From the ¶ Department of Medicine and Department
of Biochemistry and Molecular Genetics, Program in Molecular Biology,
and Colorado Cancer Center, University of Colorado Health Sciences
Center, Denver, Colorado 80262 and the ** Department of
Chemistry and Biochemistry, and
Howard Hughes Medical Institute,
University of Colorado, Boulder, Colorado 80309
Received for publication, December 20, 2002, and in revised form, February 24, 2003
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ABSTRACT |
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The Ets-binding site within the basal
transcription element (BTE) of the rat prolactin (rPRL) promoter is
critical for both basal and growth factor-regulated rPRL gene
expression. Here we report the purification and identification of the
factor that binds to the BTE. This factor was purified from GH3
pituitary nuclear extracts using ammonium sulfate fractionation,
heparin-Sepharose and Mono Q chromatography, and BTE-affinity magnetic
beads. We purified two proteins of 57 and 47 kDa and identified the
57-kDa protein by mass spectrometry as the Ets factor GABP The prolactin gene is selectively expressed in the lactotroph
cells of the anterior pituitary gland. The proximal 425 bases of the
rat prolactin (rPRL)1
promoter are sufficient to confer cell type-specific expression of the
PRL gene (1). Within this region, DNase footprinting studies have
identified three binding sites for the pituitary-specific POU
homeodomain transcription factor Pit-1 (FPI, -III, and -IV) (2). Pit-1
has been shown to play a critical role in the cell type-specific
expression of the PRL gene and the development of the lactotroph cell
lineage. However, Pit-1 has also been shown to control the expression
of the growth hormone (GH) and thyroid-stimulating hormone Further studies of the rPRL promoter have identified a region between
Recent studies have identified a binding site for the Ets family of
transcription factors within the BTE. This Ets-binding site has been
shown to be a critical cis-element for several growth factor
signaling pathways, including fibroblast growth factors 2 and 4 (10),
insulin (11, 12), insulin-like growth factor-1 (13), epidermal growth
factor (14), and thyrotropin-releasing hormone (15). By using
electrophoretic mobility shift assays (EMSA), Stanley and co-workers
(16) demonstrated that the EBS at The Ets family of transcription factors is composed of more than 30 members that contain a highly conserved DNA binding domain (18). All
members of the Ets family recognize and bind to a core 5'-GGA(A/T)-3'
DNA motif in a variety of promoters. Thus, the mechanism by which Ets
factors achieve specific transcriptional responses is of great
interest. Additional sequences flanking the core are thought to
contribute to binding specificity, in addition to the tissue-restricted
expression of certain Ets factors (18). However, the primary mechanism
for achieving specificity is thought to be through the interaction of
Ets factors with other transcription factors bound to adjacent elements
(19-21).
The rPRL promoter in GH3 pituitary cells represents an excellent model
system to study the mechanisms by which Ets factors in combination with
other classes of transcription factors at adjacent elements elicit
specific transcriptional responses. Our laboratory has previously shown
that Pit-1 selectively interacts with Ets-1 at the rPRL promoter
Ras-response element centered at In order to identify definitively the Ets factor that binds to the BTE
of the rPRL promoter, a large scale purification scheme was undertaken.
By using four purification steps, we have purified two proteins of 57 and 47 kDa from GH3 pituitary nuclear extracts. The 57-kDa protein was
identified as the Ets factor GABP EMSA
The EMSA was performed essentially as described previously (17).
Binding buffer contained 10 mM Hepes-KOH, pH 7.9, 50 mM KCl, 4% glycerol, 1 mM EDTA, 0.1% Nonidet
P-40, 200 µg/ml insulin, and 250 ng poly(dI-dC) (Amersham
Biosciences). Poly(dI-dC) was not included in gel shift reactions
containing the DNA affinity-purified fractions. The sequence of the BTE
probe used is (message strand only, linker sequences in lowercase):
tcgaCTTAATGACGGAAATAGATG. The
putative Ets-binding site (GGAA) is in boldface, and the mutant BTE
probe is identical except that the underlined sequences were changed to
an XhoI site. Protein-DNA complexes were visualized by
autoradiography, and gel shift activities were quantified using a
PhosphorImager (Amersham Biosciences).
Preparation of DNA Affinity Magnetic Beads
Complementary oligonucleotides containing three repeats of the
BTE sequence were annealed for the DNA-affinity purification step as
described previously (17). The message strand was synthesized with a
5'-biotin containing a 6-carbon (6C) spacer, and the complementary DNA strand was identical, except it did not contain a biotin moiety (Invitrogen). The sequence of the 72-nucleotide BTE oligonucleotide used is (message strand only, SalI linker sequences in
lowercase): 5'-Bio-(6C)-tcgaCTTAATGACGGAAATAGATGtcgaCTTAATGACGGAAATAGATGtcgaCTTAATGACGGAAATAGATG-3'. Approximately 3 nmol of double-stranded (BTE)3
5'-biotinylated oligonucleotide were coupled to 20 mg of M-280
streptavidin-coated magnetic beads (Dynal) according to the
manufacturer's instructions.
