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INTRODUCTION |
NUDR1 is a
transcriptional regulatory protein that was initially identified in a
monkey kidney cell (CV-1) cDNA library through protein expression
and binding to a radiolabeled retinoic acid response element (RARE)
based on the sequence in human retinoic acid receptor 
2 gene (1).
The encoded protein had 46% overall amino acid similarity to
Drosophila Deformed epidermal autoregulatory factor-1
(DEAF-1) (2) and was therefore named nuclear
DEAF-1 related (NUDR) (1). DEAF-1 has been
shown to bind to TTCG-containing motifs located adjacent to DNA binding
sites for the Deformed homeodomain protein that occur in the promoter
regions of Deformed and other Deformed-regulated genes,
indicating that DEAF-1 may act as a transcriptional cofactor of
Deformed (2). NUDR was also shown to recognize TTCG-containing motifs
(1), and the combination of sequence and functional similarities
suggests that NUDR may be the mammalian homolog of DEAF-1.
In our previous report, NUDR was shown to transcriptionally activate a
minimal proenkephalin promoter, and activation was increased by the
addition of synthetic RAREs placed 5' of the promoter (1). Because we
were unable to demonstrate NUDR binding to proenkephalin sequences in
either DNase I protection assays or mobility shift assays, we concluded
that the activation of the proenkephalin promoter was likely to occur
through protein-protein interactions (1).
Using a yeast two hybrid system, Sugihara et al. (3)
identified the mouse homolog of NUDR (called mDEAF-1) through
interaction with LMO-4, a new member of the LIM-only (LMO) family. LMOs
contain two tandem repeats of the LIM zinc finger domain, which can
associate tightly with another family of cofactors called Clims (also
referred to as Ldb or NLI) to activate (4) or inhibit (5)
transcription. Since LMO and Clim complexes have not been demonstrated
to bind directly to DNA, they have been postulated to regulate
transcription through the recruitment of DNA-binding proteins and the
assembly of transcriptional complexes (3, 6, 7). Mouse NUDR was shown
to interact with LMO-4, LMO-2, and Clim-2 in both in vitro and in vivo assays, and it was proposed that NUDR could
provide the critical DNA binding function to LMO-Clim complexes
(3).
Because NUDR showed only moderate affinity for the RARE sequence,
higher affinity sequences were selected from a library of random
oligonucleotides through binding to recombinant NUDR protein and
amplification by PCR (1). Analysis of the selected sequences revealed
the presence of one or more copies of TTCG and/or TTTCCG, and multiple
sequence alignment suggested a NUDR binding consensus sequence of
TTCGGGNNTTTCCGG (1). Comparison of the NUDR binding motifs and the RARE
sequence suggested that the original identification of NUDR was likely
through the fortuitous binding of NUDR protein to the TTCGG sequence
found between the RARE half-sites. The similarity in DNA recognition
sequences between NUDR and Drosophila DEAF-1 implied that
the DNA binding domain may be in a region of greater amino acid
homology between the proteins, namely, the zinc finger homology region
at the C terminus (56% similarity) and/or the nuclear domain (1)/KDWK
domain (2) located in the central region of the proteins (70%
similarity). The distantly related zinc finger region of the
progesterone receptor has been shown to be involved directly in the DNA
binding domain, whereas the more homologous zinc finger region of MTG8
(ETO) has recently been shown to be involved in protein-protein
interaction and the recruitment of nuclear corepressors and histone
deacetylases (8, 9). The nuclear/KDWK domains of NUDR and DEAF-1 have
similarity with proteins from the SP100 family (1, 10). SP100 proteins are localized to subnuclear structures termed "nuclear bodies" and
are thought to play a role in the etiology of acute promyelocytic leukemia (reviewed in Ref. 11). Recently it was demonstrated that
SP100B associates with non-histone chromatin components that behave as
transcriptional silencers, and when fused to a GAL4 DNA binding domain,
SP100B can repress transcription (12, 13).
In this report, we identify the DNA binding domain in the central
region of NUDR that includes the nuclear/KDWK domain and a Myc-type
helix-loop-helix structure, and we demonstrate that there are at least
two sites of protein contact with the DNA. The hnRNP A2/B1
gene, a potential early biomarker of lung cancer (14-17), is
identified as a potential target gene of NUDR regulation by the
presence of a NUDR binding consensus sequence within the promoter
region. We show that NUDR represses transcription of the hnRNP A2/B1
promoter through a DNA binding-dependent mechanism and that
NUDR binding motifs within the 5'-UTR are involved in this regulation.
We hypothesize that elevated levels of hnRNP A2/B1 found in some
cancers may be a consequence of the inactivation or deregulation of
NUDR.
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EXPERIMENTAL PROCEDURES |
Construction of Bacterial Expression Plasmids--
The bacterial
expression and purification of recombinant proteins for full-length
human NUDR (hNUDR) and monkey NUDR (sNUDR) have been described
previously (1). To facilitate the construction of various deletion
proteins and peptides, sNUDR was used to derive the peptide constructs
G, H, I, J, K, and L, whereas hNUDR was used to derive all other
constructs. The full-length proteins of sNUDR and hNUDR differ by only
five amino acids and have virtually indistinguishable binding
characteristics. For the deletion constructs B, C, D, and E, cDNA
fragments of hNUDR were excised from the parent vector pBSSK
(Stratagene, La Jolla, CA) with BspEI and SphI
(B), BspEI and HincII (C), BspEI and
AatII (D), and XcmI and EcoRI (E)
followed by T4 DNA polymerase fill-in, ligation of BamHI
linkers, and BamHI digestion. The resulting DNA fragments were ligated into the BamHI-digested pET-16b vector
(Novagen, Inc. Madison, WI) for production of N-terminal
histidine-tagged proteins. For the internal deletion construct F, the
cDNA in pBSSK was digested with NcoI and
AflII, filled in with T4 DNA polymerase, and religated. For
the internal deletion construct G, a portion of the cDNA was
excised with EcoNI and AatII and replaced with an
SV40 nuclear localization signal (18) formed by hybridization of the
following two oligonucleotides: 5'-cCCAAAAAAGAAGAGAAAGGTAgacgt-3' and
5'-cTACCTTTCTCTTCTTTTTTGGgct-3', with the lowercase letters denoting
BsmI and AatII cohesive ends. The cDNAs for
constructs F and G were excised with BspEI and
EcoRI and treated as described above to add BamHI
linkers and then subcloned into the pET-16b vector. Recombinant
histidine-tagged fusion proteins were purified as described previously
for the full-length proteins except that the pH of the renaturation
buffer was changed from 8.0 to 9.1 to adjust for differences in the
isoelectric points of the deletion proteins.
