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Molecular Biology and Genetics |
Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven and Flanders Interuniversity Institute for Biotechnology, Herestraat 49, B-3000 Leuven, Belgium
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ABSTRACT |
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INTRODUCTION |
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The PLAG1 protein contains seven canonical C2H2 zinc finger domains and a serine-rich COOH terminus that exhibits transactivation capacities when fused to the Gal4 DNA binding domain (4) , suggesting that it may act as a transcriptional regulator.
To extend our knowledge on the function of the PLAG1 gene and the mechanisms by which it causes salivary gland adenomas, we decided to further investigate functional characteristics of PLAG1 and in particular its potential transcriptional role. We determined in which subcellular compartment PLAG1 exerts its function by immunofluorescence studies; determined whether PLAG1 could bind DNA in a sequence-specific manner and identified its consensus DNA binding site by performing CASTing experiments. The zinc fingers required for sequence-specific DNA binding were determined by deletion/mutation analysis; and we used the PLAG1 consensus was used to screen the eukaryotic promoter databank. Possible target genes were studied regarding the capacity of PLAG1 to bind and activate their promoter. Finally, we analyzed the expression of such target genes in normal salivary gland tissue and in pleomorphic adenomas with or without PLAG1 overexpression to determine whether these genes could be PLAG1 targets in salivary gland adenomas.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CASTing.
To prepare a pool of random double-strand oligomers for the first round
of CASTing, 400 pmol of CAST25
(5'-CTGTCGGAATTCGCTGACGT-(N)25-CGTCTTATCGGATCCTACGT-3') were incubated
in 100 µl of polymerase reaction buffer containing 1200 pmol of
CAST-LOW (5'-ACGTAGGATCCGATAAGACG-3'), 200 µM of each
deoxynucleoside triphosphate, 2.5 µl [
-32P]dCTP
(DuPont NEN), and 10 units of Amplitaq (Perkin-Elmer-Cetus) and treated
as follows: 5 min at 94°C, 20 min at 65°C, and 20 min at 72°C.
Fifty µl were incubated with 500 ng of GST-PLAG1 (N2-C244) bound to
Glutathione Sepharose beads, 50 µg of poly
polydeoscyinosinic-deoscycytidylic acid (Sigma), and 50 µg of
BSA in 500 µl of binding buffer [10 mM Tris (pH 7.5),
200 mM NaCl, 50 µM ZnCl2, 10%
glycerol, 1 mM MgCl2, and 1 mM
DTT]. After a 30-min incubation on a rotator at room temperature the
beads were washed four times with cold binding buffer, and the
radioactivity still present on the beads was counted to monitor the
level of enrichment in each step. The oligonucleotides were eluted from
the beads by resuspending in 100 µl of water, followed by phenol
extraction and ethanol precipitation. An aliquot was used for the
subsequent amplification reaction in 100 µl of polymerase reaction
buffer containing 200 pmol of each amplimer CAST-UP
(5'-CTGTCGGAATTCGCTGACG-3') and CAST-LOW, 200 µM
deoxynucleotide triphosphates, and 2.5 units of Amplitaq (Perkin-Elmer
Cetus) with 1 µl of [-32P]-dCTP (DuPont NEN) with 25
cycles of 1 min at 94°C, 1 min at 65°C, and 1 min at 72°C. The
amplified products were subsequently used for a second round of
selection performed as described above. After four rounds of selection,
the subsequent three steps of selection were performed by EMSA with 100
ng of eluted GST-PLAG1 (N2-C244). After X-ray exposure of the dried
gel, the shifted bands were cut out of the gel, and the double-stranded
DNA was eluted 3 h at 50°C in 200 µl of polymerase reaction
buffer. An aliquot of the eluate was used for amplification. After a
total of seven amplification cycles, the oligonucleotides were cloned
into the pGEM-T Easy vector according to the manufacturer’s protocol
(Promega), and 23 independent clones were sequenced.
EMSA.
The different probes were synthesized as complementary oligonucleotides
with 4-bp sticky ends, annealed, subsequently end-labeled with
[-32P]dCTP and Klenow enzyme, and finally purified
with the QIAquick Nucleotide removal kit (Qiagen). DNA-protein binding
reactions were carried out for 10 min at room temperature in 30 µl of
EMSA binding buffer [10 mM Tris (pH 7.5), 100
mM NaCl, 50 µM ZnCl2, 10%
glycerol, 1 mM MgCl2, 1 mM DTT, 1
µg polydeoxyadenylic acid-polythymidylic acid (Sigma), 1 µg
of salmon sperm DNA, and 300 ng of BSA] with 10,000 dpm of probes
(about 0.1 ng of oligonucleotides) and an equimolar amount of proteins.