Purification of the BTE Binding Factor (BTF)
Cell Culture and Preparation of Nuclear Extracts--
GH3 cells
were grown in spinner culture at 37 °C in high glucose Dulbecco's
modified Eagle's medium (Invitrogen) supplemented with 15% horse
serum and 2.5% fetal bovine serum (Invitrogen) by the University of
Colorado Health Sciences Center Tissue Culture Core Facility. Cells
were grown to a density of 8-10 × 105 cells/ml and
collected by centrifugation at 1500 × g. Nuclear extracts were prepared from a total of 40 liters of GH3 cells (24),
with slight modifications, as described previously (17).
Ammonium Sulfate Fractionation--
BTF was purified from ~262
mg of GH3 nuclear extract derived from 40 liters of GH3 cells in four
purification steps (Fig. 2). Ammonium sulfate powder (Fisher) was added
to 50.5 ml of GH3NE on an ice bath at 4 °C, to give a final
saturation of 50% ammonium sulfate. Precipitated proteins were
collected by centrifugation at 20,000 × g at 4 °C,
and the pellet was resuspended in 20.5 ml of binding buffer (10 mM Hepes-KOH, pH 7.9, 7 mM KCl, 4% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 1 mM
Na3VO4, and 1× protease inhibitors) (Roche
Applied Science). The 50% ammonium sulfate pellet and soluble fraction
were dialyzed against binding buffer at 4 °C. The dialysate was
cleared by centrifugation at 25,000 × g. Fractions
were assayed for BTF binding activity by EMSA. Protein concentrations
were determined using the Bio-Rad protein assay.
Heparin-Sepharose Purification--
The 50% ammonium sulfate
cut containing BTF activity was divided into five samples. Each sample
was loaded onto a 5-ml Hi-Trap heparin-Sepharose column (Amersham
Biosciences) and re-circulated at 4 °C for 20 min at 2 ml/min with a
peristaltic pump. The flow-through was collected, and the column was
washed with 10 ml of binding buffer at 1 ml/min. Bound proteins were
eluted with a 50-ml 70-800 mM KCl linear gradient using
the FPLC, with a flow rate of 1 ml/min, and 1-ml fractions were
collected for each column. BTE binding activity eluted in two peaks,
and the purification of peak 1 is the focus of this study because this
peak contained unambiguous BTE binding activity. Peak 1 from five
sequential heparin-Sepharose columns was pooled and dialyzed against
binding buffer and then re-loaded onto the 5-ml heparin-Sepharose
column in order to concentrate the sample. Heparin-bound proteins were
step-eluted with binding buffer containing 0.55 M KCl, and
the concentrated peak fraction 1 was dialyzed against binding buffer.
Ion Exchange Chromatography--
The peak heparin-Sepharose
fraction 1 (5.1 mg) was loaded onto a 1-ml Mono Q column (HR 5/5,
Amersham Biosciences) pre-equilibrated with binding buffer. The column
was washed with 2 ml of binding buffer, and protein was eluted with
70-800 mM KCl linear gradient in 21 ml of binding buffer
at 0.5 ml/min using an FPLC. Peak binding activity was present in
fractions 38-41 (~300 mM KCl). These fractions were
pooled and dialyzed against 7 mM KCl binding buffer and
clarified by centrifugation.
Magnetic DNA Affinity Purification--
Twenty mg of magnetic
beads coupled to 3 nmol of double-stranded BTE3
oligonucleotide was washed three times in binding buffer (10 mM Hepes-KOH, pH 7.9, 50 mM KCl, 4% glycerol,
1 mM EDTA, 0.1% Nonidet P-40, and 200 µg/ml insulin, 1×
protease inhibitors, Roche Applied Science). Approximately one-half
(~335 µg) of the peak Mono Q fraction (~700 µg) was purified
with the (BTE)3-coupled magnetic beads in three sequential
purifications. For each purification, 20 mg of
(BTE)3-coupled magnetic beads was incubated with 40, 70, or
225 µg of the peak Mono Q fraction for 30 min at room temperature and
rotated every 3-5 min. The purification was done sequentially because
the exact capacity of the (BTE)3-coupled magnetic beads was
not known. The beads containing bound protein were washed three times,
for 5 min each, with 500 µl of binding buffer containing 100 mM KCl. For the last two washes, 15 µg of poly(dI-dC) was added. Proteins were step-eluted using 100-µl aliquots of binding buffer containing 0.2, 0.3, or 1 M KCl for 10 min each. The
peak fractions (1 M KCl) of the three sequential
(BTE)3-affinity magnetic bead purifications (40-, 75-, and
225-µg inputs) were combined.
Mass Spectrometric Analysis of Proteolytic Peptides
For gel extraction and tryptic digestion, the peak BTE
affinity-purified fractions were pooled and boiled in 2× SDS-sample buffer (60 mM Tris, pH 6.8, 2% SDS, and 10 mM
dithiothreitol) and loaded onto a 1-mm thick, 1% SDS-10%
polyacrylamide gel. The gel was stained with 0.5% Coomassie Brilliant
Blue R-250 (Sigma) in 25% isopropyl alcohol and 10% acetic
acid for 2 h and destained with 10% acetic acid for 2 h.