For construction of the glutathione S-transferase (GST)
fusion peptides H, I, J, K, and L, cDNA fragments of sNUDR were
excised from the parent plasmid with EcoNI and
AatII (H), NcoI and AatII (I),
ApaI and AatII (J), NcoI and
AflII (K), and ApaI and AflII (L),
treated as described above to add BamHI linkers, and
subcloned into the BamHI site of pGEX-2T (Amersham Pharmacia
Biotech). Recombinant GST fusion proteins were purified as described
previously for GST-sNUDR (1). To determine the concentration of each
protein preparation, the recombinant proteins were subjected to
SDS-PAGE, stained with Coomassie Blue, and compared with a bovine serum albumin standard curve using a Densitometer SI (Molecular Dynamics, Sunnyvale, CA).
Electrophoretic Mobility Shift Assays--
Recombinant proteins
were incubated on ice with either nonspecific or specific
oligonucleotide competitors (as indicated) in a 20-µl reaction
containing 500 ng of poly(dI-dC), 100 mM KCl, 20 mM HEPES (pH 8.1), 2 mM dithiothreitol, 7%
glycerol, and 0.05% Tween 20. The glucocorticoid response element
(GRE) oligonucleotide used for nonspecific competitor (Fig.
2B) was formed by hybridization of two synthetic
oligonucleotides, 5'-TCGACTGTACAGGATGTTCTAGCTACT-3' and
5'-TCGAAGTAGCTAGAACATCCTGTACAG-3' (19), and the N42-78 oligonucleotide was formed by hybridization of
5'-cgggatccTTCGGACTGATTCGGCTTCCCACTTCG-3' and
5'-cgggatccCGAAGTTCCCCGAAGTGGGAAGCCGAA-3'. The lowercase letters denote BamHI restriction sites used for subcloning.
Radioactive oligonucleotide probes were produced by fill-in reactions
with Klenow and [
-32P]dATP. After 15 min, the
reactions were mixed with 120-240 fmol of 32P-labeled
probe and incubated an additional 15 min at 25 °C. Protein-DNA complexes were separated on 4% nondenaturing polyacrylamide gels (acrylamide:bis, 40:0.8, in 1× Tris-borate EDTA) at 120 volts for
3 h, and results were imaged using a 445 SI PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
DNase I Protection Assays--
The following DNA fragments were
isolated and radiolabeled by fill-in reaction with
[-32P]dATP and Klenow DNA polymerase:
EcoRI/BspMI fragment of hNUDR8 cDNA (Fig.
2A), EcoRI/HpaI fragment of N42-78
that was inserted in the BamHI site of pBLCAT5 (Fig.
2C), HindIII/SmaI fragment of the
hnRNP A2/B1 gene from hnRNPCAT (Fig. 5A), and
EcoRI/HincII fragment of the hnRNP A2/B1 gene
from hnRNP in pBSKSII (Stratagene) (Fig. 5B). DNase I
protection assays were performed as described (1), except that
poly(dI-dC) was not used in Fig. 5.
Protein-DNA Photocross-linking--
The synthesis of the
photoreactive nucleotide analog AB-dUTP (Fig. 4A) has been
described elsewhere (20, 21). The N42-78 oligonucleotide of hNUDR8 was
subcloned into the BamHI site of pBLCAT5 and then excised
with HindIII and EcoRI. After biotinylating the
DNA fragment, one of the DNA strands was selectively immobilized on
paramagnetic beads and used as the template in the synthesis of the
photoaffinity probe (22). Approximately one pmol of template was
used in a reaction containing 6 pmol of the specific
oligonucleotide 5'-CGGCTTCCCACTTCGGGG-3', ~4.5 pmol
[-32P]dCTP, 0.6 µM AB-dUTP, 0.6 µM dATP, and 0.25 units of exonuclease-free Klenow
fragment of DNA polymerase I (Amersham Pharmacia Biotech) in a final
volume of 20 µl. After 5 min at 37 °C, 2.5 µl of 5 mM unlabeled dNTPs were added and incubated at 37 °C for
an additional 10 min. A second oligonucleotide, complementary to the
multiple cloning region of pBLCAT5 and 5' of the first oligonucleotide, was annealed to the immobilized DNA, and double-stranded DNA was synthesized with T4 DNA polymerase and subsequent treatment with T4 DNA
ligase to seal the nicks. The double-stranded DNA photoaffinity probe
was removed from the solid support by digestion with HincII.
The binding reaction conditions for cross-linking were identical to
those described for electrophoretic mobility shift assays (EMSAs),
except that 2 fmol of the photoreactive probe were used. The
cross-linking of the DNA and protein was performed by irradiation with
UV light at 380 µW/cm2 for 2 min at a distance of 20 cm.
The cross-linked samples were treated with DNase I and S1 nuclease as
described (23) to remove all but the four labeled pyrimidines attached
to the protein (labeled Intact, Fig. 4D). An
aliquot of the cross-linked sample was treated with 70% formic acid
and 2% diphenylamine at 70 °C for 20 min to cleave the acid-labile
Asp-Pro linkage at position 195-196 (labeled Asp-Pro cleavage, Fig.
4D). Samples were separated by SDS-PAGE on 10%
polyacrylamide gels, followed by autoradiography.
Construction of Reporter Plasmids and Mammalian Expression
Plasmids--
A 743-bp DNA fragment containing the hnRNP A2/B1
promoter (positions 1844-2586) was amplified by 35 cycles of PCR
(GeneAmp 9600, Perkin-Elmer) using 600 ng of genomic DNA isolated from the human JEG-3 cell line, and the primers 5'-ACTTTCAGCAGCGAACTCTCC-3' and 5'-AGTCGCTTCAGCCCGATTTC-3'. The PCR product was subcloned into the
EcoRV site of pBSKSII (Stratagene) before excision with BamHI and HindIII and ligation into the
BamHI/HindIII site of pBLCAT6 (24) to produce the
reporter plasmid, hnRNPCAT. The hnRNP PCR product in pBSKSII was
digested with BspEI, followed by a fill-in reaction and
ligation of BamHI linkers. The DNA fragment containing the
hnRNP promoter was excised with BamHI and HindIII and ligated into the BamHI/HindIII site of
pBLCAT6 to produce the reporter hnRNP
ICAT. The reporter plasmids
hnRNPIICAT and hnRNPI,IICAT were produced by excision of a
XhoI DNA fragment from the plasmids hnRNPCAT and
hnRNPICAT and subsequent religation.
