Molecular Interactions between Single-stranded DNA-binding
Proteins Associated with an Essential MCAT Element in the Mouse
Smooth Muscle
-Actin Promoter*
Robert J.
Kelm Jr.
,
John G.
Cogan§,
Paula K.
Elder
,
Arthur
R.
Strauch§, and
Michael J.
Getz
¶
From the
Department of Biochemistry and Molecular
Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905 and the
§ Department of Physiology, Ohio State University, College
of Medicine, Columbus, Ohio 43210
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ABSTRACT |
Transcriptional activity of the mouse vascular
smooth muscle
-actin gene in fibroblasts is regulated, in part, by a
30-base pair asymmetric polypurine-polypyrimidine tract containing an essential MCAT enhancer motif. The double-stranded form of this sequence serves as a binding site for a transcription enhancer factor
1-related protein while the separated single strands interact with two
distinct DNA binding activities termed VACssBF1 and 2 (Cogan, J. G., Sun, S., Stoflet, E. S., Schmidt, L. J., Getz, M. J., and Strauch, A. R. (1995) J. Biol. Chem. 270, 11310-11321; Sun, S., Stoflet, E. S., Cogan, J. G., Strauch,
A. R., and Getz, M. J. (1995) Mol. Cell. Biol.
15, 2429-2936). VACssBF2 has been recently cloned and shown to consist
of two closely related proteins, Pur
and Pur
(Kelm, R. J.,
Elder, P. K., Strauch, A. R., and Getz, M. J. (1997)
J. Biol. Chem. 272, 26727-26733). In this study, we
demonstrate that Pur
and Pur
interact with each other via highly
specific protein-protein interactions and bind to the purine-rich strand of the MCAT enhancer in the form of both homo- and heteromeric complexes. Moreover, both Pur proteins interact with MSY1, a
VACssBF1-like protein cloned by virtue of its affinity for the
pyrimidine-rich strand of the enhancer. Interactions between Pur
,
Pur
, and MSY1 do not require the participation of DNA. Combinatorial
interactions between these three single-stranded DNA-binding proteins
may be important in regulating activity of the smooth muscle
-actin MCAT enhancer in fibroblasts.
 |
INTRODUCTION |
Eukaryotic gene transcription requires the coordinated assembly of
upstream cis-element binding proteins, intermediary
cofactors, and components of the basal transcription machinery into a
multicomponent complex competent to initiate transcription. During this
process, sequence-specific DNA-binding transcriptional activators
and/or repressors play a pivotal role in modulating the cell-type
specific expression of genes. While most such proteins bind to
double-stranded DNA target sequences, a small but intriguing subclass
has been identified that show enhanced affinity and specificity for
either the sense or antisense strands of certain
cis-regulatory elements required for promoter-specific
activation (1-4) or repression (5-9). We have recently cloned and
identified two single-stranded DNA
(ssDNA)1-binding proteins,
Pur
and Pur
, that interact with the purine-rich strand of an
essential transcription control sequence upstream of the mouse vascular
smooth muscle (VSM)
-actin gene promoter (10).
The involvement of ssDNA-binding proteins in VSM
-actin gene
transcription was discovered as a consequence of promoter mapping studies that led to the identification of a conserved 5'-flanking sequence required for both activation and repression of promoter activity in fibroblasts and undifferentiated myoblasts (11, 12). This
proximal promoter element (PE) sequence (
195 to
165) exhibited
polypurine-polypyrimidine asymmetry, an inverted muscle-specific MCAT
(AGGAATG) enhancer element, and bound at least three distinct DNA
binding activities in a sequence and strand-specific manner. The two
ssDNA binding activities, formerly designated vascular actin single-strand
binding factors, VACssBF 1 and 2, appeared to
play a role in repression (11) while a transcription enhancer factor
1-related protein was implicated in activation (12, 13). Although the
mechanism of repression remains to be formally established, a
hypothetical model involving VACssBF-mediated disruption of MCAT
element base pairing and competition for transcription enhancer factor
1 binding was proposed (11). Interestingly, an additional binding site
for VACssBF2 was later identified on the purine-rich coding strand of a
GGAATG-containing sequence element located in a downstream VSM
-actin exon (14). This coding element sequence functioned as a
VACssBF2-dependent repressing element when positioned 5'
and adjacent to a transcription enhancer factor 1- or activator protein
1-dependent enhancer element in chimeric promoter
constructs (14). Because the noncoding strand of the coding element
sequence lacked detectable VACssBF1 binding affinity (14), these data suggested that VACssBF2 binding was necessary and sufficient for repression. Screening of a mouse lung cDNA expression library with
the exonic VACssBF2-binding site ultimately resulted in the isolation
of two clones encoding the purine-rich ssDNA-binding proteins, Pur
and Pur
(10). Biochemical analyses of the cloned mouse Pur proteins
expressed in fibroblasts confirmed that Pur
and Pur
corresponded
to the p46 and p44 components of VACssBF2 that bind to the
purine-rich strand of the PE and presumably down-regulate VSM
-actin gene expression (10).
In the present study, we used a similar binding site screen to confirm
the identity between VACssBF1 and the mouse Y-box protein, MSY1. By
utilizing recombinant proteins and isoform-specific immune reagents, we
demonstrate highly specific protein-protein interactions between
Pur
, Pur
, and MSY1 which seem likely to have functional significance. These include the binding of Pur proteins to ssDNA as
both homo- and heterodimeric complexes and the formation of heterotrimeric complexes between all three proteins in the absence of
DNA. These data suggest that protein-protein interactions between Pur
, Pur
, and MSY1 play an important role in regulating
transcriptional activity of the VSM
-actin gene in fibroblasts.
