related transcription factors in the complex. This observation was
confirmed by using an Sp1 consensus oligonucleotide as competitor (30)
sharing only the Sp1 core sequence with Xmrk
67/
32, which also
inhibited formation of complexes C and C
. The unrelated binding
consensus of transcription factor AP1 (31) as competitor showed no
effect. Accordingly, no protein binding was detectable in an EMSA using the Xmrk
67/
32 mutant as a probe (Fig. 5C).
Recent studies indicate that the zinc finger protein Sp1 is one of
several members of a differentially expressed gene family (32, 33). In
order to determine which of their corresponding fish homologues might
bind to the Xmrk oncogene promoter, we performed supershift
analyses with different antibodies raised against human Sp proteins.
Whereas addition of a polyclonal anti-Sp1 antiserum as well as an
antibody raised against residues 520-538 of the human Sp1 protein
(data not shown) left both complexes unaltered, addition of an anti-Sp3
antiserum led to loss of complex C and a strong reduction of C,
indicating the presence of a fish Sp3 homologue in the complexes
(Fig. 6). Addition of an antiserum against human Sp4, a
nuclear factor with a very limited expression pattern (32), as well as
preimmune serum as control also showed no effect on complex formation
(data not shown).
Sp-related Proteins in Xiphophorus
In various studies Sp1 has
been reported to be ubiquitously expressed (29), and therefore its lack
of detectability in the supershift assay was unexpected. The inability
of both anti-Sp1 antisera to reduce or supershift complexes C and C
could either result from the absence of Sp1 in the nuclear extracts or
a lack of cross-reactivity of the antibodies toward fish Sp1 under EMSA conditions. To test this we conducted Western analyses of nuclear extracts prepared from PSM cells using different antibodies for detection (Fig. 7). An antibody raised against a peptide
outside the zinc finger region of human Sp1 (
Sp1(PEP2)), which
identifies the human Sp1 95- and 106-kDa polypeptide species,
recognized one major protein of about 80 kDa in melanoma cell nuclear
extracts. A polyclonal serum against full-length Sp1 (
Sp1) also
detected a protein of approximately 80 kDa and in addition species of
85 and 75 kDa molecular mass. Immunodetection using anti-Sp3 antiserum also identified a prominent band of about 85 kDa. No such bands were
observed using preimmune sera in control experiments. These results
demonstrate the existence of fish homologues of human Sp1 and Sp3 in
PSM cells. Hence, it seems likely that the inability of anti-Sp1
antibodies to supershift the complex formed over Xmrk
67/
32 was
rather due to a lack of cross-reactivity under EMSA conditions than to
the general absence of Sp1 in the fish cells.
Functional Analysis of the GC Box within Xmrk
To
determine whether the GC box constitutes an important functional
cis element within the Xmrk promoter, the region
between 67 and
32 was analyzed in more detail in a set of transient transfections in PSM cells (Fig. 8). Oligonucleotide
Xmrk
67/
32 and its corresponding mutant carrying two nucleotide
exchanges within the GC box (Xmrk
67/
32 mut) were inserted directly
upstream of the tk TATA box and evaluated functionally in this
heterologous promoter context. To avoid interference with the
endogenous Sp1 site within the HSV tk promoter, the tk-derived TATA box
was used. Xmrk
67/
20 and Xmrk
67/
32 in either orientation
stimulated CAT activity to comparable levels, demonstrating that these
sequences are sufficient to enhance reporter gene expression. Mutation
of two base pairs within the GC box (Xmrk
67/
32 mut) resulted in a
complete loss of activity. In accordance with these findings an
isolated Sp1 site (30) sharing only an 8-bp core sequence with Xmrk
67/
32 (Fig. 5A) was able to drive CAT expression to a
level which did not differ significantly from the Xmrk
promoter fragment. These results clearly indicate that the GC box is
the functional element within Xmrk
67/
32 and therefore critical for
the activity of the Xmrk oncogene promoter.