The 5'-UTR DNA was excised from hNUDR8 with
EcoRI/BspEI, followed by a fill-in reaction and
the addition of BamHI linkers. The 356-bp DNA fragment was
ligated into the BamHI site or BglII site of
pBLCAT5 (24) to produce the reporter constructs (h8N1-356)TKCAT and
TK(h8N1-356)CAT, respectively. The 121-bp
EcoRI/BspMI fragment of hNUDR8 was treated
similarly and ligated into the BglII site of pBLCAT5 to
produce TK(h8N1-121)CAT.
To achieve high levels of protein expression in mammalian cells, the
cDNAs for hNUDR, sNUDR, and hNUDR-R302T/K304T were subcloned into
an expression plasmid that utilized the human cytomegalovirus immediate
early gene promoter (CMV), as described previously (1). The zinc finger
homology region of hNUDR was deleted by HpaI and Bsu36I digestion of the cDNA in pBSSKII, followed by
fill-in and religation of the plasmid. The cDNA was excised with
EcoRI, and BamHI linkers were added, digested
with BamHI, and subcloned into the BglII site of
pCMVNeo for the construct, CMVhNUDRaa1-505. The
NcoI/EcoRI DNA fragment containing the C-terminal
portion of hNUDR was subcloned into the BglII site as above
to produce the construct CMVhNUDRaa243-565. The primers
5'-CGCGGATCCACCATGGCAGCTCCCCTCAC-3' and
5'-CTACCGGATCCTAGACGTCGCCCTGGGC-3' were used in a 20-cycle PCR reaction
with hNUDR as the template. The PCR product was digested with
BamHI and subcloned into the BglII site of
pCMVNeo for the construct, CMVhNUDRaa167-368. An EcoRI
fragment of hNUDR from construct G in pBSSK was treated as above for
the construct CMVhNUDR255-367/SV40NLS. For each construct, the
orientation and DNA sequence of the sites flanking an insertion or
deletion were determined. Protein expression was confirmed in
transfected CV-1 cells by immunofluorescence detection of NUDR, and the
percentage of cells showing nuclear or cytoplasmic localization was
estimated: hNUDRaa243-565 was 100% nuclear; hNUDR, hNUDRaa1-505 and
hNUDRaa167-368 were 80% nuclear; hNUDR255-367/SV40NLS was 64%
nuclear; and hNUDR-R302T/K304T was 100% cytoplasmic
(immunofluorescence data not shown).
Chloramphenicol Acetyl Transferase Assay--
CV-1 cells were
transfected with various reporter constructs and expression plasmids,
cell extracts were prepared in 250 µl of homogenization buffer, and
chloramphenicol acetyl transferase (CAT) activities were determined and
normalized as described previously (1). The normalized CAT activity
determined for the indicated reporter construct was set at 100% and
the effects of cotransfecting different expression plasmids with the
reporter are shown relative to this activity.
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RESULTS |
NUDR Binding Sequences Are Found in the 5'-UTRDNA of
NUDR cDNAs--
In a search for genes that contain the NUDR
binding consensus sequence or TTCG motifs, we noted that multiple TTCG
motifs occurred in the cDNA corresponding to the 5'-untranslated
region (5'-UTRDNA) of human NUDR8 and monkey NUDR. Computer
analysis of the NUDR cDNA for TTCG motifs demonstrates the presence
of 14 motifs in the 5'-UTRDNA of hNUDR8, four motifs in the
coding region, and the absence of motifs in the 3'-UTRDNA
(Fig. 1A). To test whether
NUDR protein could bind these motifs, we performed an EMSA using
radiolabeled DNA sequences from the 5'-UTRDNA (99 bp) and
3'-UTRDNA (130 bp) of hNUDR8. Low mobility complexes were observed with the 5'-UTRDNA probe when combined with 10 and
30 pmol of recombinant hNUDR protein, whereas no complexes were formed with the 3'-UTRDNA probe (Fig. 1B). These data
indicate that NUDR protein could potentially bind multiple TTCG motifs
within its own 5'-UTRDNA in vivo.
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Fig. 1.
NUDR protein binds to sequences within the
5'-UTRDNA of hNUDR8. A, the cDNA
sequence for hNUDR8 was analyzed for TTCG sequences by the WINDOW
program and plotted with the STATPLOT program from the Wisconsin
sequence analysis package by Genetics Computer Group, Inc. (GCG,
Madison, WI). The window size was 50 bp, and the shift increment was 3 bp. B, A 99-bp EcoRI/BspMI fragment
(5'UTR) and a 130-bp SmaI/EcoRI
(3'UTR) fragment of hNUDR8 were radiolabeled by fill-in
reaction, and each fragment was incubated with no protein (lanes
1 and 4) or with 10 pmol (lanes 2 and
5) and 30 pmol (lanes 3 and 6) of
recombinant hNUDR protein before separation of DNA-protein
complexes and free probe on a 4% nondenaturing polyacrylamide gel.
Results were visualized with a PhosphorImager.
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To examine the specific sequences within the 5'-UTRDNA that
NUDR was binding, we utilized DNase I protection assays. In the presence of NUDR protein, a large 74-base pair region in the upper half
of the 5'-UTRDNA was protected from nuclease digestion
(Fig. 2A). This protected
region contained, but was not limited to, the TTCG motifs. Within this
large region was a smaller area that was protected by the lowest
protein concentration and contained two sets of TTCG pairs separated by
six nucleotides (shown in bold capitals letters in Fig.
2D). Comparison of the two sets showed that 9 of the 15 nucleotides of each set were identical to each other and can be
represented by the sequence, TTCGGNNNNNTTCGN. In addition, 9 nucleotides within each set of TTCG pairs (15 nucleotides in length)
were identical to the derived NUDR binding consensus sequence,
TTCGGGNNTTTCCGG. The six-nucleotide spacing between a pair of TTCGs
would align the TTCG sequences on the same face of the DNA double helix
within one turn and may allow optimal binding or interaction of one or
more NUDR molecules.
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Fig. 2.