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EXPERIMENTAL PROCEDURES |
Cloning of a cDNA Encoding a PE-MCAT Strand Binding Protein,
MSY1--
A mouse lung cDNA expression library (Stratagene) was
screened for cDNA-encoded proteins that interact with the
pyrimidine-rich strand of the VSM
-actin MCAT enhancer element using
a binding site cloning methodology described previously (10). Eight
independent clones were isolated from 250,000 plaques initially
screened. DNA sequencing by semi-automated dideoxy termination
indicated that all eight clones encoded the mouse Y-box protein, MSY1
(15).
Construction of Bacterial Expression Vectors and Purification of
6xHis-tagged Pur
, Pur
, and MSY1--
The cDNAs encoding the
open reading frame minus the start methionine of mouse Pur
, Pur
(10), and MSY1 (clone 7-1, this study) were amplified by polymerase
chain reaction using primers which generated 5' BamHI and 3'
KpnI cloning sites. The polymerase chain reaction products
were gel purified, cut with restriction enzymes, and subcloned into
pQE-30 (Qiagen) to generate fusion constructs encoding a N-terminal
6xHis tag. The resultant plasmids were transformed into
Escherichia coli strain JM109 and the orientation and
fidelity of the polymerase chain reaction-amplified cDNA inserts were determined by DNA sequencing. For protein preparation, 1 liter of
terrific broth containing 100 µg/ml ampicillin was inoculated (1:50)
with an overnight bacterial culture and incubated for 5-7 h at
37 °C. Recombinant protein synthesis was induced by the addition of
isopropyl-
-D-thiogalactopyranoside to 2.0 mM
and an additional 4-h growth period. E. coli were collected
by centrifugation at 5000 × g for 10 min and
resuspended in 14 ml of cold 50 mM sodium phosphate, pH
8.0, 300 mM NaCl (lysis buffer) supplemented with 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 µg/ml each
pepstatin, leupeptin, and aprotinin. Cells were lysed by sequential
30-min incubations on ice with lysozyme added to 1.0 mg/ml followed by Triton X-100 added to 0.13% (v/v). Lysates were centrifuged at 100,000 × g for 30 min. Supernatants were collected,
combined with 4 ml of packed Ni-NTA agarose resin (Qiagen) equilibrated in lysis buffer, and mixed overnight at 4 °C. The resin was washed sequentially at room temperature with 50 mM sodium
phosphate, pH 8.0, buffer containing 0.3, 1.0, and 2.0 M
NaCl. The resin was then packed into a column and washed with 50 mM sodium phosphate, 2.0 M NaCl, pH 8.0, until
the A280 nm of the flow-through was
0.02.
His-tagged protein was eluted with a step gradient of 50 mM
phosphate, 2.0 M NaCl containing 20, 40, 80, and 500 mM imidazole. Fractions were analyzed by SDS-polyacrylamide
gel electrophoresis and Coomassie Blue R-250 staining. Pur protein or
MSY1-enriched fractions were pooled, dialyzed versus 50 mM sodium phosphate, 1.0 M NaCl, pH 8.0, and
chromatographed a second time on Ni-NTA agarose to ensure optimal
purity. His-tagged protein eluates were dialyzed against 25 mM HEPES, 0.5 M NaCl, pH 8, aliquoted, and stored at
20° C. Recombinant proteins were also purified as above but in buffers supplemented with 8 M urea. Purification
under such conditions enhanced the yield of His-tagged protein. No
differences were observed in the in vitro protein-binding
properties between His-tagged proteins purified under nondenaturing or
denaturing conditions after dilution and/or dialysis into aqueous
buffer. Recombinant protein concentration was estimated by optical
density measurement using molar extinction coefficients and molecular weights of 18,610 and 35,000 for Pur
, 18,610 and 34,000 for Pur
, and 26,170 and 36,000 for MSY1. In some cases, proteins were quantified by dye-binding assay (Bio-Rad) using bovine serum albumin as a standard.
Preparation of Peptide-specific Polyclonal Antibodies against
Mouse Pur
, Pur
, and MSY1--
Peptides corresponding to amino
acids 42-69, 210-229 and 302-324 of mouse Pur
(10), 149-175 and
291-313 of mouse Pur
(10), and 85-110, 139-165, 242-267,
and 276-302 of MSY1 (this study) were synthesized using modified
Merrifield solid-phase chemistry and purified by reverse-phase high
performance liquid chromatography by the Mayo Protein Core Facility.
The composition of each peptide was confirmed by amino acid analysis.
Each peptide was synthesized with a cysteine residue at either the N or
C terminus to facilitate coupling to maleimide-activated KLH (Pierce)
and iodoacetyl-agarose (Sulfolink, Pierce). KLH-coupled peptides were
used as immunogens and rabbit polyclonal antisera production was
carried out by a commercial vendor (Cocalico) using a 60-day standard
protocol. Polyclonal rabbit IgGs were affinity purified using
peptide-coupled agarose columns. Briefly, whole IgG was enriched from
rabbit antisera by 40% ammonium sulfate precipitation. Following
centrifugation for 10 min at 5000 × g, the IgG-rich
pellet was reprecipitated, dissolved in phosphate-buffered saline, and
dialyzed. Rabbit IgG was then applied to the appropriate 2-ml
peptide-agarose (0.5-1.0 mg of peptide/ml) column equilibrated with
phosphate-buffered saline. The flow-through fraction was collected and
reapplied. After washing the column with phosphate-buffered saline,
peptide-bound IgG was eluted with 0.1 M glycine, pH 2.5, and immediately neutralized with 1 M Tris, pH 9.5. Affinity
purified IgG was precipitated by the addition of solid ammonium sulfate
to 75% saturation. The pellet was collected by centrifugation,
dissolved in 50% (v/v) glycerol/phosphate-buffered saline, and stored
at
20° C. Rabbit IgG from pooled preimmune serum was purified on
Protein A/G-agarose (Calbiochem). IgG concentration was estimated by
optical density measurement based upon a molar extinction coefficient
and molecular weight of 210,000 and 150,000, respectively.