Protein bands and a control "blank" piece were excised from the
gel, and in-gel trypsin digests were performed as described previously
(25). Tryptic peptides were extracted from gel slices, concentrated,
and desalted as described previously (25). For the LCQ analysis, the
desalted extracts were completely dried in a vacuum centrifuge and
resuspended in 5 µl of 0.1% formic acid.
Mass Spectrometry Analysis
MALDI-TOF mass spectrometry (Voyager-DE STR, PerkinElmer Life
Sciences) in delayed extraction mode was used to identify the unknown
proteins by peptide mass fingerprinting, as described previously (25).
Briefly, one-tenth (0.5 µl) of each desalted extract was
co-crystallized with matrix
( Liquid chromatography electrospray ionization mass spectrometry with a
quadrupole ion trap mass spectrometer (LCQ, Finnigan) coupled to a
Magic 2002 high pressure liquid chromatography pump and stream splitter
(Michrom BioResources) was used to obtain amino acid sequence, as
described previously (25). Briefly, peptide fragmentation spectra were
obtained for the 57-kDa protein as peptides eluted on a 100-µm (inner
diameter) fused Silica C18 column (LC Packings) using an acetonitrile
gradient at a flow rate of 300 nl/min. The amino acid sequences
identified from the peptide fragmentation data were used to search the
NCBI nonredundant protein data base using the Sequest software
(Finnigan) (26).
Western Blot Analysis and UV Cross-linking Studies
Western blot analysis of GH3NE (50 µg), the 50% ammonium
sulfate cut (50 µg), the peak heparin-Sepharose (20 µg), Mono Q (8 µg), and pooled BTE-affinity fractions was performed as described previously (17). UV cross-linking studies of these same peak fractions
were performed as described previously (17). The BTE probe used for UV
cross-linking has been detailed previously (17). Where indicated,
competitor oligonucleotides were included at 400-fold molar excess.
Plasmid Constructs and Transient Transfections
The expression plasmids pSG5c-Ets-1, pSG5c-Ets-2, pTLElk-1, and
pTL2Net, and the luciferase reporter constructs pA3-425rPRLluc and
pA3-36rPRLluc have been described previously (23, 27). Other
expression constructs used included the following: pCDNA3Ehf (Frank
Burton, University of Minnesota, Minneapolis, MN) (28); pCMXGABP siRNA Transfections and Western Blot Analysis
Double-stranded synthetic oligonucleotides directed against
lamin A/C, GABP Purification of BTF--
The BTE is a key element of the proximal
rPRL promoter that regulates both basal and growth factor-stimulated
promoter activity (1, 6-10, 13-15, 33) (Fig.
1). We have characterized previously (17)
a specific protein complex (complex A) present in GH3 nuclear extract
that binds to the BTE of the rPRL promoter. This previous characterization suggested that an Ets factor is a component of complex
A. However, the identity of the functionally relevant factor(s) that
bind to the BTE remains unclear. In order to identify precisely the
endogenous protein(s) that bind(s) to the BTE of the rPRL promoter, we
set up a large scale protein purification scheme to purify BTF from GH3
pituitary cells. The four-step purification scheme used to purify BTF
is shown in Fig. 2. Nuclear extracts were
prepared from 40 liters of GH3 cells and purified by ammonium sulfate
fractionation, heparin-Sepharose and Mono Q chromatography, and
BTE-affinity magnetic beads. The presence of BTF binding activity in
each fraction was monitored by EMSA using a radiolabeled BTE probe.
As an initial purification step, GH3 nuclear extract (GH3NE) was
precipitated by a 50% saturated ammonium sulfate cut. BTF binding
activity was enriched ~1.75-fold in the 50% ammonium sulfate precipitate, and no detectable gel shift activity was present in the
soluble fraction (Table I and data not
shown). The 50% ammonium sulfate fraction was divided into five equal
fractions, and each fraction was loaded sequentially onto a
heparin-Sepharose column, and proteins were eluted using a 50-ml
70-800 mM linear KCl gradient. A representative protein
elution profile and EMSA are shown in Fig.
3. Gel shift analysis of the
heparin-Sepharose purified fractions shows that BTF binding activity is
present in the input but not the flow-through fractions (Fig. 3B,
lanes 1 and 2). BTF binding activity eluted from the
heparin-Sepharose column at two different salt concentrations, the
first peak of eluting in fractions 20-26 (peak 1, ~300
mM KCl) (Fig. 3, A and B, lanes
6-12), and a second broader peak (peak 2) eluting at a higher
salt in fractions 30-42 (peak 2, ~500-600 KCl) (Fig. 3,
A and B, lanes 17-29). Cumulative purification
was ~10-fold for peak 1 and ~0.4-fold for peak 2 (Fig.
3A and Table I). Peaks 1 and 2 from the heparin-Sepharose
column were pooled separately. Further purification of peak 2 did not
yield specific BTF binding activity, and Western blot analysis showed
that GABP was not present in peak 2 (data not shown). The purification
of peak 1 is the focus of this study.