DNA-protein complexes were analyzed on nondenaturing polyacrylamide
gels [6% acryl-bisacrylamide (19:1), 0.5x TBE (1x TBE = 89
mM Tris base, 89 mM boric acid, 2
mM EDTA pH 8.0) and 5% glycerol]. Electrophoresis
was performed at 4°C at 14 V/cm.
Plasmid Constructions.
The PLAG1 expression vector pCDNA3-PLAG1 was constructed by inserting
into EcoRI-XhoI-digested pCDNA3 (Invitrogen) the
complete open reading frame of PLAG1 preceded by its own
Kozak consensus translation start site. This fragment was generated by
PCR using Pfu polymerase (Stratagene) with the 5' primer
G8N-3 (5'-CCCGAATTCTAGGCTGCGATGGCCACTGT-3') and the 3' primer G8C500
(5'-GGGCTCGAGCTACTGAAAAGCTTGATGGAAAC-3'). The same blunt-ended fragment
was also cloned in the blunt-ended EcoRI site of the pCAGGS
vector (5)
to get a second expression construct for PLAG1
(pCAGGS-PLAG1) with higher levels of expression in transfected cells.
The three mutant PLAG1 proteins (PLAG1-F2mut, PLAG1-F3mut, and
PLAG1-F7mut) were produced by replacing in pCDNA3-PLAG1 the first
histidine in the C2H2 motif of the
corresponding zinc finger (His81, His110, and His231, respectively)
with an alanine. For this, we applied the QuickChange Site-directed
Mutagenesis kit (Stratagene) according to the instructions of the
supplier. All constructs were sequenced to confirm the fidelity of the
PCR and the site-specific mutagenesis. The full-length PLAG1 protein as
well as the three mutants were expressed by in vitro
transcription and translation using the TnT kit (Promega). Quality of
translation was monitored by SDS gel analysis of
[35S]Met-labeled proteins. For the EMSA experiments, 3
µl of translation reaction products were used per lane.
The PDGF-B (-389/+22) luciferase reporter construct has been made by digesting the vector pA0166 (6) by SstI and HindIII and inserting this fragment in SstI/HindIII-digested pGL2basic (Promega). The GOS24 (-384/+27) luciferase reporter construct has been obtained by cloning in PGL2basic (Promega) the fragment obtained by PCR amplification on genomic DNA using the primers 5'-GGCGAGCTCTCCCCGCCCCCATCCGTCT-3' and 5'-CCGCTCGAGAGTGGGAGCGCTGAAGTC-3' derived from the sequence retrieved from Genbank (accession number M92844). The IGF-II-P3 (-1229/+140) luciferase reporter construct [called Hup3 (7) ] is a generous gift of Dr. P. E. Holthuizen (1. Universiteit, Utrecht, the Netherlands) and has been obtained by cloning the IGF-II promoter 3 into the pSLA3 luciferase vector (8) . The c-Ha-Ras (-325 to +58 in the first intron) luciferase reporter construct was obtained by inserting the 384-bp fragment XmaIII blunt-ended/SacI deriving from the pbc-N1 vector (Ref. 9 ; ATCC) into the pGL2basic with a splice acceptor site added. The prohormone convertase 2 (-789/+137) luciferase reporter construct (10) and the somatostatin (-192/+50) luciferase reporter construct [called pSRIF-Luc (11) ] are generous gifts of Dr. E. Jansen (University of Leuven and Flanders Interuniversity Institute for Biotechnology, Belgium) and Dr. B. Peers (University of Liege, Belgium), respectively. (WT)6-TK-luc, (mCO)6-TK-luc, (mCLU)6-TK-luc, and mCLUmCO)6-TK-luc have been obtained by inserting into pTK81luc (12) six copies of the corresponding ds oligonucleotides: WT, 5'-CTAGAAGGGGCTCTAGAAAGGGTAA-3'; mCO, 5'-CTAGAATGCACTCTAGAAAGGGTAA-3'; mCLU, 5'-CTAGAAGGGGCTCTAGAAA-TACTAA-3'; and mCOmCLU, 5'-CTAGAATGCACTCTAGAAATACTAA-3'.