Screening of Peptide Affinity-purified Antibodies by
ELISA--
His-tagged mouse Pur
, Pur
, or MSY1 diluted to 50 nM in 25 mM HEPES, 150 mM NaCl, pH
7.5 (HBS), containing 5.0 µg/ml crystalline grade bovine serum
albumin (Roche Molecular Biochemicals), was applied to polystyrene
microtiter wells (100 µl/well) (Corning ELISA plate number 25805) and
incubated 16-20 h at 4° C. The resultant Pur
-, Pur
-, or
MSY1-coated wells were washed once with HBS containing 0.05% (v/v)
Tween 20 (HBST), and blocked for 1 h with 0.2% (w/v) bovine serum
albumin in HBS (250 µl/well). Wells were washed once and rabbit
anti-mouse Pur or MSY1 peptide antibody (1.0-0.016 µg/ml, 100 µl/well) diluted in HBST containing 0.1% bovine serum albumin was
applied for 2 h at room temperature. Primary antibody solution was
aspirated and wells were washed three times with HBST. Goat anti-rabbit
IgG-HRP (Santa Cruz) diluted 1:2000 in HBST was then applied for 1 h. Wells were washed as above and 100 µl of ABTS chromogenic
substrate (Roche Molecular Biochemicals) was added. Absorbance readings
at 405 nm were determined after 5-6 min using a 96-well microplate spectrophotometer.
Screening of Peptide Affinity-purified Antibodies by Western
(Immuno)blotting--
Cellular Pur
, Pur
, or MSY1 were
enriched from 1 mg of AKR-2B fibroblast nuclear protein by selective
capture on biotinylated-ssDNA (PE-F for Pur proteins and PE-R for MSY1)
coupled streptavidin-paramagnetic particles as described previously
(10). DNA-bound proteins were eluted with 1% SDS, resolved on a 10%
(29:1) polyacrylamide mini-curtain gel, and electrotransferred to a
polyvinylidene difluoride membrane (Immobilon-P) for 90 min (300 mA) in
25 mM Tris, 192 mM glycine, 20% (v/v) methanol
at 4° C. After blocking overnight in 25 mM Tris, 150 mM NaCl, pH 7.5 (TBS), with 5% (w/v) Carnation nonfat dry
milk at 4° C, the membrane was fitted into a multiscreen apparatus (Mini-PROTEAN II, Bio-Rad) and selected channels were loaded with 0.6 ml of rabbit anti-Pur or MSY1 peptide antibody diluted to 2.0, 0.5, and
0.1 µg/ml in 2% nonfat dry milk/TBS. Following a 1-h incubation at
ambient temperature with gentle mixing, the antibody solutions were
aspirated, and each channel was washed once with TBS, containing 0.05%
Tween 20 (TBST). The initial wash solution was aspirated, the apparatus
disassembled, and the entire blot washed three more times (5 min/25-ml
wash). Goat anti-rabbit IgG-HRP (Santa Cruz) diluted 1:2000 in TBST was
then applied for 1 h. The blot was washed four times (30 min
total) and chemiluminescence reagent (ECL, Amersham) was applied for 1 min. Immune complexes were visualized on x-ray film (XAR-5, Kodak)
following a 5-10-s exposure.
Electrophoretic Mobility Shift Assay for Protein-DNA
Binding--
Band shift assays were performed as described previously
(11, 14). For antibody supershift experiments, rabbit IgGs (0.25-1.0 µg) were preincubated for 20 min with AKR-2B nuclear protein (10) in
binding buffer containing poly(dI-dC) (11). A 32P-ssDNA
probe corresponding to the purine-rich coding strand of the PE was then
added (~1 nM final) and mixtures were incubated for an
additional 20 min prior to electrophoresis on a 6% nondenaturing polyacrylamide gel.
ELISA for Protein-Protein Interaction--
His-tagged Pur
,
MSY1, or dihydrofolate reductase (DHFR)-coated microtiter wells (50 nM application as described above) were incubated with
varying amounts of AKR-2B nuclear protein (10) (100 µl/well) diluted
in binding buffer (HBST with 0.1% bovine serum albumin) for 16-18 h
at 4° C. Wells were aspirated and washed 3 times with HBST and 100 µl of anti-Pur or anti-MSY1 peptide IgG diluted to 1.0 µg/ml in
binding buffer was applied for 1 h at room temperature. Primary
antibody solution was aspirated and wells were washed three times with
HBST. Goat anti-rabbit IgG-HRP (Santa Cruz) diluted 1:2000 in HBST was
then applied for 1 h. Secondary antibody solution was aspirated
and wells were washed four times with HBST. Immune complexes were
detected using 100 µl of ABTS chromogenic substrate. Absorbance
readings at 405 nm were determined after 45 min.
Immunoprecipitation Assay for Protein-Protein
Interaction--
Mouse AKR-2B fibroblasts were transiently transfected
with mouse Pur
and Pur
expression vectors as described previously (10). All subsequent steps including cell synchronization,
serum-stimulation, harvest, extraction, and protein assay have been
detailed previously (10, 14). Whole cell protein extract (100 µg)
from transfected cells or nuclear extract from nontransfected rapidly
growing AKR-2B fibroblasts (10) was combined with 2.5 µg of selected
rabbit anti-Pur or MSY1 peptide IgGs in a final volume of 250 µl.