NUDR protein binds with high affinity to
nucleotides 42-78 of hNUDR8. A, the lower strand of an
EcoRI/BspMI fragment of hNUDR8 was radiolabeled
and treated with DNase I in the absence (0) or presence of
increasing amounts of recombinant hNUDR protein (15, 50, and 100 pmol,
indicated by the wedge). Samples were separated on
denaturing 6% acrylamide gels, and the results were visualized with a
PhosphorImager. The NUDR-protected sequence is indicated by the
striped bar to the right of panel A and below the
corresponding nucleotide sequence in D. B, a
double-stranded oligonucleotide consisting of nucleotides 42-78 of
hNUDR8 cDNA (bold capitals letters in D) was
radiolabeled (probe) and incubated with 10 pmol of recombinant hNUDR
protein alone (hNUDR) and in the presence of increasing
amounts of unlabeled oligonucleotide (indicated by the
wedge), that was either the N42-78 sequence
(specific) or a glucocorticoid response element
(nonspecific). Samples were analyzed as in Fig.
1B. C, the upper DNA strand of N42-78
oligonucleotide was radiolabeled and either untreated (U) or
treated with DNase I in the absence (0) or presence of
increasing amounts of recombinant NUDR protein (10 and 145 pmol
indicated by the wedge) and analyzed as in A. The
NUDR-protected sequence is indicated by the gray bar to the
right of the panel and above the nucleotide sequence in
D. D, the nucleotide sequence of the
EcoRI-BspMI fragment of hNUDR8 is shown, with the
bars indicating the sequences protected from DNase I by NUDR
protein binding. Nucleotide positions 42-78 are shown in bold
capital letters.
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Other areas in the lower half of the 5'-UTRDNA of hNUDR8
were also protected from nuclease digestion by NUDR binding; some of
these contained TTCG motifs whereas others did not (data not shown).
Because the sequences and boundaries of the DNA protected by NUDR
binding were not limited to TTCG or TTCG-like motifs, protein-protein
interactions may extend the protection from DNase I to flanking
sequences or may alter the DNA binding specificity.
To facilitate subsequent studies, an oligonucleotide spanning the two
sets of TTCG pairs from the 5'-UTRDNA of hNUDR8
(nucleotides N42-78, shown in bold capital letters in Fig.
2D) and including BamHI restriction sites at both
ends was synthesized. In EMSA, radiolabeled N42-78 oligonucleotide was
shifted by the addition of recombinant NUDR protein (Fig.
2B). DNA binding specificity of NUDR was shown by DNA
binding competition with an excess of unlabeled N42-78, whereas no
competition was observed with an excess of unlabeled oligonucleotide
containing a glucocorticoid response element (Fig. 2B). The
N42-78 oligonucleotide was subcloned into a plasmid, and a DNA
fragment containing this sequence was used in DNase I protection
assays. As shown in Fig. 2C, NUDR protein protected the
entire N42-78 sequence from nuclease digestion. In addition, NUDR
protein binding also produced DNase I hypersensitive sites in the
sequence flanking N42-78 (Fig. 2C) and in small regions of
the 5'-UTRDNA of hNUDR8 (data not shown).
Characterization of the DNA Binding Domain of NUDR--
We
examined NUDR for a potential DNA binding domain, and the
cysteine-rich, C terminus of the protein appeared as the most likely
candidate. There are at least 20 protein sequences in the GenBankTM
data base that have homology to this region of NUDR (1), and several
investigators have suggested that this arrangement of cysteines and
histidines may constitute a zinc finger motif capable of interacting
with DNA (2, 25-29).
To investigate the region(s) of the protein responsible for DNA
binding, we constructed various N-terminal, C-terminal, and internal
deletions of NUDR (Fig. 3) and inserted
them into bacterial expression vectors to produce fusion proteins with
GST or an N-terminal histidine tag (see "Experimental Procedures").
Recombinant proteins were purified and assayed for their ability to
bind the radiolabeled N42-78 sequence in EMSAs. We found
deletion of the last 84 amino acids, which includes the potential zinc
finger motif, had little effect on the DNA binding of NUDR (Fig. 3,
construct B). Similarly, deletion and site-directed
mutations of the zinc finger motif in DEAF-1 had no effect on its DNA
binding properties (2). Furthermore, removal of up to 195 amino acids
from the C terminus of NUDR (Fig. 3, constructs B-D) and up
to 138 amino acids from the N terminus (data not shown) had little
effect on NUDR binding of the N42-78 probe. DNA binding was
compromised but not abolished by the deletion of the first 187 amino
acids from the N terminus of NUDR (Fig. 3, construct E),
suggesting that the area between amino acids 138 and 187 of NUDR may be
involved in DNA binding. The recombinant proteins produced with
internal deletions of amino acids 242-289 and 254-368 showed reduced
binding (Fig. 3, constructs F and G). These data
indicate that the region of NUDR between amino acids 242-368 are also
important for DNA binding.
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Fig. 3.
Deletion analysis to identify the NUDR DNA
binding domain. At the top is a schematic representation of the
full-length NUDR protein with salient features and potential functional
domains indicated including two proline rich regions (PR1, 44% proline
and PR2, 28% proline), a zinc finger homology domain (ZFH),
and a nuclear localization signal sequence (NLS) (1). The
open rectangle is a region with similarity to Myc-type,
helix-loop-helix dimerization domain signature (HLH
signature) that was identified in a search of the PROSITE data
base (32). The open triangle denotes an Asp-Pro linkage that
is susceptible to acid cleavage. The line drawings represent
various deletions and peptide portions of recombinant NUDR protein that
were produced as bacterial fusion proteins with either glutathione
S-transferase or an N-terminal histidine tag (see
"Experimental Procedures"). The numbers listed to the right of each
construct indicate the amino acids of NUDR included in the fusion
protein, and those preceded by a are amino acids deleted from
full-length NUDR. Forty and 80 pmol of each recombinant protein
(constructs A-L) were assayed for their ability to bind
radiolabeled N42-78 in an EMSA, as in Fig. 1B. Note that in
some cases increased levels of protein produced decreased mobility of
complexes, thus indicating potential multimerization of NUDR
proteins.
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In an attempt to transfer the DNA binding properties of NUDR to a
non-DNA-binding protein, we constructed a series of NUDR peptides fused
to GST. A peptide containing amino acids 267-368 (Fig. 3,
H) was not sufficient for DNA binding, but the inclusion of
an additional 25 N-terminal amino acids (amino acids 243-368) conferred some binding activity (Fig. 3, peptide I). Full
DNA binding activity was achieved by peptide J, composed of amino acids
167-368. The N-terminal half of peptide J, amino acids 167-289 (peptide L), showed similar DNA binding as the C-terminal half, amino
acids 243-368 (peptide I), with both peptides having reduced binding
capacities compared with peptide J. The region shared by peptides L and
I did not constitute the DNA binding domain, since construct F, which
lacks this region, was still able to bind DNA, and a shorter peptide K
(amino acids 243-328), which includes this region, was unable to bind
DNA. The reduced binding activities of the peptides (I and L) and the
deletion constructs (F and G) suggest there may be cooperativity or
synergy among the peptide regions to achieve the stronger DNA binding
activity displayed by peptide J and the full-length protein (Fig. 3).