Transfections and Luciferase Assay.
The human fetal kidney epithelial cell line 293 (ATCC; CRL 1573) was
cultured according to the suppliers’ protocols. Cells (6-well plates)
were transiently cotransfected in triplicate with 200 ng of the
expression vector DNA, 200 ng of reporter plasmid, and 200 ng of
internal control Rous sarcoma virus ß-galactosidase DNA using 3 µl
of FuGENE 6 Transfection Reagent (Boehringer Mannheim) according to the
manufacturer’s protocol. Cells were harvested 40 h after the
transfection, and luciferase reporter enzyme activity was measured
using a Monolight 2010 luminometer (Analytical Luminiscence Laboratory)
and performing end point assays.
Preparation of RNA and Northern Blot Analysis.
Total RNA was extracted from primary tumors and analyzed by Northern
blot analysis as described previously (2)
. The human
IGF-II exon 9 probe, common to the four different
transcripts P1, P2, P3, and P4, was generated by PCR and contained
nucleotides 7970 to 8774 of the published gene sequence (Ref. 13
;
GenBank/EMBL, accession number X03562). A human IGF-II exon
5 probe specific for the P3 transcript was a kind gift of Dr. P. E.
Holthuizen. The human GOS24 probe was generated by PCR and contained
nucleotides 1828 to 2345 of the sequence submitted to GenBank
(accession number M92844). The human c-Ha-Ras probe was generated by
isolation of 1.2-kb HindIII coding fragments from
RSV-ras(leu61) vector (14)
. The human PDGF-B probe was
generated by purification of the 1.7-kb BamHI fragment
isolated from the pAO73 plasmid (15)
.
Immunofluorescence Analyses.
COS-1 kidney fibroblast cells (ATCC, CRL1650) were grown on glass
chamber slides (Nunc) and transfected with 1 µg of pCAGGS-PLAG1 and 1
µg of PM3 plasmid, which encodes the Gal4 DNA binding domain (amino
acids 1–147; Ref. 16
). Twenty-four h after transfection, the cells
were washed twice with PBS and fixed with 4% paraformaldehyde in PBS
for 30 min at room temperature, followed by two washes with PBS. Slides
were then incubated in PBS-BT [PBS/0.5% blocking reagent (Boehringer
Mannheim)/0.2% Triton X-100] for 30 min at room temperature. To
simultaneously detect the PLAG1 and Gal4 proteins, cotransfected cells
were incubated at room temperature for 1 h in PBS-BT containing
the rabbit polyclonal anti-PLAG1 together with the mouse monoclonal
anti-GAL4 (SC-510; Santa-Cruz). The polyclonal anti-PLAG1 was obtained
by immunizing rabbits with the peptide FSSTSYAISIPEKEQPL (amino acids
336–352 in PLAG1) and the specificity of the antibody was verified by
Western blot analysis (data not shown). After three washes with PBS-T
(PBS/0.2% Triton X-100), the slides were incubated in PBS-BT with
FITC-labeled swine anti-rabbit (DAKO, F0205) and Texas red-labeled
sheep antimouse (Amersham, N2031). This allowed simultaneous
visualization of the PLAG1 (FITC, green) and GAL4 (Texas red) proteins.
After three washes in PBS-T, slides were mounted in Citifluor
containing 0.5 µg/µl of 4',6-diamidino-2-phenylindole (DAPI) and
analyzed with a Zeiss Axiophot microscope equipped with UV optics.
Images were recorded with a CE200A CCD camera (Photometrics), using
Smart capture (Digital Scientific) and Iplab Spectrum (Signal
Analytics) software.
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RESULTS |
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, which perfectly coincides with the DAPI nuclear DNA staining (Fig. 1B)
. The PLAG1 protein also colocalizes with the
well-characterized nuclear protein Gal4 (Fig. 1, C and D)
. Nuclear localization of the endogenous PLAG1 protein was
also established by immunofluorescence analysis of the fetal kidney 293
cell line and independently confirmed by Western blot analysis of 293
nuclear extracts (data not shown).