After a 1-h incubation at room temperature, ~107 sheep
anti-rabbit IgG-coupled magnetic dynabeads (Dynal) were added and the
mixtures incubated for an additional 90 min. In some experiments, goat
anti-rabbit IgG-biotin (Santa Cruz) coupled to streptavidin-coated
paramagnetic beads (Promega) were used. The beads were then captured
with a magnet and washed three times with HBS. Rabbit IgG-bound protein
was specifically eluted by adding a vast excess of free peptide (20 µl at 50 µM) and incubating for 30 min at room
temperature. Eluates were supplemented with Laemmli SDS sample
preparation buffer and 5% (v/v)
-mercaptoethanol, and subjected to
electrophoresis on a 10% (29:1) polyacrylamide mini-gel.
Immunoprecipitates were evaluated for the presence Pur proteins and
MSY1 via immunoblotting as described above.
 |
RESULTS |
Development and Characterization of Isoform-specific Immune
Reagents to Pur
and Pur
--
A repertoire of immune reagents
based upon cDNA-deduced amino acid sequences of Pur
and Pur
were produced to assist in defining determinants of protein-DNA and
putative protein-protein interactions. Synthetic peptides corresponding
to both conserved and unique sequences within the Pur proteins were
used as immunogens in rabbits (Fig. 1).
IgGs were enriched from rabbit antisera and then subjected to affinity
purification using peptide-coupled agarose columns. The resultant
affinity purified IgGs were tested for reactivity using both
recombinant (His-tagged) and cellular Pur
and Pur
(Figs.
2 and 3).
Assessment of antibody binding to immobilized recombinant Pur
and
Pur
by ELISA (Fig. 2) indicated that several antibodies possessed
remarkable specificity for either Pur
(anti-A291-313) or Pur
(anti-B210-229 and anti-B302-324) while another antibody directed
against a conserved region (anti-B42-69) cross-reacted with Pur
and
Pur
(Fig. 2). The specificity of these antibodies was also evaluated
by Western blotting of cellular Pur proteins enriched from an AKR-2B
fibroblast nuclear extract by selective capture on paramagnetic
particles coupled with the purine-rich strand of the VSM
-actin MCAT
element, PE-PrMss, or PE-F (10). Consistent with ELISA data,
anti-B42-69 demonstrated similar reactivity toward fibroblast-derived
Pur
(p46, Mr ~ 46,000) and Pur
(p44, Mr ~ 44,000) which migrate as a closely spaced
doublet (Fig. 3, lanes 4-6). Anti-A291-313 recognized only
the slower migrating Pur
(p46) isoform (Fig. 3, lanes
16-18) while both anti-B210-229 and anti-B302-324
preferentially detected the faster migrating Pur
(p44) isoform (Fig.
3, lanes 10-12 and 13-15). As expected, preimmune rabbit IgG failed to detect the Pur proteins in both screening assays.

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Fig. 1.
Peptides used in the preparation of
polyclonal antibodies to Pur and Pur . Five peptides
corresponding to selected amino acid sequences (underlined)
were synthesized, coupled to KLH, and used as immunogens. Polyclonal
IgGs were enriched from rabbit antisera by ammonium sulfate
fractionation and then subjected to affinity purification on
peptide-coupled agarose columns.
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Fig. 2.
Screening of rabbit anti-Pur peptide IgGs
against recombinant Pur proteins. Purified 6xHis-tagged mouse
Pur or Pur at 50 nM was applied to polystyrene
microtiter wells (100 µl/well) and incubated 16 h at 4° C.
Following washing and blocking steps, rabbit anti-mouse Pur peptide IgG
(1.0-0.016 µg/ml, 100 µl/well) was applied to the Pur
protein-coated wells. After a 2-h incubation at room temperature,
solid-phase immune complexes were detected by ELISA using a goat
anti-rabbit IgG-HRP conjugate as the secondary antibody.
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Fig. 3.
Screening of anti-Pur peptide antibodies
against fibroblast-derived Pur proteins. Pur and Pur were
enriched from 1 mg of AKR-2B fibroblast nuclear protein by selective
capture on biotinylated-ssDNA (PE-F)-coupled streptavidin-paramagnetic
particles (10). DNA-bound proteins were eluted, resolved on a 10%
SDS-polyacrylamide mini-curtain gel, and electrotransferred to a
polyvinylidene difluoride membrane. Pur (p46 band) and Pur (p44
band) were detected by immunoblotting using a multiscreen apparatus
(Bio-Rad) in which selected channels were loaded with anti-Pur peptide
antibody diluted to 2.0, 0.5, and 0.1 µg/ml.
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Effect of Anti-Pur Antibodies on Protein-DNA Complex
Formation--
Band shift assays were conducted in the presence of
anti-Pur peptide antibodies to confirm the identity of Pur
protein-ssDNA complexes previously suggested by overexpression studies
(10). Initial experiments utilized nuclear protein from AKR-2B
fibroblasts as a source of cellular Pur
and Pur
and a
32P-oligonucleotide probe corresponding to the purine-rich
coding strand of the PE (PE-PrMss or PE-F) (11). As shown in earlier studies, Pur
and Pur
-ssDNA complexes migrate as a closely spaced doublet (Fig. 4A, lane 2). A
slower migrating complex (NS) that is also detected is composed of an
unrelated, nonspecific DNA-binding protein (11, 14). As illustrated in
Fig. 4A, three out of the four anti-Pur antibodies tested
(lanes 4-6) were found to selectively supershift the two
major Pur protein-ssDNA complexes into two slower migrating complexes,
designated SS1 and SS2. These supershifted complexes were not formed
when preimmune IgG or anti-B42-69 were included in the reaction
mixtures (lanes 2 and 3). The inability of
anti-B42-69 to supershift suggests that this antibody is unable to
bind Pur proteins in their native (i.e. nondenatured) state since immobilization on polystyrene (Fig. 2) or denaturation by SDS
(Fig. 3) did not interfere with epitope recognition.

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Fig. 4.