Alternatively, the reduced binding activities of the peptides could be
a consequence of improper folding of the recombinant proteins and not
simply an elimination of the amino acids involved in binding.
Nonetheless, these results indicate that the DNA binding domain resides
in the central region of the protein and is represented by peptide J.
To verify that this central region of the protein was in direct contact
with DNA, we performed DNA-protein photocross-linking. A DNA
photoaffinity probe was synthesized by incorporating the photoreactive
deoxyuridine analog AB-dUTP (Fig.
4A) and radioactive deoxynucleotides into the N42-78 sequence (Fig. 4B). A
short-chain-length tether between the photoreactive aryl azide and the
deoxyuridine (AB-dUTP, ~10.0 Å) was used to label the protein at
sites of DNA contact (20, 21, 30). The double-stranded DNA
photoaffinity probe was used in an EMSA under reduced lighting
conditions to show that NUDR protein was able to bind the modified DNA
(Fig. 4C). After photocross-linking with UV light, the
protein-DNA complex was treated with DNase I and S1 nuclease to reduce
the DNA attached to the protein to the four radiolabeled and modified
nucleotides. The radioactive protein was cleaved with formic acid, and
the peptide fragments were resolved by SDS-PAGE (Fig. 4D).
Two radiolabeled peptides were observed with a combined molecular mass
that approximately equaled the uncut protein (Fig. 4D).
Because NUDR contains only one pH 2.5 acid-labile bond at amino acid
position 195/196 (shown schematically in Fig. 3), the cross-linking
data indicates that amino acids on both sides of the cleavage site are
in contact with the DNA. Considering the EMSA results, the
cross-linking of the 37-kDa N-terminal peptide would indicate that DNA
is in contact with NUDR between amino acids 167 and 195, and this may be further refined to a position between amino acids 167 and 187 based
on the decreased DNA binding of deletion construct E (amino acids
188-565) compared with peptide J (Fig. 3). The cross-linking of the
57-kDa C-terminal peptide would be consistent with additional protein-DNA contacts between amino acids 195 and 368. Together these
data indicate that the DNA binding domain of NUDR, as delineated by
amino acids 167-368 (peptide J), consists of at least two regions of
the protein in contact with the DNA that interact together to enhance
DNA binding.
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Fig. 4.
Photocross-linking of NUDR protein to
radiolabeled DNA indicates at least two DNA contacts.
A, the chemical structure of the photoreactive deoxyuridine
analog AB-dUTP consists of a photoreactive aryl azide group attached to
the C-5 position of deoxyuridine triphosphate. B, the
sequence of the N42-78 oligonucleotide was modified by incorporation
of [-32P]dCTP (shown by an asterisk) and
AB-dUTP (shown by a verticle arrow) at the positions indicated.
C, the modified N42-78 double-stranded oligonucleotide
probe (2 fmol) was incubated under low light conditions in the absence
or presence of hNUDR protein, and binding was analyzed by EMSA as in
Fig. 1B. D, the modified N42-78 double-stranded
oligonucleotide probe (6 fmol) was incubated with hNUDR protein,
irradiated with UV light, and either left intact or incubated with 70%
formic acid before separation by 10% SDS-PAGE. The results were
visualized with a PhosphorImager. Approximate molecular masses of the
labeled proteins were estimated from the mobility of prestained
molecular weight markers (not shown).
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NUDR Protein Binds and Regulates the hnRNP A2/B1 Promoter--
A
data base search for genes that may contain the NUDR binding consensus
sequence revealed an exact match within the human hnRNP A2/B1 promoter
(see sequence in bold, Fig.
5C). DNA fragments containing
various regions of the hnRNP A2/B1 promoter were used in DNase I
protection assays to determine whether NUDR protein would bind. As
shown in Fig. 5, NUDR bound three specific regions of the promoter. The
first region (shaded bar, Fig. 5A) included the
NUDR binding consensus sequence, a second region (open bar, Fig. 5A) appeared to have limited homology to the consensus
(6 nucleotides matched out of the 15 defined nucleotides), and a third
region located 3' of the transcription initiation site included two
closely spaced TTCG motifs (closed bar, Fig. 5B).
Closer examination of the first region revealed that the entire
protected sequence (34 nucleotides in length) was a large inverted
repeat, whereas the third protected region occurs in the
5'-UTRDNA of hnRNP, a position analogous to the NUDR
binding sequences found in the 5'-UTRDNA of NUDR.
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Fig. 5.
NUDR protein binds to sequences in the human
hnRNP A2/B1 promoter. A, a
HindIII/SmaI fragment from the hnRNPCAT plasmid
was radiolabeled and either untreated (U) or treated with
DNase I in the absence (0) or presence of increasing amounts
of recombinant NUDR protein (20 and 148 pmol, indicated by the
wedge) and analyzed as in Fig. 2A. The
NUDR-protected sequences are indicated by the different bars
adjacent to the panel, and their corresponding
bars above the nucleotide sequences in C.
B, a EcoRI/HincII fragment from the
5'-UTRDNA of hnRNP was radiolabeled and treated as in
panel A (10 and 50 pmol, indicated by the wedge).
C, the numbering of the human hnRNP A2/B1 gene
sequences has been adjusted so that the transcription initiation site
is (+1) (based on exon 1 starting at position 2427 in GenBankTM
accession number D28877). The NUDR binding consensus sequence is shown
in bold, capital letters.
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To determine whether NUDR might regulate the expression of hnRNPA2/B1,
a 742-bp DNA fragment containing the promoter was ligated to the
reporter gene CAT in the construct, hnRNPCAT. Cotransfection of
hnRNPCAT with the expression vector for full-length hNUDR resulted in a
65-70% reduction in CAT activity compared with the reporter alone
(Fig. 6 and
7). The transcriptional repression of
this promoter was somewhat surprising, since previous transfection
studies had shown that NUDR activated transcription of the
proenkephalin promoter by 26-fold (1). However, in contrast to the
current studies, we had been unable to demonstrate direct NUDR binding
to proenkephalin sequences, and we therefore suggested that
transcriptional activation by NUDR may occur through additional
protein-protein interactions.