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. We found that the fusion protein GST-PLAG1 (N2-C244) binds strongly to
the consensus (Fig. 3B
, Lane 1), whereas mutations in the
G-cluster (Fig. 3B
, Lane 2) reduce drastically the binding
(
8-fold) and mutations in the Core nearly completely abolish it
(37-fold reduction; Fig. 3B
, Lane 3) as well as mutations
in both (Fig. 3B
, Lane 4). The distance between the
G-cluster and the core is also important because PLAG1 binds weakly to
the probe containing the G-cluster separated by 2 bp instead of 7 bp
from the core (WT2ml; Fig. 3B
, Lane 5). These results
indicate that both motifs in the consensus are important for the
binding of PLAG1, with an importance more pronounced for the core
compared with the G-cluster.
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, WT2 competes much more efficiently than mCLU2 or mCO2,
confirming the importance of the two motifs in the consensus. mCLU2
competes poorly but nevertheless better than mCO2, confirming by this
way that the destruction of the G-cluster is not so deleterious for the
binding as the destruction of the core. As expected, no competition
could be observed, with mCOmClu2 presenting a mutation in both motifs.
Fingers 6 and 7 of PLAG1 Bind to the Core, Whereas Finger 3
Interacts with the G-Cluster.
To determine which of the zinc fingers contributes to the binding to
the consensus sequence, we examined the binding of bacterially
expressed GST fusion proteins containing different combinations of zinc
fingers (Fig. 3B
, Lanes 6–25). A similar binding pattern is
observed with the protein containing fingers 3 to 7 compared with the
protein F1–F7, which contains the complete zinc finger region (compare
Lanes 6–10 with Lanes 1–5), suggesting that
fingers 1 and 2 are not directly involved in the binding. In contrast,
the protein containing fingers 4–7 binds 3-fold less to the WT2 probe
(Fig. 3B
, Lane 11). More importantly, protein F4-F7 binds
nearly as well to the probe, presenting a mutation in the G-cluster
(mCLU2; Fig. 3B
, Lane 12) as to the WT2 probe, indicating
that the G-cluster is not important for the binding of F4–F7. This is
a good indication for the requirement of finger 3 for the interaction
with this cluster. The F6–F7 protein binds nearly with the same
affinity as F4–F7 to all of the different probes (Fig. 3B
,
compare Lanes 16–20 to Lanes 11–15), suggesting
that fingers 4 and 5 are not directly involved in the binding. This
F6–F7 protein still binds clearly to all of the probes containing an
intact core motif (Fig. 3B
, Lanes 16, 17, and
20), indicating that fingers 6 and 7 are sufficient for the
interaction with the core. Finally, the protein F1–F5 does not show
any clear binding (Fig. 3B
, see Lanes 21–25),
indicating the absolute requirement of the two last fingers F6 and F7.
All of these results suggest that finger 3 of PLAG1 interacts with the
G-cluster, whereas the core is recognized by fingers 6 and 7.
To confirm this model, we performed additional EMSAs using
full-length PLAG1 protein translated in vitro in
reticulocyte lysates instead of the GST fusion proteins expressed in
bacteria. Three mutant PLAG1 proteins (F2mut, F3mut, and F7mut) were
also produced by replacing with an alanine the first histidine of the
corresponding C2H2 motif. This mutation hinders
the coordination of the zinc and has been shown to prevent the
formation of a functional zinc finger (17)
. The
full-length PLAG1 protein expressed in reticulocyte lysate binds with
the same specificity as the bacterial F1–F7 protein (compare Fig. 3D
with 3B, Lanes 1–5). The destruction of the
zinc finger 2 (PLAG1/F2mut) does not affect the binding specificity of
this protein but decreases its affinity 3-fold (Lanes
6–10). In contrast, the finger 3 destruction decreases
drastically the affinity of this protein as F3mut binds 17-fold less to
the WT2 probe than the natural PLAG1 protein (compare Lane
11 with Lane 1). The specificity was also completely
modified since F3mut binds equally well to WT2 (Lane 11),
mCLU2 (Lane 12), and WT2ml (Lane 15). Thus, the
presence or absence of a G-cluster does not affect the binding of
F3mut, indicating that finger 3 is actually the finger required for the
interaction with this motif. This conclusion is in agreement with the
conclusions drawn from the EMSA experiments performed with the
bacterial protein. Finally, destruction of finger 7 completely prevents
any binding (Lanes 16–20), confirming the absolute
requirement of this finger.
PLAG1 Can Stimulate Transcription Through Its Consensus Binding
Site.