Anti-Pur peptide antibodies selectively
supershift specific Pur protein-ssDNA complexes (A and
B) and deoxycholate blocks Pur protein-ssDNA complex
formation (C). A, B, and
C, band shift assays were performed using the purine-rich
forward (coding) strand of the PE (PE-F, ~1 nM) as the
32P-ssDNA probe (11). A, the indicated rabbit
IgGs (0.5 µg) were preincubated for 20 min with AKR-2B nuclear
protein (3 µg). Probe was then added and mixtures were incubated for
an additional 20 min prior to electrophoresis. B, the
indicated rabbit IgGs (0.5 µg) were preincubated for 20 min with
lysed whole cell protein (2 µg) obtained from AKR-2B fibroblasts
transiently transfected with pCI-Pur (10 µg), pCI-Pur (10 µg), or both (5 µg each) expression vectors (10). Reaction mixtures
were supplemented with probe and incubated as above. Lane 1 contains lysed cell protein from empty vector (pCI) transfected cells
and thus represents ssDNA-binding of endogenous Pur proteins.
C, AKR-2B nuclear protein (2 µg) was preincubated for 20 min with varying concentrations of sodium deoxycholate (DOC)
or Triton X-100 (TX-100) prior to the addition of the ssDNA
probe. Arrows and labels designate Pur , Pur , and
antibody supershifted (SS1 and SS2) protein-ssDNA
complexes. The "*" highlights a minor Pur -containing complex
(A) which is enhanced upon forced expression of Pur
(B).
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Closer inspection of the band shift patterns obtained using
isoform-specific antibodies provided unexpected results. While anti-A291-313 appeared to only partially supershift the major Pur
-ssDNA complex (Fig. 4A, lane 6, arrow), a minor and
slower migrating complex (denoted by a *) was completely supershifted by this antibody. Moreover, anti-B210-229 and anti-B302-324 clearly supershifted the most rapidly migrating Pur
complex and,
surprisingly, the major (middle) Pur
-containing complex
as well (Fig. 4A, lanes 4 and 5). These data
suggested that the major (middle) Pur
complex is
heterogeneous and likely contains Pur
while the minor, slowest migrating complex is composed exclusively of Pur
. Owing to the low
abundance of this minor Pur
-containing complex, we also performed antibody supershift analyses using extracts from AKR-2B fibroblasts transfected with mouse Pur
and/or Pur
expression vectors (10). As
shown in Fig. 4B, overexpression of Pur
enhanced
formation of the minor (slowest migrating) Pur
-ssDNA complex
(lane 2, *) which was supershifted by anti-A291 (lane
5) but not by anti-B302 (lane 8). Overexpression of
Pur
enhanced formation of the major (most rapidly migrating)
Pur
-ssDNA complex (Fig. 4B, lane 3) which was
supershifted by anti-B302 (lane 9) but not by anti-A291 (lane 6). Co-expression of both proteins enhanced formation
of the two major (middle and most rapidly migrating) Pur protein complexes but not the minor (slowest migrating) Pur
complex (Fig. 4B, lane 4). Both major complexes were efficiently
supershifted by anti-B302 (compare lanes 4 and
10) while only the middle Pur
/
complex was effected by
anti-A291 (compare lanes 4 and 7). Importantly, formation of all three Pur protein-ssDNA complexes was abolished by low
concentrations of sodium deoxycholate, a mild ionic detergent known to
disrupt protein-protein interactions (16) (Fig. 4C, lanes
2-5). In contrast, the nonspecific ssDNA-binding complex (NS) was
largely unaffected by deoxycholate while the Pur protein-ssDNA complexes were unaffected by Triton X-100 (lanes 6-9). It
is important to note that no faster migrating ssDNA complex was
detected in samples treated with deoxycholate implying that monomeric
binding of the Pur proteins to ssDNA does not occur under these
conditions. Together, these data provide strong evidence that mouse
Pur
and Pur
bind to the purine-rich strand of the MCAT enhancer
in the form of homo- and heterodimers. No evidence was obtained for the existence of monomeric ssDNA-binding complexes. These data are consistent with a recent finding that human Pur
binds to a
recognition element in the myelin basic protein gene promoter as a
homodimer (17) and suggest that dimerization between Pur proteins may be a necessary prerequisite for functional activity.
Pur
and Pur
Associate in the Absence of ssDNA--
To
determine whether Pur protein dimerization requires coincident
interaction with a ssDNA recognition element, we evaluated the ability
of the Pur proteins to associate in the absence of ssDNA. Initially, we
performed immunoprecipitation experiments using whole cell extracts of
AKR-2B fibroblasts transiently co-transfected with Pur
and Pur
expression vectors (10). Isoform-specific anti-Pur IgGs were used to
specifically capture Pur proteins from the cell extract while
anti-B42-69, which cross-reacts with both Pur
and Pur
, was
employed to assess the composition of immunoprecipitates by Western
blotting. As shown in the top panel of Fig.
5, both anti-B210-229 (lane
3) and anti-B302-324 (lane 4) were found to co-immunoprecipitate Pur
and Pur
. Anti-A291-313 was also able to
co-immunoprecipitate Pur
and Pur
(Fig. 5, lane 5)
albeit with reduced efficiency relative to the anti-Pur
antibodies
(lanes 3 and 4). As expected, preimmune rabbit
IgG failed to immunoprecipitate the Pur proteins (Fig. 5, lane
2). Owing to the isoform specificity of the precipitating
antibodies, these results indicate that Pur
and Pur
can associate
via protein-protein interaction.

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Fig. 5.
Detection of a Pur -Pur complex by
immunoprecipitation and ELISA. Top, AKR-2B fibroblasts
were transiently transfected with 2 µg each of pCI-Pur and
pCI-Pur expression vectors (10). Transfectants were rendered
quiescent and serum-stimulated for 6 h. Cell extracts were
prepared and 100 µg of lysed cell protein was supplemented with 2.5 µg of the indicated rabbit IgG and incubated for 1 h. Following
an additional incubation with sheep anti-rabbit-coupled magnetic beads,
immune complexes were captured on a magnet, washed, and rabbit IgG
bound protein eluted with free peptide. Eluates were analyzed by
immunoblotting using anti-B42-69 to detect Pur and Pur .