As a third purification step, peak 1 from the heparin-Sepharose column
was pooled, dialyzed, and separated by Mono Q anion-exchange chromatography (Fig. 4). A linear
gradient of KCl (70-800 mM) was applied to the Mono Q
column (Fig. 4A) and BTF binding activity eluted in
fractions 38-41 (~400 mM KCl) (Fig. 4B, lanes
6-9). BTF binding activity was not detected in the flow-through
or washes (data not shown). At this stage of the purification, a
19-fold increase in specific activity was obtained with a cumulative
yield of 4% (Fig. 4A and Table I).
For the final purification step, the peak Mono Q fractions (38-41)
were pooled, dialyzed to 50 mM KCl, and approximately
one-half of the pooled Mono Q fraction (340 µg) was incubated with
magnetic beads coupled to three tandem copies of the BTE
oligonucleotide. After incubation with the BTE-affinity magnetic beads,
the flow-through was collected, and the beads were washed three times
with buffer containing 100 mM KCl. To improve the
efficiency of the purification, excess poly(dI-dC) was included in the
last two washes. Proteins were step-eluted from the BTE-affinity beads
with buffer containing 0.2, 0.3, or 1 M KCl. EMSA analysis
of the BTE-purified fractions shows that BTE binding activity was
retained on the DNA affinity beads (Fig.
5A, lane 1 versus 2), and a small amount eluted in Wash 1 (Fig. 5A, lane 3). BTE binding activity was not
present in the last two washes or the 0.2 M KCl fractions
(Fig. 5A, lanes 4-7) but began to elute in the
0.3 M KCl fraction 1 and ended in the 1 M KCl fraction 1 (Fig. 5A, lanes
8-10). Approximately 25,000-fold purification was achieved by
this step (Table I).
The purity of the BTE-affinity fractions was assessed by SDS-PAGE
followed by silver staining. The silver stain of the BTE-affinity purified fractions shows that the input, flow-through, and washes contained a complex mixture of proteins (Fig. 5B,
lanes 1-5). Analysis of the proteins present in the input
versus the flow-through fractions revealed that two proteins
of 47 and 57 kDa were retained on the BTE-affinity beads. The 47-kDa
protein eluted between 0.2 M KCl and 1 M KCl
fraction 1 (Fig. 5B, lanes 6-10). The 57-kDa protein eluted between 0.3 M KCl fraction 1 and 1 M KCl fraction 1 (Fig. 5B, lanes
8-10). The majority of gel shift activity was present in the 0.3 M KCl fractions 1 and 2 and the 1 M KCl
fraction 1, where both the 47- and 57-kDa proteins were present (Fig.
5A). Binding activity was not detected in the 0.2 M KCl fractions where only the 47-kDa protein was present
(Fig. 5, A and B). These data suggest that the
47-kDa protein does not bind to DNA, or that both proteins are required
for efficient DNA binding.
UV Cross-linking of Purified Fractions--
In order to determine
which protein binds to the BTE probe, UV cross-linking studies were
done. An equal volume of each peak fraction was incubated with a BTE
probe substituted with 5-bromodeoxyuridine and exposed to UV light, as
described under "Experimental Procedures." As observed previously
(17), UV cross-linking of GH3NE with the BTE probe resulted in two
different protein-DNA complexes: a distinct and specific protein-DNA
complex that migrated with an apparent molecular mass of 64 kDa,
and a more diffuse, nonspecific protein-DNA complex that migrated at
~50 kDa (Fig. 6, lane 2). Both protein complexes were present in the 50% ammonium sulfate fraction and the peak heparin-Sepharose and Mono Q fractions (Fig. 6,
lanes 3-5). UV cross-linking of the BTE affinity-purified
fraction shows that only the 64-kDa protein complex was present (Fig.
6, lane 6) and that this protein was specific to the BTE
because addition of wild-type but not mutant BTE oligonucleotide
competed for binding (Fig. 6, lanes 6-8). In estimating the
molecular weight of the protein binding to the BTE probe, we assumed
that during the denaturation of the protein-DNA complex in SDS sample
buffer that only one strand of the BTE probe (~7 kDa) remained
cross-linked to the protein. Therefore, we estimate the true molecular
mass of the cross-linked protein to be ~57 kDa, which corresponds to the 57-kDa protein purified by DNA-affinity chromatography and visualized by silver staining (Fig. 5B, lanes 8-10). The
co-purification of the 47- and 57-kDa proteins through this extensive
purification scheme indicates that both proteins are likely to be
involved in binding to the BTE, although UV cross-linking studies show that only the 57-kDa protein directly binds DNA.
Mass Spectrometry Identification of BTF--
In order to identify
the 47- and 57-kDa proteins that bind to the BTE, the peak fractions
from the BTE-affinity purification were pooled and visualized by
Coomassie staining. The 47- and 57-kDa proteins were excised from the
gel and digested with trypsin in situ. The tryptic peptides
were extracted from the gel slices and analyzed by mass spectrometry.