To investigate whether PLAG1 binding sites could mediate a
transcriptional activation by PLAG1, six copies of the minimal
consensus (WT) were cloned upstream the herpes simplex virus thymidine
kinase promoter, followed by the luciferase reporter gene. This
reporter construct was then transfected into the fetal kidney 293 cell
line in the presence or absence of the expression vector pCAGGS-PLAG1.
Table 1
shows that PLAG1 stimulates expression of this reporter construct
19-fold. This stimulation is completely abolished by mutations in
the G-cluster or in the core since no stimulation was detectable with
six copies of mCLU or mCO. This demonstrates that the activation is
completely dependent of the presence of the two motifs.
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presents the list of all of the human promoters present in the EPD
containing at least two potential binding sites for PLAG1. This list
includes several proto-oncogenes and growth factors. Four promoters,
i.e., IGF-II promoter 3, GOS24, c-Ha-ras, and PDGF-B, have
been selected for further investigation. By transient transfection
experiments, we next investigated whether these promoters are
responsive to PLAG1. To that end, we generated four reporter plasmids
where each promoter has been cloned independently in front of the
luciferase gene. Table 2
shows that the activity of the promoter 3 of
IGF-II was highly stimulated by cotransfection of a PLAG1 expression
vector (8-fold). In contrast, a weaker stimulation was obtained on the
GOS24 promoter, and the promoters c-Ha-Ras and PDGF-B did not respond
significantly to PLAG1. As expected, no stimulation was observed of
promoters that do not contain any PLAG1 binding sites. We thus focused
our attention on IGF-II and investigated whether we could really
observe a binding of PLAG1 to the putative PLAG1 binding sites in the
promoter 3 of IGF-II. For that, EMSAs were performed on a probe located
from -192 to -172 in the P3 promoter, which contains one putative
PLAG1 binding sites. This element called P3-4 has been shown to be
essential for P3 activity (19)
. As shown in Fig. 4A
, a clear binding of PLAG1 is seen on the probe P3-4. The affinity of
PLAG1 for this site is comparable with the one for WT2 because
approximately the same level of competition is obtained with P3-4 and
WT2 in EMSA competition experiments (Fig. 4B)
.
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,Lanes 1, 5, and 8). In contrast, PLAG1
expression is readily detectable in tumors carrying a chromosomal
translocation affecting the PLAG1 gene (Fig. 5
, Lanes
2, 3, 6, and 7). This overexpression is the result of
promoter substitution between PLAG1 and ubiquitously
expressed genes such as those coding for ß-catenin (cg644
and cg682) or the leukemia inhibitory factor receptor (c895
and c904; Refs. 1 and 2
). The two tumors carrying chromosome
aberrations outside the PLAG1 locus (cg650 and cg601; Refs. 20
and 21
) do not show any PLAG1 expression (Fig. 5
,
Lane 4 and Fig. 5B
). We next investigated
IGF-II expression in these tumors using a probe specific for
exon 9 of IGF-II, which is a common part of all the
IGF-II transcripts. No IGF-II expression could be
detected in normal salivary gland tissues (Fig. 5
, Lanes 1,
5, and 8) and in tumors without PLAG1
overexpression (lane 4 and cg601 in Fig. 5B
). In contrast, a
drastic up-regulation of IGF-II was observed in tumors with
PLAG1 overexpression (Fig. 5
, Lanes 2, 3, 6, and
7). The detected 6-kb IGF-II transcript
corresponds to the transcript deriving from the P3 promoter, because
hybridization with a probe specific for the P3 transcript (exon 5
probe) detects exactly the same band (data not shown). Thus, we have a
perfect correlation between PLAG1 and IGF-II gene
expression, suggesting that IGF-II is one of the PLAG1
targets. In contrast, no significant expression of c-Ha-Ras
and PDGF-B could be detected in tumors and in normal
salivary gland tissue, whereas no alteration in GOS24
expression was observed (Fig. 5B)
.