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Fig. 6.
NUDR represses expression from the hnRNP
A2/B1 promoter. CV-1 cells were cotransfected with 2 µg of the
hnRNPCAT reporter and 1 µg of a CMV expression vector without
(open bar) or with a NUDR cDNA for wild-type or one of
the deletion constructs (shaded bars) and shown
schematically to the left of the graph. The nuclear localization signal
is indicated by NLS, and an X indicates a double mutation
(R302T/K304T) in the NUDR NLS. The internal deletion construct that
removed the NUDR NLS was modified to include the NLS from the SV40
large T-antigen (see "Experimental Procedures"). The results are
presented as percent CAT activity, with the activity of the hnRNPCAT
reporter alone set at 100%, and are the average of triplicate
measurements from two independent experiments ±S.D.
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Fig. 7.
NUDR repressor activity on the hnRNP A2/B1
promoter is through sequences downstream of transcription
initiation. CV-1 cells were cotransfected with 2 µg of the
indicated hnRNP promoter containing CAT reporter and 1 µg of either
CMVNeo (control) or CMVhNUDR (+hNUDR) expression
vectors. Regions I and II represent regions of the hnRNP promoter that
were protected from DNase I by NUDR protein (see Fig. 5) and have been
deleted where indicated. The results are presented as percent CAT
activity with the activity of the hnRNPCAT reporter alone set at 100%
and are the average of triplicate measurements from two independent
experiments ±S.D.
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The establishment of NUDR binding to sequences in the hnRNP promoter
provided the rationale to use transcriptional repression of hnRNP as an
in vivo approach to assess the functional activity of NUDR
proteins with various mutations and deletions (see "Experimental Procedures," Fig. 3). Mutations in the NLS of NUDR (R302T/K304T) that
resulted in the protein being localized exclusively to the cytoplasm
(1) failed to repress transcription of the hnRNP promoter (Fig. 6).
Since point mutations in the NLS did not impair the DNA binding
activity of NUDR (data not shown), these results indicate that nuclear
localization is required for transcriptional repression. Similarly, the
N-terminal half of NUDR (amino acids 1-242) also had no effect on
transcription (data not shown). A C-terminal-truncated NUDR protein
(amino acids 1-505) that lacked the zinc finger homology region was
less effective as a repressor compared with the full-length protein
(34% reduction versus 65%). This result suggests a
potential role of the zinc finger homology region in repressor function
but may also reflect the somewhat reduced DNA binding activities
observed for recombinant NUDR proteins that lack this region (Fig. 3).
The C-terminal half of NUDR (amino acids 243-565) was unable to
repress transcription of the hnRNP promoter. Since recombinant NUDR
proteins and peptides missing amino acids 167-243 show reduced DNA
binding activities (Fig. 3), the lack of repression by the C-terminal
protein may also be a consequence of reduced DNA binding. We tested
peptide J (amino acids 167-368) for repressor activity, because this
peptide was shown to have DNA binding activity similar to full-length
NUDR (Fig. 3). Peptide J repressed transcription of hnRNPCAT to a
similar level as the full-length protein, suggesting this region of
NUDR is sufficient for repression of the hnRNP promoter. To further establish that the central region of NUDR was involved in
transcriptional repression, we constructed a chimeric protein of NUDR
in which the NLS of NUDR and part of the DNA binding domain (deletion
of amino acids 254-368, similar to construct G in Fig. 3)
were replaced with the NLS from the SV40 large T-antigen (see
"Experimental Procedures"). The chimeric protein was localized to
the nucleus (not shown) but was unable to repress the expression of
hnRNPCAT. Together these results indicate that the repressor domain
colocalizes with the DNA binding domain in the central portion of NUDR
(amino acids 167-368) and that DNA binding is required for repressor function.
Because DNase I protection assays had demonstrated NUDR protein binding
to DNA sequences within the hnRNP promoter (Fig. 5), we examined the
importance of these sequences for transcriptional activity and NUDR
repression. Deletion of region I from the hnRNP A2/B1 promoter
(nucleotides 
583 to 471) removed a major portion of the two binding
sequences identified upstream of transcription initiation, whereas
deletion of region II (nucleotides 51 to 160, hnRNPIICAT) removed
NUDR binding sequences identified in the 5'-UTRDNA. As
shown in Fig. 7A, deletion of region I (hnRNPICAT) resulted in a slight reduction (25%) in basal CAT activity compared with hnRNPCAT; however NUDR repressed transcription of hnRNPICAT as
effectively as full-length hnRNPCAT (30% of basal activity). These
results indicate that NUDR repression is not mediated through sequences
in region I. Deletion of region II from the hnRNP promoter in the
reporter constructs, hnRNPIICAT and hnRNPI,IICAT resulted in
significantly reduced basal CAT activities when compared with the
parent construct hnRNPCAT (Fig. 7). However, the transcriptional activities of these promoters were not eliminated and were
approximately 10-fold higher than those produced by the thymidine
kinase (TK) promoter in pBLCAT5 (data not shown). The reduced activity
could signify the removal of a cis-acting element in the
5'-UTRDNA region of the promoter. Importantly, the
overexpression of NUDR did not repress the basal activities of these
two reporters. These data support the hypothesis that NUDR repression
of the hnRNP promoter occurs through NUDR binding to sequences located
3' and proximal to the transcription initiation site.
Because NUDR repression mapped to the 5'-UTRDNA of the
hnRNP promoter, and since we had also observed multiple NUDR binding motifs in the 5'-UTRDNA of the hNUDR8 cDNA clone, we
sought to determine whether placement of NUDR sequences on a
heterologous promoter and in positions comparable with those found in
the hnRNP promoter would affect basal transcription and confer NUDR
protein regulation. We inserted sequences from the
5'-UTRDNA of hNUDR8 into pBLCAT5 in positions 5' of the TK
promoter ((h8N1-356)TKCAT) and 3' of the transcription initiation site
of the CAT gene (TK(h8N1-356)CAT and TK(h8N1-121)CAT) and examined
the effects of NUDR coexpression. The transcriptional activity of
(h8N1-356)TKCAT showed low basal activity similar to pBLCAT5 and was
not regulated by NUDR (Fig. 8). We also
found no effect on CAT activity when the 5'-UTRDNA sequences were placed in a promoterless CAT vector, pBLCAT6 (data not
shown). In contrast, the reporter TK(h8N1-356)CAT with the 5'-UTRDNA sequences inserted 3' of the TK transcription
initiation site produced significant increases in the basal CAT
activity, and this activity was repressed by NUDR protein
overexpression (89% reduction). Since we had localized the majority of
NUDR binding to sequences in the upper third of the
5'-UTRDNA of hNUDR8 (Fig. 2), we tested a second reporter,
with nucleotides 1-121 of the h8NUDR cDNA inserted 3' of the TK
transcription initiation site (TK(h8N1-121)CAT). The TK(h8N1-121)CAT
reporter also showed an elevated basal level of CAT activity, and this
activity was repressed by NUDR overexpression (84% reduction).