Overproduction of the Xmrk-encoded receptor tyrosine
kinase has been shown to be responsible for melanoma formation in
Xiphophorus hybrids (7, 11). Resulting from high steady
state levels of the corresponding mRNA, this elevated expression is
most likely due to a transcriptional deregulation of the
Xmrk promoter in tumor cells. In order to contribute to an
understanding of the mechanisms leading to tumor formation in
Xiphophorus, it was important to identify regulatory
elements that account for this high promoter activity. Preliminary
experiments had revealed that a fragment comprising 277 bp upstream of
the transcriptional start site of the Xmrk oncogene was able
to drive the expression of a reporter gene (11). In this report we have
isolated sequences further upstream of the originally described
promoter and identified a potent cis element and its
corresponding transacting factors enhancing transcription of the
Xmrk oncogene. An Xmrk promoter fragment spanning
the region 675/+34 was shown to be highly active in two different
Xiphophorus cell lines of melanoma and nonmelanoma origin.
In addition, the analyzed upstream sequences proved not only to be
functional in tissue culture cells but also in fish embryos after
transient gene transfer. Comparison with CMVtkCAT demonstrated the high
level activation potential of the Xmrk promoter in
vivo in a developing embryo. The variability of CAT activity observed in individual embryos could be explained by the mosaic distribution of the injected plasmid (10) leading to CAT expression only in a subset of tissues exhibiting responsiveness to the
Xmrk promoter.
Whereas deletion analysis suggested the presence of two distinct
positive regulatory elements downstream of positions 277 and
125,
respectively, subsequent evaluation of various promoter fragments in a
heterologous promoter context revealed the existence of only one potent
positive regulatory element. The failure to detect enhancer activity
within the more distal region in this type of analysis could be due to
the requirement for an authentic Xmrk promoter context of
this putative regulatory element to function. Such specific
enhancer-promoter interactions have been described for several other
genes and may represent a mechanism ensuring transcriptional
specificity (34, 35). Within the proximal region, however, a 48-bp
segment adjacent to the Xmrk TATA box proved to be able to
confer strong transcriptional activity to a heterologous promoter.
By copper-orthophenanthroline footprinting analysis the DNA-protein
complex formed over this region was shown to be positioned over a core
sequence, 5-CCCGCCCC-3
, that is identical to the binding motif
described for the zinc finger protein Sp1 (29). Mobility shift assays
substantiated the finding that GC-binding proteins interacting
specifically with the Xmrk GC box are present in
Xiphophorus nuclear extracts. The cross-competition observed between the Xmrk-derived binding site and the Sp1 consensus
oligonucleotide, which are unrelated outside the core region, clearly
demonstrates the crucial role of the GC box for the protein-DNA
interaction. This is supported by the complete loss of protein binding
upon mutation of two base pairs within the core consensus. In addition to the members of the Sp protein family (32, 33), other GC box binding
proteins have been identified. Whereas the BTE binding factors seem to
recognize a variant sequence motif deviating from the Sp1 consensus
site analyzed here (36, 37, 38), the only other proteins described so far
binding to this consensus are the yeast MIG1 repressor (39) and its
fungal homologue CREA (40). MIG1 interacts specifically with the
pentanucleotide motif 5
-GCGGG-3
; however, it requires flanking
AT-rich sequences adopting a particular geometry for high affinity
binding (41), making it unlikely that a factor of this nature binds to
the Xmrk GC box.
Supporting evidence for the identity of the GC box binding factors in
PSM cells is provided by the Western blot experiments indicating the
presence of proteins that share structural features with members of the
mammalian Sp family of transcription factors even outside the highly
conserved zinc finger region. Consistent with these findings are
supershift assays in which addition of an anti-Sp3 antiserum led to
reduced formation of the respective DNA-protein complexes, indicating
the involvement of different forms of a fish Sp3 homologue in complexes
C and C, which is in accordance with observations made in mammalian
cells (26). Anti-Sp1 antiserum was, in contrast, not able to exhibit an
effect in this type of assay, although Sp1-related proteins are present in Xiphophorus. It is conceivable, however, that an antibody
may only under certain conditions be able to cross-react with its corresponding polypeptide, in particular when crossing species border.