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) is corroborated by the
stereochemical rules governing the DNA contacts of individual zinc
fingers (22)
. These rules are based on different types of
studies: (a) structural studies have shown that each finger
module folds to form a compact ßß structure with the -helix
fitting into the major groove (reviewed in Ref. 23
). Residues -1, 2,
3, and 6 (numbering with respect to the start of the -helix)
typically make key base contacts that are responsible for defining
sequence specificity; (b) by phage-display selections and
site-directed mutagenesis, correlations were established between the
amino acids present in key positions -1, 2, 3, and 6 and the
nucleotide sequences of their optimal binding sites. This led to a
recognition code governing zinc-finger/DNA interactions. As depicted in
Fig. 6A
, the consensus we found by CASTing shows good agreement with the binding
site predicted by such a recognition code. It is interesting to note
that the fingers that are not involved in DNA recognition
(e.g., fingers 1, 4, and 5) do not present in key position
amino acids known to interact with DNA. The distance between the
G-cluster and the core is also in good agreement with the presence of
two noninteracting zinc fingers that are predicted to cover six
nucleotides. However, alignment of all the sequences selected by seven
rounds of CASTing indicates that the distance between the G-cluster and
the core seems to vary from six to eight nucleotides (see Fig. 2
). One
explanation to this variability could be the presence of a longer
linker region between fingers 5 and 6 (14 amino acids instead of the
usual 7 amino acids). This longer linker may generate sufficient
flexibility to allow an interaction with a cluster at different
positions.
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. However,
no G-cluster was identified, suggesting that Zac1/PLAGL1 does not
interact in the same way as PLAG1. Both PLAG1 and Zac1/PLAGL1 are
possibly involved in tumorigenesis, PLAG1 as a putative oncogene that
contributes to pleomorphic adenomas whereas Zac1/PLAGL1 as a tumor
suppressor candidate that regulates apoptosis and cell cycle arrest
(26)
. It is thus interesting to further investigate the
differences of specificities between these two related proteins to
determine whether the opposite functions are the result of different
target genes or different actions on the same set of genes. The PLAG1 DNA binding consensus is highly GC-rich, which is a hallmark of most promoters of genes controlling cell growth. It is thus tempting to speculate that PLAG1 will exert its oncogenic effect via the activation of growth factors. The IGF-II is an excellent candidate because IGF-II is a peptide growth factor that plays an important role in embryonic development and also in carcinogenesis (28) . The human IGF-II is a complex transcription unit that is regulated by activation of multiple promoters designated P1 to P4. Promoter 1 activity has been demonstrated only in adult liver, whereas promoters 2, 3, and 4 are coexpressed in a variety of fetal tissues, notably fetal liver, and at a lower level, in many adult tissues with the exception of adult liver. Promoters 3 and 4 are also highly active in numerous tumor tissues, suggesting that transcriptional up-regulation of P3 and P4 activities may be importantly involved in tumorigenesis. Our study is the first demonstration of IGF-II up-regulation in tumors of the salivary glands. This up-regulation is the result of a drastic up-regulation of promoter 3 activity, as demonstrated by the hybridization performed with a P3-specific probe. Several lines of evidence strongly suggest that IGF-II up-regulation in salivary gland tumors results from transcriptional activation by PLAG1: (a) five potential binding sites were found in promoter 3 of IGF-II; (b) PLAG1 binding was effectively demonstrated on the site P3-4; (c) IGF-II promoter 3 activity is up-regulated by PLAG1; (d) IGF-II is highly expressed in tumor cells that overexpress PLAG1 but could not be detected in tumors without PLAG1 up-regulation or in normal salivary gland tissues. Our study suggests that the oncogenic activity of PLAG1 results from its positive regulation of IGF-II expression, known to potently stimulate cell proliferation in human tumors through autocrine or paracrine mechanisms (28, 29, 30) .
The PLAG1 consensus sequence is also reminiscent of G-rich sequences
recognized by an important group of zinc finger proteins that include
Sp1, Zif268/Egr1, and WT-1. As shown in Fig. 6B
, the PLAG1
consensus binding motif overlaps with the consensus binding sequence
for the transcription factor Sp1 and the tumor suppressor WT-1,
suggesting that these proteins could at least partly regulate the same
set of genes. In fact, the growth factor gene IGF-II has
been shown to be also one of the targets of the tumor suppressor WT-1.
High levels of IGF-II in Wilms’ tumor were attributed to
the loss of function of WT-1, which normally represses
IGF-II transcription (31)
. The WT-1 protein
binds within the IGF-II P3 promoter to multiple sites with
some overlapping with potential PLAG1 binding sites. This suggests that
one control for IGF-II expression during development could
be provided by a balance between activators (like PLAG1) and repressors
(like WT-1) acting on overlapping sequences. Deregulation of one of
these factors is probably an important step in tumor formation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the "Geconcerteerde
Onderzoekacties 1997–2001" and the "Fonds voor Wetenschappelijk
Onderzoek Vlaanderen" (FWO). M. V. is a post-doc of the Flanders
Interuniversity Institute for Biotechnology. K. K. is a post-doc of
the FWO. 