Fig. 7A shows the MALDI-TOF spectrum of the 57-kDa protein. Twelve tryptic peptides with masses of
627.4, 755.5, 893.5, 941.6, 1036.5, 1090.5, 1139.6, 1307.7, 1465.8, 1475.7, 1699.7, and 1716.8 matched mouse GABP
The MALDI-TOF identification of GABP Western Blot Analysis of the Purified Fractions--
To confirm
the MALDI-TOF and MS/MS identification of GABP Functional Analysis of GABP--
Previous studies (12, 17) have
indicated that GABP
To determine the functional role of GABP
To test the specificity of Ets-factor activation of the BTE minimal
promoter, other Ets factors that are expressed in pituitary cells were
tested for their ability to activate the (BTE)8 minimal promoter in GH3 pituitary cells. As shown in Fig.
11A, the Ets factors Ets-1,
Ets-2, ER81, Ehf, Elk-1, and Net failed to activate the
(BTE)8 promoter, whereas co-transfection of
GABP
To establish further the role of GABP in the regulation of rPRL gene
expression, small interfering RNA (siRNA) was used to knock down GABP
protein levels, as described under "Experimental Procedures."
Because cycloheximide studies indicated that GABP protein turnover is
prolonged (96 h, data not shown), extended siRNA incubations were
necessary in order to reduce the expression of endogenous protein.
Western blot analysis of GH3 cells transfected with GABP siRNA showed a
significant reduction of GABP This study demonstrates a key role for GABP The protein purification procedure used in the studies described here
yielded an ~25,000-fold purification with a cumulative yield of 5%
(Table I), which was sufficient to identify the 57-kDa protein by
MALDI-TOF and MS/MS sequencing as GABP The identification of BTF as GABP Although GABP The BTE site, however, not only contributes to basal rPRL promoter
activity, it is also critical for a variety of hormonal and growth
factor-regulated responses. The BTE contributes to the cAMP (1, 6, 8),
FGF (10), insulin (11, 12), IGF-1 (13), epidermal growth factor (14),
and thyroid-releasing hormone (15) responses. Although the role
of the BTE site in mediating these responses is unquestionable, the
identity of the precise transcription factors that transduce these
signaling events directly through the BTE is less clear. The
transcription factors that have been implicated in mediating the
insulin response include GABP (11, 12) or Elk-1 in combination with
C/EBP. Western blot analysis identified the 47-kDa protein as GABP
1.
Co-transfection of dominant-negative GABP
1 blocks prolactin promoter
basal activity by 85-88% in GH3 cells in the presence or absence of
FGF-4. Additionally, expression of wild-type GABP
/
1 selectively
activates a minimal BTE promoter 24-28-fold in GH3 cells, and this
activation is dependent on the Ets-binding site. Finally, small
interfering RNA depletion of GABP in GH3 cells results in the loss of
prolactin protein. Thus, we have identified GABP
/GABP
1 as a
critical and functionally relevant Ets factor that regulates rPRL
promoter activity via the BTE site.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
genes in
somatotroph and thyrotroph cells, respectively (3-5). Thus, other
cis-elements and transcription factors must be involved in
the cell type-specific expression of the PRL gene.
112 and
85 that is critical for the basal activity of the promoter
(6-8). This basal transcription element (BTE) was subsequently shown
to overlap with a cAMP-response element (1, 6, 8); however, it was
shown that the proto-typical target CREB is unlikely to be involved at
this site (8, 9). Similarly, studies (9) using the human PRL promoter
have shown that the homologous A-site is critical for both basal and
cAMP human PRL promoter activity, and that a ubiquitous 100-kDa
protein distinct from CREB and Pit-1 binds to this site.
96 can bind to the Ets factors
Elk-1, Sap-1, and GABP
/GABP
1; this group has also shown that the
CCAAT/enhancer-binding protein (C/EBP
) also binds to the BTE.
However, the functional significance of any of these factors in
pituitary cells remains unclear. We have characterized previously the
Ets factors that bind to the BTE and have shown by EMSA and antibody
supershift analysis that Ets-1 and GABP
/GABP
1 can bind to the EBS
of the BTE (17). In addition, UV cross-linking studies with GH3 nuclear
extract and the BTE probe suggested that a protein of ~49-57 kDa
binds to the BTE (17). However, transient transfection studies of a
variety of Ets factors have failed to identify the functionally relevant protein. Thus, the exact identity of the protein that binds to
the BTE and regulates rPRL gene expression remains unknown.
212, and this interaction may
contribute to the cell type-specific expression of the PRL gene (22,
23). Thus, the identification and characterization of the Ets factor
that binds to the BTE should further our understanding of the cell
type-specific expression of the PRL gene.
by MALDI-TOF and MS/MS sequencing,
and the 47-kDa protein was identified as GABP
1 by Western blotting.