Although TK(h8N1-356)CAT and TK(h8N1-121)CAT produced high levels of
CAT activity relative to pBLCAT5, it should be noted that these levels
are modest relative to hnRNPCAT. These results are analogous to those
observed for the hnRNP A2/B1 promoter and support the hypothesis that
NUDR represses transcription through DNA binding at sites located 3' proximal to the transcription start site.
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Fig. 8.
Analysis of hNUDR8 5'-UTRDNA
sequences using the thymidine kinase promoter. Sequences from the
5'-UTRDNA of hNUDR8 were inserted into pBLCAT5 as described
under "Experimental Procedures." CV-1 cells were cotransfected with
2 µg of the indicated reporter constructs, drawn schematically at the
left, and 1 µg of CMVNeo (control) or CMVhNUDR
(+hNUDR) expression vectors. The results are presented as
percent CAT activity with the activity of pBLCAT5 alone set at 100%
(multiplied by 102) and are the average of triplicate
measurements from two independent experiments ±S.D.
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DISCUSSION |
In this report, we continue the characterization of NUDR as a
DNA-binding protein and transcriptional regulator. We have localized the DNA binding domain of NUDR to the central region of the protein by
demonstrating that a peptide comprised of amino acids 167-368 (peptide J, Fig. 3) has in vitro DNA binding
activities comparable with the full-length protein. The DNA binding
domain appears to be composed of smaller regions that individually have
weak DNA interactions yet can interact cooperatively to achieve full
binding activity. The DNA binding domain overlaps with the previously identified nuclear domain (1)/KDWK domain (2) and is the region that
displays the highest homology between NUDR and Drosophila DEAF-1 (70%) (1). Consequently, it is not surprising that the DNA
sequences recognized by NUDR and DEAF-1 are similar and consist of at
least one and usually multiple TTCG motifs (1, 2). Although we have
derived a NUDR binding consensus of TTCGGGNNTTTCCGG from the analysis
of oligonucleotides selected by NUDR binding and PCR amplification (1),
we have also noted that NUDR binds preferentially to sequences with
mulitiple copies of TTC(C/G)G without a strict requirement for specific
spacing between the motifs.
The observations that NUDR protects large DNA regions in DNase I
protection assays and the presence of multiple, low mobility complexes
in EMSA are indicative of potential protein-protein interactions (see
Figs. 1 and 2). The concept of NUDR homodimerization or multimerization
is supported by the observation that Drosophila DEAF-1 may
form multimers (2). In addition, MTG8 (also known as ETO) has homology
to NUDR in the zinc finger region and has been observed to form
homomeric complexes (31), whereas the mouse homolog of NUDR (mDEAF-1)
was shown to interact with itself, albeit weakly, in a yeast two-hybrid
system (3). A difficulty in resolving this issue is that we have yet to
resolve a minimal DNA element recognized by NUDR. NUDR binds DNA
fragments with multiple TTC(C/G)G-like sequences with greater affinity
and at lower protein concentrations than DNA fragments with fewer
TTC(C/G)Gs (data not shown). And, at least in vitro, NUDR
appears to bind somewhat promiscuously and with low affinity to other
sequences flanking these motifs, especially at higher protein
concentrations (Fig. 2 and 5; Ref. 1). We suggest that optimal binding
of NUDR to DNA occurs when multiple binding elements are in close proximity to one another to enhance the cooperativity among NUDR multimers.
Utilizing EMSA and DNase I protection assays, we have demonstrated that
NUDR protein binds to sequences within its own 5'-UTRDNA and to sequences within the hnRNP A2/B1 promoter both 5' and 3' of
transcription initiation. In transient transfection assays, NUDR was
shown to repress transcription from the hnRNP A2/B1 promoter. Furthermore, the peptide encompassing the DNA binding domain (peptide J, amino acids 167-368) was shown to be almost as effective (55% reduction) as the full-length protein in repressing hnRNPCAT activity, suggesting that the majority of NUDR repressor activity also appears to
reside within this domain.
The finding that amino acids 167-368 of NUDR could contain a repressor
domain is intriguing because this region has sequence homology with
SP100B and LYSP100B (1) and was identified as a "Myc-type,
helix-loop-helix dimerization domain signature" (amino acids
319-358) in a comparison of the PROSITE data base (32). SP100 proteins
are localized to subnuclear structures termed nuclear bodies and are
thought to play a role in the etiology of acute promyelocytic leukemia
(reviewed in Ref. 11). SP100 was shown to associate with non-histone
chromatin components that behave as transcriptional silencers, and when
fused to a GAL4 DNA binding domain, SP100B was able to repress
transcription (12, 13).
Helix-loop-helix (HLH) motifs are often dimerization domains, and when
accompanied by an adjacent region rich in basic amino acids (basic
helix-loop-helix or bHLH), they can interact directly with DNA (33).
Typically the two helices in HLH domains are amphipathic and create a
hydrophobic interface to stabilize the interaction between dimers.
Although secondary structure algorithms (e.g. Chou and
Fasman (34)) predict NUDR to have two helices separated by a turn in a
region near the basic amino acids of the NLS, other programs that plot
the peptide sequence as a helical wheel do not recognize these helices
as amphipathic (not shown). In mobility shift assays, most of the
recombinant NUDR proteins that included the potential HLH motif
displayed decreased mobility complexes with increased levels of protein
(Figs. 1B and 3), indicating the possible multimerization of
NUDR proteins. We have shown that the region encompassing the NLS and
potential HLH is required for full DNA binding activity in
vitro (Fig. 3), and the repressor activity of NUDR also maps to
this region. We have not yet determined whether these activities are
separable. It is interesting to note that the inhibitory domain of an
ets oncogene family member also bears strong resemblance to
a HLH motif (35), strengthening the potential contribution of the NUDR
HLH region in repressor function.