2 To whom requests for reprints should be
addressed, at Laboratory for Molecular Oncology, Center for Human
Genetics, University of Leuven and Flanders Interuniversity Institute
for Biotechnology, Herestraat 49, B-3000 Leuven, Belgium. Phone:
32-16-346041; Fax: 32-16-346073; E-mail: marianne.voz{at}med.kuleuven.ac.be
3 The abbreviations used are: PLAG1,
pleomorphic adenoma gene 1; CASTing, cyclic amplification and selection
of target sequences; GST, glutathione S-transferase; EMSA,
electrophoretic mobility shift assay; ATCC, American Type Culture
Collection; PDGF, platelet-derived growth factor; EPD, Eukaryotic
Promoter Databank; IGF, insulin-like growth factor.
Received 7/20/99. Accepted 10/28/99.
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 M.-C. W. Yang, J. C. Weissler, L. S. Terada, F. Deng, and Y.-S. Yang Pleiomorphic Adenoma Gene-Like-2, a Zinc Finger Protein, Transactivates the Surfactant Protein-C Promoter Am. J. Respir. Cell Mol. Biol., January 1, 2005; 32(1): 35 - 43. [Abstract] [Full Text] [PDF]
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 H. Werner and J. Katz The Emerging Role of the Insulin-like Growth Factors in Oral Biology Journal of Dental Research, November 1, 2004; 83(11): 832 - 836. [Abstract] [Full Text] [PDF]
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F. Van Dyck, E. L. D. Delvaux, W. J. M. Van de Ven, and M. V. Chavez Repression of the Transactivating Capacity of the Oncoprotein PLAG1 by SUMOylation J. Biol. Chem., August 20, 2004; 279(34): 36121 - 36131. [Abstract] [Full Text] [PDF]
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 L. H. Castilla, P. Perrat, N. J. Martinez, S. F. Landrette, R. Keys, S. Oikemus, J. Flanegan, S. Heilman, L. Garrett, A. Dutra, et al. Identification of genes that synergize with Cbfb-MYH11 in the pathogenesis of acute myeloid leukemia PNAS, April 6, 2004; 101(14): 4924 - 4929. [Abstract] [Full Text] [PDF]
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 M Pia-Foschini, J S Reis-Filho, V Eusebi, and S R Lakhani Salivary gland-like tumours of the breast: surgical and molecular pathology J. Clin. Pathol., July 1, 2003; 56(7): 497 - 506. [Abstract] [Full Text] [PDF]
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 M. Martinelli, F. Martini, E. Rinaldi, L. Caramanico, E. Magri, E. Grandi, F. Carinci, A. Pastore, and M. Tognon Simian Virus 40 Sequences and Expression of the Viral Large T Antigen Oncoprotein in Human Pleomorphic Adenomas of Parotid Glands Am. J. Pathol., October 1, 2002; 161(4): 1127 - 1133. [Abstract] [Full Text] [PDF]
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C. V. Braem, K. Kas, E. Meyen, M. Debiec-Rychter, W. J. M. Van de Ven, and M. L. Voz Identification of a Karyopherin alpha 2 Recognition Site in PLAG1, Which Functions As a Nuclear Localization Signal J. Biol. Chem., May 24, 2002; 277(22): 19673 - 19678. [Abstract] [Full Text] [PDF]
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K. Hensen, I. C. C. Van Valckenborgh, K. Kas, W. J. M. Van de Ven, and M. L. Voz The Tumorigenic Diversity of the Three PLAG Family Members Is Associated with Different DNA Binding Capacities Cancer Res., March 1, 2002; 62(5): 1510 - 1517. [Abstract] [Full Text] [PDF]
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M. K. Hibbard, H. P. Kozakewich, P. D. Cin, R. Sciot, X. Tan, S. Xiao, and J. A. Fletcher PLAG1 Fusion Oncogenes in Lipoblastoma Cancer Res., September 1, 2000; 60(17): 4869 - 4872. [Abstract] [Full Text]
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