By using transient transfection studies, we show that expression of
dominant-negative GABP
1 blocks basal rPRL promoter activity in the
presence or absence of FGF-4. Co-transfection of wild-type
GABP
/GABP
1 activates the rPRL promoter in GH3 pituitary cells and
selectively activates a minimal BTE promoter. Finally, siRNA against
GABP results in the selective reduction of PRL protein levels. These
studies show that GABP
/GABP
1 is the critical nuclear factor that
binds to the BTE and regulates basal rPRL promoter gene expression.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyano-4-hydroxy-trans-cinnamic acid, Hewlett-Packard)
on a gold-coated sample plate, and the monoisotopic masses of
des-Arg1-bradykinin (904.47 Da) and
Glu1-fibrinopeptide B (1570.67 Da) were used to calibrate
the instrument. The sum of 150 acquisitions for each sample was used to
identify the peptide masses for each unknown protein. Proteins were
identified using the Profound program
(prowl.rockefeller.edu/cgi-bin/ProFound) searching the rodent, human,
or eukaryotic NCBI nonredundant protein data bases.
and
pCMXGABP
1 (Tom Brown, Pfizer) (29); pSG5ER81 (Ralf Janknecht, The
Salk Institute, La Jolla, CA) (30); and MSVGABP
(D)DN
(Laurent Schaeffer, Laboratory de Biologie Moleculaire et Cellulaire UMR, Lyon, France) (31). The minimal
36 rPRL promoters containing wild-type and mutant BTE inserts were constructed by inserting multiple
copies of each double-stranded BTE oligonucleotide containing a
SalI overhang into the SalI site of pA3-36luc,
as described previously (23). The wild-type and mutant BTE
oligonucleotides used were the same as that used for EMSA as described
above. The minimal promoters were sequenced by the University of
Colorado Health Sciences Center DNA Sequencing Facility. Adherent GH3
or GHFT1 pituitary cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 12.5% horse serum and 2.5% fetal calf serum and maintained at 37 °C in 5% CO2. GH3
cells were transiently transfected by electroporation, harvested, and luciferase/
-galactosidase activities assayed as described previously (23). Cells were treated with or without 2 ng/ml FGF-4, 6 h prior to harvest as described previously (10).
, and GABP
1 were prepared according to the
manufacturer's instructions (Dharmacon Research Inc.). The targeted
sequences of GABP
and GABP
1 were 5'-AAGGAUGCUCGAGACUGUAUU-3' and
5'-AACAUAUGGCAAGCCCUCAAU-3', respectively. The sequence of the lamin
A/C oligonucleotide was as described previously (32). Adherent GH3
cells were seeded at 1 × 106 cells per 12-well plate.
GH3 cells were transfected with 200 pmol of both GABP
and -
1
oligonucleotides or 400 pmol of the lamin A/C oligonucleotide or no
siRNA oligonucleotide using 10 µl of Effectine reagent (Qiagen).
Cells were harvested in EB as described previously (10), and 75 µg of
lysate was subjected to Western blot analysis as described above. Blots
were probed with antibodies against
-tubulin (Oncogene), GABP
(provided by Dr. Tom Kristie, National Institutes of Health), or
prolactin (provided by Dr. A. F. Parlow at the National Hormone
and Pituitary Program). Protein levels were determined using an Alpha Imager.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structural organization of the proximal rPRL
promoter. The region between nucleotides 220 and +73 is shown.
Pit-1-binding sites (FPI, -III, and -IV) are shown by
rectangles; Ets-binding sites are shown by solid
ovals, and the FPII repressor site and basal transcription element
are denoted by the circle and triangle,
respectively. P indicates the regulation of Ets factors by
phosphorylation.
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Fig. 2.
Purification scheme for BTF. BTF was
purified from GH3NE using a four-step purification scheme.
Summary of BTF purification
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Fig. 3.
Heparin-Sepharose purification of GH3NE.
A, elution profile of the heparin-Sepharose chromatography.
The 50% ammonium sulfate fraction was purified by heparin-Sepharose,
as described under "Experimental Procedures." Proteins were eluted
with a 50-ml linear gradient from 70 to 800 mM KCl, and
1-ml fractions were collected. Protein concentrations were determined
by the Bradford protein assay and are shown by closed
circles. Salt concentrations were determined by conductivity
measurements using KCl standards, and the KCl gradient is depicted by
closed squares. Specific activity units are shown by the
open circles. B, gel shift assay of peak
heparin-Sepharose fractions. One µl of input and 2 µl of each
eluted fraction were incubated with the BTE probe to assay for BTF
binding activity. Protein-DNA complexes were separated by nondenaturing
gel electrophoresis. Peak binding activity was present in fractions
20-26 (~300 mM KCl). Specific protein-DNA complexes are
indicated by BTF; NS denotes nonspecific bands,
and Free indicates free probe.
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Fig. 4.
Mono Q purification of GH3NE.
A, elution profile of the Mono Q purification. Peak
heparin-Sepharose fraction 1 (5.1 mg) was loaded onto a 1-ml Mono Q
column. Proteins were eluted with a 21-ml gradient from 70 to 800 mM KCl, and 0.5-ml fractions were collected. Protein
(closed circles), salt concentrations (closed
squares), and specific activities (open circles) were
determined as described in Fig. 4. B, gel shift assay of
peak Mono Q fractions. Each Mono Q fraction (1 µl) was incubated with
the BTE probe to assay for binding activity as above. Peak BTF binding
activity was present in fractions 38-41 (~300 mM KCl).