Lane 1 shows Pur protein immunoreactivity observed in 2 µg
of whole cell extract prior to immunoprecipitation. Bottom,
His-tagged Pur and DHFR-coated microtiter wells (50 nM
application) were incubated with varying amounts of AKR-2B nuclear
protein for 16 h at 4° C. Wells were aspirated, washed 3 times,
and solid-phase Pur -Pur complexes were detected by ELISA using
anti-PurA 291-313 as the primary antibody. Absorbance readings at each
point were corrected by subtracting a background A405
nm reading generated with His-tagged protein-coated wells and
binding buffer.
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As a more quantitative test for Pur protein interaction, we examined
the ability of recombinant Pur
to selectively bind cellular Pur
in a discontinuous, solid-phase binding assay. Polystyrene microtiter
wells coated with His-tagged Pur
or His-tagged DHFR were incubated
with varying amounts of nuclear protein extracted from rapidly growing
AKR-2B fibroblasts. After removal of unbound nuclear protein,
solid-phase Pur
-Pur
complexes were detected by ELISA using a
Pur
-specific antibody, anti-A291-313. As shown in the bottom
panel of Fig. 5, the colorimetric signal obtained from
Pur
-coated wells was dose-dependent and saturable. The
specificity of this interaction is demonstrated by the total absence of
color generated by DHFR-coated control wells. These data reinforce the conclusion drawn from the immunoprecipitation and band shift
experiments and confirm that Pur
and Pur
can form a specific and
stable protein-protein complex. Moreover, these data demonstrate that the Pur-protein complex formation does not require the presence of
exogenous ssDNA.
Identification of MSY1 as a PE-MCAT Strand-binding
Protein--
Screening of a mouse lung cDNA expression library
with a 32P-end-labeled tetramer of the pyrimidine-rich
strand of the PE yielded eight independent PE-MCATss-binding clones.
Each clone was tested for its ssDNA binding specificity using wild-type
and mutant oligonucleotide competitors in a tertiary filter binding
assay. All eight clones produced identical results. Fig.
6 shows the data for one of the clones,
7-1, where excess wild-type oligonucleotide (PE-MCATss) completely
inhibited the binding of the 32P-tetramer in comparison to
a mutant oligonucleotide (PE-MCATmu2) lacking VACssBF1 binding affinity
(11). DNA sequencing revealed that each clone contained overlapping
nucleotide sequences that were virtually identical to the cDNA
sequence encoding MSY1 previously reported by Tafuri and co-workers
(15). The full-length cDNA sequence of clone 7-1 and the published
MSY1 cDNA sequence differ by only a single nucleotide within the
open reading frame. Alignment of the deduced amino acid sequences
illustrates that clone 7-1 encodes a glycine residue rather than an
alanine residue at codon 29 (Fig. 7). The
reason for this discrepancy is probably due to a polymorphism although
the glycine codon is conserved in the rat and human Y-box homologues,
EFIA (18), dpbB (19), and YB-1 (20) (Fig. 7).

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Fig. 6.
Cloning of a cDNA encoding a PE-MCAT
strand binding protein. Screening of a mouse lung cDNA
expression library (250,000 plaques) with a 32P-end labeled
tetramer of the pyrimidine-rich strand of the PE yielded eight
independent PE-MCATss-binding clones. Each clone was tested for its
ssDNA-binding specificity in a filter binding assay. All eight clones
produced identical results. The data for one of the clones, 7-1, is
shown. Excess (150-fold molar) wild-type oligonucleotide (right
side) completely inhibited the binding of the
32P-tetramer while a mutant oligonucleotide deficient in
VACssBF1 binding did not (left side).
|
|

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Fig. 7.
Clone 7-1 encodes the mouse Y-box protein,
MSY1. The cDNA-deduced amino acid sequences of clone 7-1 and
the Y-box homologues, MSY1 (15), rat EFIA (18), human dbpB
(19), and human YB-1 (20) are shown. Clone 7-1 encodes a glycine
residue rather than an alanine residue at codon 29. This glycine
residue is conserved in the other Y-box homologues. The highly
conserved "cold shock" or DNA-binding domain is
underlined.
|
|
Synthetic peptides corresponding to several sequences of predicted
antigenicity were used as immunogens to derive MSY1-specific antibodies. Affinity purified rabbit IgGs were tested for specificity as described for the panel of anti-Pur antibodies (data not shown), and
two, anti-MSY242-267 and anti-MSY276-302, were selected for use in
further experiments. Importantly, neither antibody exhibited detectable
cross-reactivity with either of the Pur proteins. These antibodies were
used to test whether cellular, as opposed to recombinant, MSY1 would
bind to the pyrimidine-rich strand of the MCAT enhancer. As shown in
Fig. 8, an MSY1 immunoreactive species
was captured from a crude AKR-2B fibroblast nuclear extract by
paramagnetic particles coupled with the pyrimidine-rich, reverse strand
of the enhancer (PE-R, lane 3) but not by particles coupled
with the opposing, purine-rich forward strand (PE-F, lane
2). In contrast, the purine-rich forward strand, but not the
pyrimidine-rich reverse strand, effectively captured Pur
and Pur
(compare lanes 5 and 6). These data validate the
expression cloning results and provide strong evidence that the
ssDNA-binding complex previously termed VACssBF1 (11, 12), is
identical, or closely related to the mouse Y-box protein, MSY1. The
anomalous electrophoretic mobility of MSY1
(Mr~55,000) is consistent with previous
findings for other Y-box proteins (21).

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Fig. 8.