It is likely that the lack of immunoreactivity of anti-Sp1 antiserum in
the supershift experiments was rather due to its failure to cross-react
with the corresponding fish protein in this specific type of experiment
than to the general absence of Sp1 in PSM cell extracts.
In summary, it can be assumed that homologues of human Sp proteins, namely Sp1 and Sp3, are present in Xiphophorus and that at least one of them is able to bind to the GC box contained within the Xmrk promoter. To this date homologues of human Sp proteins have only been described for mouse and rat (37, 42), and several distant relatives have been isolated from Drosophila (6, 43, 44). Whereas all known mammalian Sp1 proteins are nearly identical to human Sp1 in their complete primary sequence, the respective Drosophila proteins show fairly low overall similarity except for their zinc finger region. These proteins are functionally related to Sp1 (44) but may not necessarily represent the Drosophila Sp1 orthologue. To our knowledge, we report here for the first time that at least some members of the Sp family may be highly conserved in a nonmammalian vertebrate. Notably, this conservation seems to extend even outside the DNA-binding domain, as is demonstrated by immunoreactivity toward an antibody raised against a region outside the zinc finger domain of human Sp1. The size difference between fish and human Sp proteins observed hereby is not unusual considering the evolutionary distance between these two species.
It cannot be ruled out that there might even be a larger number of GC-binding proteins present in fish that were not detected under our experimental conditions. This speculation is supported by the inability of either antibody used to completely prevent complex formation in the supershift assays even at high concentrations.
Transient transfection analyses of wild-type and mutant
oligonucleotides containing residues 67 to
32 of the
Xmrk promoter demonstrated that the GC box is not only able
to interact with nuclear proteins in vitro but is also
functional in an in vivo system. Since mutation within the
GC box completely abolished the transcriptional activation potential of
67/
32, it seems to be the only functionally relevant element within
this region. Our data provide evidence that Sp-like transcription
factors bind to this sequence within the Xmrk promoter
in vitro and might thus play a role in the observed
transcriptional activation. However, we cannot exclude the possibility
that other so far uncharacterized GC box binding proteins are involved,
for which a similar DNA target recognition would have to be
postulated.
It is conceivable that other, possibly closely positioned, positive and
negative elements went undetected by our set of deletions. Based upon
the observation that, when compared with the tk promoter, Xmrk
675/+34 activated CAT expression in PSM cells by about 16-fold, but
fragment
67/
20 enhanced transcription of a heterologous promoter
only 5-7-fold, it can be assumed that the coordinate action of a
greater number of regulatory elements outside the region investigated
in detail is required for full promoter activity. It will be
interesting in the future to determine the quantitative effect of the
GC box on the overall promoter activity, e.g. by mutagenizing this sequence within the Xmrk
675/+34 construct. This,
however, will only be fully informative if other functional elements
present upstream of the Xmrk gene have been
characterized.
The notion that high transcriptional activity of the Xmrk
promoter accounts for the overexpression of the Xmrk
oncogene in melanoma tissue is strongly supported by the presence of a
potent positive regulatory element identified within the
Xmrk 675/+34 promoter region. Since this element is not
only functional in transformed but also nontransformed cells that are
normally not susceptible to melanoma development, the existence of
additional silencer elements positioned outside the investigated region
leading to transcriptional inactivation of Xmrk in
nonpigment cells has to be postulated. It is conceivable that the
genetically defined R locus, suppressing the melanoma
inducing potential of the Xmrk oncogene in wild-type
Xiphophorus, might act through such regulatory elements.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U60766[GenBank].
We thank G. Hagen and G. Suske for the gift of the Sp protein antisera and G. Suske and I. Schlupp for critical reading of the manuscript, discussion, and suggestions.