BTF indicates specific protein-DNA complexes; NS
indicates nonspecific binding, and Free indicates free
probe.
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Fig. 5.
DNA affinity purification of GH3NE.
A, gel shift assay of BTE affinity-purified fractions.
One-half of the peak Mono Q fraction (~340 µg) was purified with
the BTE-affinity magnetic beads. A representative gel shift assay from
the BTE affinity purification using 225 µg of the peak Mono Q
fraction as input is shown. One µl of each BTE-purified fraction and
5 µl of each wash were incubated with the BTE probe in a gel shift
assay. BTF activity elutes in the 0.3 and 1 M KCl
fractions. BTF indicates BTF activity, and Free
indicates free probe. B, silver stain of the BTE
affinity-purified fractions. Fifteen µl of each BTE-purified fraction
was separated on an SDS-10% polyacrylamide gel, and proteins were
visualized by silver staining. Two proteins of ~47 and 57 kDa are
enriched in the 0.3 and 1 M KCl fractions. Molecular mass
markers are indicated in kDa at the left.
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Fig. 6.
UV cross-linking of the peak purified
fractions. One µl of GH3NE and ammonium sulfate fraction and 2 µl of each remaining peak fraction were UV-cross-linked to the
32P-BTE probe. Cross-linked protein-DNA complexes were
visualized by autoradiography, as described under "Experimental
Procedures." Molecular weight markers are indicated on the
left.
within 0.2 Da with
18% coverage. Insufficient data were obtained to identify the 47-kDa
protein by MALDI-TOF (data not shown).
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Fig. 7.
Mass spectrometry identification of
GABP . A, MALDI-TOF
identification of GABP
. The peak 1 M KCl fraction from
the BTE-affinity purification were pooled and separated by SDS-PAGE.
The 57-kDa protein was subjected to in-gel digestion with trypsin, and
the extracted peptides were analyzed by MALDI-TOF. A resolution between
6000 and 9000 was obtained, and 12 peptide masses (627.4, 755.5, 893.5, 941.6, 1036.5, 1090.5, 1139.6, 1307.7, 1465.8, 1475.7, 1699.7, and
1716.8) matched mouse GABP
within 0.2 Da with 18% sequence
coverage. T indicates tryptic peptides derived from trypsin.
Unlabeled peaks correspond to keratin contamination or were
contaminating peptides also present in the blank. B, MS/MS
sequencing of GABP
. The MS/MS sequence of the 1139.6 MH+
parent ion is shown and the observed b (249) and y (235) ions are
indicated. The m/z values for the
b6-10 ions were 695.4, 794.4, 865.5, 993.5, and 1121.6 (
parent), respectively, and the m/z values for
the y3-4 and y7-9 ions were 346.5, 445.3, 784.4, 912.5, 1026.5, and 1139.6, respectively.
was confirmed by MS/MS
sequencing. As shown in Fig. 7B, one sequence tag
(LNQPELVAQK), corresponding to the 1139.6 tryptic peptide identified by
MALDI-TOF (Fig. 7A), matched the human and mouse GABP
proteins in the nonredundant data base, confirming the identity of the
57-kDa protein as GABP
.
and to determine
whether the 47-kDa protein is GABP
1, Western blots of the purified
fractions with specific antibodies to GABP
and -
1 were performed.
Western blot analysis with GABP
antisera reveals that GABP
is
detected as a faint band (57 kDa) in GH3NE (50 µg), as observed
previously (17). GABP
is enriched in the 50% ammonium sulfate
fraction (50 µg) and further enriched in the peak fractions from the
heparin-Sepharose (20 µg), Mono Q (8 µg), and BTE affinity (25 µl) purifications (Fig. 8). Similarly, Western blot analysis with specific antisera to GABP
1 shows that a
faint band of 47 kDa is contained in GH3NE and is enriched in the
ammonium sulfate fraction, as observed previously (17). GABP
is
further enriched in the heparin-Sepharose, Mono Q and BTE affinity peak
fractions. These data confirm the mass spectrometry identification of
the 57-kDa protein as the ETS factor GABP
and identify the 47-kDa
protein as the ankyrin-related protein GABP
1.
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Fig. 8.
Western blot identification of
GABP and -
1.
GH3NE (50 µg), the 50% ammonium sulfate cut (50 µg), the peak
Heparin-Sepharose fraction (20 µg), the peak Mono Q fraction (8 µg), and the pooled, peak BTE-affinity purified fraction (25 µl)
were resolved on an SDS-10% polyacrylamide gel and transferred to an
Immobilon-P. Blots were probed with antibodies against GABP
or
-
1, where indicated.
/GABP
1 binds to the EBS within the BTE;
however, the functional role of GABP in growth factor- and basal-rPRL
promoter activity at the BTE has been unclear and has been shown
previously (12) to block insulin induction of the rPRL. The role of
GABP
/GABP
1 in basal and FGF-regulated rPRL promoter activity was
tested by overexpression of a recently developed dominant-negative
GABP
1 (DN-GABP
1) construct (31, 34). Deletion of the
transactivation domain of GABP
1 results in a dominant-negative
effector of GABP
/GABP
1-dependent responses.