Pur proteins and MSY1 bind to opposing
strands of the VSM -actin MCAT element
(PE). Parallel reaction mixtures containing equivalent amounts of
AKR-2B fibroblast nuclear protein (200 µg) and biotinylated
oligonucleotide (100 pmol) were incubated under conditions that
simulated a band shift assay. DNA-bound proteins were captured on
streptavidin-coupled paramagnetic particles, washed, and eluted with
1% SDS. Eluates were first assayed for MSY1 by Western blotting with a
mixture of anti-MSY1 peptide antibodies (left panel). The
MSY1 blot was stripped and reprobed with anti-B42-69 to detect Pur
and Pur (right panel). Each lane represents the amount of
Pur , Pur , or MSY1 captured from 100 µg of nuclear
protein.
|
|
Interaction of Pur
and Pur
with MSY1--
The identification
of MSY1 as a pyrimidine-rich strand, VSM
-actin MCAT
enhancer-binding protein was particularly intriguing given a previous
report implicating the human homolog, YB-1, as a transient
Pur
-binding protein in the context of a different promoter element
(22). As an independent evaluation of the potential for protein-protein
interaction between mouse Pur
and/or Pur
and MSY1, quantitative
binding studies were conducted with purified, recombinant proteins. The
binding of cellular Pur
and Pur
to His-tagged MSY1 passively
immobilized on polystyrene microtiter wells was evaluated by ELISA.
Fluid-phase AKR-2B nuclear protein was incubated with both MSY1 and
DHFR-coated wells. After removal of unbound nuclear protein,
solid-phase Pur
-MSY1 and Pur
-MSY1 complexes were detected using
antibodies that specifically recognize the C-terminal region of either
Pur
or Pur
. While virtually no signal was obtained from wells
coated with DHFR, the colorimetric signal generated with MSY1-coated
wells was dose-dependent and saturable (Fig.
9A). Similar results were
obtained when the assay was performed using His-tagged Pur
or Pur
as the solid-phase ligands and an MSY1-specific antibody to detect
complex formation (Fig. 9B). Although MSY1 binding was not
completely saturable in this orientation (owing to decreased
accessibility of binding sites, lower affinity of the detecting
antibody, and/or limiting fluid-phase MSY1), the immobilized Pur
proteins were nonetheless indistinguishable in terms of their ability
to specifically partner with cellular MSY1. These data demonstrate that
both Pur
and Pur
can form a stable, heterotrimeric complex with
MSY1 in the absence of ssDNA.

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Fig. 9.
Detection of cellular Pur and Pur
binding to recombinant MSY1 by ELISA. A, His-tagged
MSY1 and DHFR-coated microtiter wells (50 nM application)
were incubated with varying amounts of AKR-2B nuclear protein for
16 h at 4° C. Wells were aspirated, washed 3 times, and
solid-phase Pur -MSY1 or Pur -MSY1 complexes were detected by ELISA
using anti-PurA 291-313 or anti-PurB 302-324, respectively, as the
primary antibodies. B, His-tagged Pur , Pur , or
DHFR-coated microtiter wells (50 nM application) were
incubated with varying amounts of AKR-2B nuclear protein for 16 h
at 4° C. Wells were aspirated, washed 3 times, and solid-phase
MSY1-Pur or MSY1-Pur complexes were detected by ELISA using
anti-MSY1 242-267 as the primary antibody. Absorbance readings at each
point were corrected by subtracting a background A405
nm reading generated with His-tagged protein-coated wells and
binding buffer.
|
|
To test whether or not such a heterotrimeric complex could be detected
in a nuclear extract without disturbing the equilibrium by exposure to
immobilized recombinant ligand, immunoprecipitation experiments were
performed with anti-Pur and anti-MSY1 antibodies and a nuclear extract
from nontransfected AKR-2B fibroblasts. The Pur protein and MSY1
composition of each immunoprecipitate was analyzed by Western blotting
using anti-PurB42-69 to detect both Pur isoforms, followed by
anti-MSY276-302 to detect MSY1. Consistent with the results obtained
using extracts from transfected fibroblasts (Fig. 5), Pur
and Pur
were co-immunoprecipitated by the Pur
-specific antibodies,
anti-B210-229 and anti-B302-324 (Fig.
10, lanes 1 and
2). The Pur
-specific antibody, anti-A291-313, preferentially captured Pur
and a marginal amount of Pur
(lane 3). Importantly, an anti-MSY1 immunoprecipitate
(M242-267) included Pur
and a small but detectable amount of Pur
(lanes 6). Subsequent immunoblotting for MSY1 revealed the
presence of an additional Mr ~ 55,000 band
(MSY1) in the anti-MSY242-267 and anti-B302-324 immunoprecipitates
(lanes 7 and 8) but not the anti-B210-229 and anti-A291-313 immunoprecipitates (data not shown). The
Mr ~ 60,000 band present in all lanes, except
the no IgG control lane (Fig. 10, lane 4), is a nonspecific
band (NS) likely corresponding to the heavy chain of rabbit IgG. It is
noteworthy that these heterodimeric and heterotrimeric complexes
composed of Pur
-Pur
and Pur
-Pur
-MSY1 could be detected
without manipulating the cellular levels of Pur proteins and/or MSY1
via the use of expression vectors.

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Fig. 10.