The C-terminal, zinc finger homology region of NUDR shares amino acid
homology with the repressor domain of MTG8/ETO (1, 8). The zinc finger
region of MTG8/ETO (previously described as the MYND domain (2)) was
established as a crucial site of interaction with the nuclear
corepressor N-CoR, and deletion of this region impaired transcriptional
repression (8). In acute myeloid leukemias, the chromosomal
translocation t (8, 21) converts the transcriptional activator AML-1
into a transcriptional repressor by producing an AML-1/ETO fusion
protein (28). This fusion protein most likely disrupts normal
hematopoietic differentiation by recruitment of nuclear corepressors
(i.e. N-CoR and Sin3), which then repress genes essential
for normal differentiation (9, 36, 37). The high degree of homology
between MTG8/ETO and NUDR suggests that the C terminus of NUDR may also
be involved in the recruitment or interaction with corepressors.
Deletion of the zinc finger domain of NUDR resulted in a protein that
was not as effective as the full-length protein in transcriptional repression, implying that this region of NUDR may indeed recruit corepressors. However, since peptide J, which lacks the zinc finger domain, approached the level of repression achieved by the full-length protein, the majority of the repressor activity appears to reside within the central DNA binding domain of NUDR, with possible minor contributions by the C-terminal zinc finger domain.
The C-terminal region of the mouse homolog of human NUDR (mDEAF-1) has
been shown to interact with LMO proteins (3). Using a yeast two-hybrid
interaction assay, Sugihara et al. (3) showed that the
region between amino acids 334 and 518 of mDEAF-1 interacted with the
LIM domain. Although LMO proteins do not directly bind DNA, they are
thought to interact with DNA-binding proteins and form complexes
involved in transcriptional regulation (38). LMOs have been identified
at sites of chromosomal translocations, and their ectopic expression
has been associated with childhood T-cell acute leukemias (39). Since
the LMO interaction and DNA binding domains overlap, it is interesting
to speculate that LMO interaction with NUDR could alter the DNA binding
specificity of NUDR, perhaps allowing recognition and transcriptional
activation of the proenkephalin promoter.
The hnRNP A2/B1 gene was first indicated as a potential NUDR
regulated gene in a data base search by the identification of a perfect
match of the NUDR binding consensus sequence upstream of the proximal
promoter. Although NUDR does bind to this consensus sequence, NUDR
binding sites downstream of the transcription initiation were shown to
be responsible for the NUDR repression of transcription (Fig. 7). The
position-dependent repression by NUDR was confirmed in a
heterologous promoter by transferring NUDR binding sequences from the
5'-UTRDNA of hNUDR8 into sites downstream of the TK
promoter (Fig. 8). NUDR repressed transcription of TK(h8N1-356)CAT by
89% but did not repress transcription of the reporter with NUDR
binding sequences inserted upstream of transcription initiation
((h8N1-356)TKCAT). The position-dependent repression by
NUDR suggests that a potential mechanism of NUDR repression may be
through blocking of the RNA polymerase II complex and inhibition of
elongation rather than inhibition of transcription initiation. Pausing
of RNA polymerase II at sites downstream of transcription initiation
have been demonstrated for c-Myc (40, 41), c-Myb (42), c-Fos (43),
adenosine deaminase (44), and Drosophila hsp70 (45).
Recently, the 1,25-dihydroxyvitamin D3-induced transcriptional
elongation block of c-Myc was found to be linked to HOXB4 binding site
within intron 1 of the c-myc gene (46), suggesting a role
for developmental factors in c-Myc regulation.
Multiple sequence elements in the 5'-UTR of the bcl-2 gene
have been shown to be responsible for the decreased expression from the
bcl-2 P1 promoter, and transfer of these sequences to a 5'-UTR position
in a CMVNeo construct resulted in decreased expression from the CMV
promoter (47). In contrast, NUDR binding sequences in the
5'-UTRDNA position of hnRNPCAT and TK(h8N1-356)CAT resulted in elevated levels of basal reporter activity, suggesting the
possible recruitment of a position-dependent activator
protein(s). Thus, an alternative explanation for
NUDR-dependent repression may be through competition or
inhibition of an activator at or near NUDR binding sites. Mechanisms of
transcriptional repression by competition and displacement have been
noted for several transcription factors, including Sp1, EGR-1, and WT1
(48), although in most cases the repression, both direct and indirect,
occurs through sequences upstream of the promoter (49-52). Finally,
the interactions of glucocorticoid receptor and the transcription
factor AP-1 (Jun and Fos heterodimer) illustrate another possible
mechanism for repression. Although in most cases glucocorticoid
receptor and AP-1 behave as transcriptional activators, in some
promoters the colocalization of glucocorticoid receptor and AP-1 on DNA
sequences can repress transcription (53).
A comparison of DNA sequences recognized by NUDR and other
transcription factors revealed that NUDR binding sequences may overlap
with the DNA recognition sequence of some ETS domain-containing proteins, many of which have been shown to be transcriptional activators (54-57). The sequence recognized by several ETS domain proteins is 5' A/GCCGGAA/T
3', with the ETS core binding sequence underlined (56). This sequence
contains a potential NUDR binding motif (shown in bold), which is more
readily visualized on the complementary strand, 5'
A/TTCCGGC/T 3'. Whether ETS domain proteins can bind to sequences in the 5'-UTRDNA
of hnRNP A2/B1 or the NUDR gene and up-regulate their
expression remains to be determined, as does the precise mechanism of
NUDR repression.
The identification of NUDR binding sites within the hnRNP A2/B1
promoter in vitro and the demonstration that NUDR represses expression from this promoter in transactivation assays indicate a
potential in vivo role for NUDR in the regulation of the
hnRNP A2/B1 gene. hnRNP A2/B1 mRNA and protein have been
shown to display dynamic patterns of expression during mammalian lung
development, with highest levels in primitive alveoli, and lowest
levels in mature lung (58). Although low levels of hnRNP A2/B1 protein were detected in normal bronchial epithelium, elevated levels of
protein were detected in a variety of lung cancer cell lines (15).
hnRNP A2/B1 has been proposed as an early marker in the detection of
lung cancer (14-17), and the results of ongoing clinical trials have
shown that up-regulation of hnRNP A2/B1 expression can accurately
predict the subsequent development of lung cancer (17, 59, 60). Based
on our data, we would predict that deregulation of NUDR expression
and/or mutations in NUDR that inactivated the DNA binding or repressor
activities could contribute to the higher expression levels of hnRNP
A2/B1 found in some cancers.
,
TOP
ABSTRACT
INTRODUCTION