Immunoprecipitation of a Pur -Pur -MSY1
complex from an AKR-2B fibroblast nuclear extract. Nuclear extract
from rapidly growing AKR-2B fibroblasts (136 µg of protein) was
combined with selected rabbit anti-Pur or MSY1 peptide IgGs (2.5 µg)
and incubated for 1 h. Following an additional incubation with
goat anti-rabbit IgG-biotin coupled streptavidin-paramagnetic
particles, immune complexes were captured with a magnet, washed, and
rabbit IgG bound protein eluted with free peptide. Eluates were assayed
by immunoblotting with anti-B42-69 to detect Pur and Pur
followed by anti-MSY 276-302 to detect MSY1.
|
|
 |
DISCUSSION |
Because Pur
and/or Pur
appear to function as repressors of
VSM
-actin MCAT enhancer activity (11, 14) and because Pur
has
not previously been characterized, we felt it important to study
potential molecular interactions between these two ssDNA-binding proteins. Recombinant mouse Pur proteins and rabbit polyclonal antibodies directed against specific domains both common and unique to
Pur
and Pur
were used in these studies (Figs. 1-3).
Surprisingly, we found that several antibodies specific for the Pur
isoform supershifted protein-ssDNA complexes composed of both Pur
and Pur
implying that the Pur proteins can transiently associate via
protein-protein interaction (Fig. 4). This conclusion was validated by
both immunoprecipitation and ELISA-based, protein-protein binding
experiments which indicated that a specific and stable Pur
-Pur
complex can form in the absence of a ssDNA recognition element (Fig.
5). This interaction likely underlies the previously unrecognized
ability of Pur
and Pur
to form heterodimeric ssDNA-binding complexes and is likely of consequence given the diverse functional roles attributed to Pur
. For example, Pur
has been previously implicated in DNA replication of several viral genomes (23-25) and
transcriptional activation of both viral (22, 26) and mammalian
promoters (27-32). In contrast, our results suggest that mouse Pur
and/or Pur
are able to repress the activity of a nearby enhancer in
the context of both natural and chimeric promoters (11, 14). While it
is not uncommon for a transcription factor to function as either an
activator or repressor depending on promoter context, it is equally
likely that the properties of a homodimeric Pur
ssDNA-binding
complex differ substantially from a homodimeric Pur
complex, or from
a Pur
/Pur
heterodimer. This could easily explain why the VSM
-actin MCAT enhancer is negatively regulated despite the presence of
a transcriptional activator like Pur
. This possibility is lent
additional credence by the structural differences between Pur
and
Pur
, most notably the absence of a C-terminal polyglutamine
sequence, a potential transactivation domain, in Pur
(Fig. 1).
In an earlier study, a binding site screen of a human astroglioma cell
cDNA expression library was used to tentatively identify a member
of the Y-box family of nucleic acid-binding proteins as the
pyrimidine-rich ssDNA binding activity previously termed VACssBF1 (33).
In the present study, we confirmed this result using a cDNA library
from a mouse tissue enriched in smooth muscle. This screen yielded
eight independent clones, all encoding the mouse Y-box protein MSY1.
Moreover, MSY1 was selectively captured from fibroblast nuclear
extracts by the pyrimidine-rich strand of the MCAT enhancer coupled to
paramagnetic beads (Fig. 8). Together, these studies provide convincing
evidence that MSY1 does indeed represent the ssDNA binding activity
which interacts with the strand of the MCAT enhancer opposing the Pur
protein recognition site. Thus, it may be highly significant that MSY1
specifically interacts with both Pur
and Pur
in vitro
(Fig. 9), and indeed, can be co-immunoprecipitated from fibroblast
nuclear extracts in the form of an MSY1-Pur protein complex (Fig.
10).
While the functional significance of these interactions to
transcriptional regulation of the VSM
-actin gene remains to be established, it is noteworthy that the human Y-box protein homologue, YB-1, has been shown to similarly interact with Pur
to reciprocally modulate each others binding to the JC polyomavirus lytic control element (22). Similar cooperative interactions seem likely to occur
within the context of the MCAT enhancer element. Because Pur
and/or
Pur
appear capable of repressing MCAT enhancer activity independently of MSY1 (14), protein-protein interactions with MSY1
might serve to antagonize this effect by virtue of sequestering one or
both of the Pur proteins into an inactive complex. Alternatively, MSY1
may potentiate the effect(s) of the Pur proteins owing to the fact that
vertebrate Y-box proteins have been functionally implicated in both
transcriptional activation (18, 34-36) and repression (37, 38). Other
possibilities can easily be envisioned but can only be resolved through experimentation.
While potential combinatorial interactions between Pur
, Pur
,
MSY1, and their respective ssDNA recognition motifs are numerous, they
are not the only interactions which may be important to the regulation
of MCAT enhancer activity. In particular, the binding of Pur
to a
ssDNA recognition element has also been shown to be modulated by
specific protein-protein interaction with the retinoblastoma tumor
suppressor protein, Rb (39). Whether Rb similarly interacts with Pur
is not known. However, a Pur
sequence implicated in Rb binding,
termed the "psycho" motif (39, 40), is largely conserved in Pur
,
albeit with modification (10). We are currently exploring the potential
involvement of Rb in regulating VSM
-actin gene transcription.
Irrespective of the outcome of these experiments, it seems quite clear
that complex combinations of protein-protein and protein-ssDNA
interactions are likely to be important to the ability of Pur
,
Pur
, and MSY1 to modulate the activity of the VSM
-actin MCAT
enhancer element. Delineation of the effects of such interactions on
both the topology and activity of this element is an important priority.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant R01 HL54281 (to M. J. G.), Minnesota Affiliate of the
American Heart Association Grant MN-97-F-20 (to R. J. K.),
and the Mayo Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Mayo Clinic/Foundation, 200 First St. Southwest, Rochester, MN 55905. Tel.: 507-284-2875; Fax:
507-284-3383; E-mail: getz.michael{at}mayo.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
ssDNA, single-stranded DNA;
ABTS, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid);
KLH, keyhole
limpet hemocyanin;
HRP, horseradish peroxidase;
PVDF, polyvinylidene
difluoride;
DHFR, dihydrofolate reductase;
ELISA, enzyme-linked
immunosorbent assay;
VSM, vascular smooth muscle;
PE, promoter
element.
 |
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