Specifically, GABP
, which lacks a transactivation domain, is still
able to bind DNA and interact with DN-GABP
1 but unable to activate
transcription, because the transactivation domain of GABP
1 is
deleted (31, 34). Transfection of DN-GABP
1 results in a highly
specific and selective inhibitor of GABP-dependent responses because GABP
is the only known cofactor of GABP
1 (35). Fig. 9 shows that co-transfection of
DN-GABP
1 blocks basal rPRL promoter activity by 85% and blocks rPRL
promoter activity in the presence of FGF-4 by 88%. Of note, basal
transcription of the ancestrally related and
Pit-1-dependent rat growth hormone promoter is not affected
by expression of DN-GABP
1 (data not shown). Taken together, these
data demonstrate that GABP
/
1 plays a key role in both basal and
FGF-regulated rPRL promoter activity.
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Fig. 9.
The effect of dominant-negative
GABP 1 on basal and FGF-mediated rPRL promoter
activity. GH3 cells were transfected with 5 µg of
pA3-425rPRLluc, 0.5 µg of pCMV
gal, and 50 µg of
MSVGABP
(D)DN or MSV empty vector where indicated. Cells
were treated ±2 ng/ml FGF-4, 6 h prior to harvest. Western blot
analysis showed that expression of DNGABP
1 was at least 10-fold
lower than that of endogenous GABP
1 (not shown). Results presented
are a representative experiment from three transfections done in
duplicate.
/GABP
1 in the regulation
of the rPRL promoter via the BTE, three or eight copies of the
wild-type and three copies of the mutant BTE were cloned upstream of
the unresponsive
36rPRL promoter and transfected into GHFT1 pituitary
cells. As shown in Fig. 10,
co-transfection of wild-type GABP
/GABP
1 robustly activated both
the (BTE)8 and (BTE)3 promoters 28- and
24-fold, respectively. Co-transfection of GABP
/GABP
1 only
modestly activated the (mutBTE)3 promoter and failed to
activate the minimal
36rPRL promoter (Fig. 10). Thus, GABP activation
of the BTE-containing promoters is dependent on exogenous GABP, the
BTE, and an intact EBS within the BTE.
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Fig. 10.
.
GABP /GABP
1 activation
of a minimal BTE promoter. GHFT1 pituitary cells were transfected
with 5 µg of pA3-36(BTE)8, pA3-36(BTE)3, or
pA3-36(mutBTE)3 along with 0.5 µg of pCMV
gal, with or
without 5 µg each of CMXGABP
and CMXGABP
1, where indicated.
Cells were harvested and assayed, and the results presented are a
representative experiment from three transfections in duplicate.
/GABP
1 activated the (BTE)8 promoter ~6-fold.
To ensure that the lack of Ets-factor activation of the
(BTE)8 promoter was not due to lack of effector expression
in GH3 cells, these same Ets constructs were transfected and tested for
their ability to activate the proximal
425 rPRL promoter, which
contains several consensus Ets-binding sites. Fig. 11B shows
that Ets-1, Ets-2, GABP, and ER81 all activated the
425 rPRL promoter
1.5-4.5-fold, with the strongest activation mediated by GABP. However,
as observed previously (23, 27), Net, Elk, and Ehf did not
significantly activate the
425 rPRL promoter. Taken together, these
data show that although several Ets factors, including GABP, Ets-1,
Ets-2, and ER81, significantly activate the
425 rPRL promoter (Fig.
11), GABP is the only Ets factor capable of activating a minimal BTE
promoter (Figs. 10 and 11). These data demonstrate that
GABP
/GABP
1 is the specific Ets factor that regulates basal rPRL
promoter activity via the EBS within the BTE.
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Fig. 11.
Activation of a minimal BTE promoter and the
425 proximal rPRL promoter by Ets factors. A,
GABP
/GABP
1 selectively activates a minimal BTE promoter. GH3
cells were transfected with 5 µg of pA3-36(BTE)8, 0.5 µg pCMV
gal, and 10 µg of Ets-1, Ets-2, ER81, Ehf, Elk, or Net,
or 5 µg each of GABP
and GABP
1, where indicated. Results are
the mean ± S.D. of three transfections in triplicate.
B, activation of the proximal
425rPRL promoter by Ets
factors. GH3 cells were transfected with pA3-425rPRLluc and pCMV
gal
as described in Fig. 10A. Cells were harvested and assayed
as described above, and the results are the mean ± S.D. of 2-3
transfections done in triplicate.
protein levels by 30% at 168 h
and by 50 and 60% at 216 and 240 h post-transfection, respectively (Fig. 12, A and
C). To determine the effect of knocking down GABP on PRL
expression, blots were probed with an antibody against PRL. Fig. 12,
A and D, shows that PRL protein levels are reduced by 50, 70, and 80% at 168, 216, and 240 h
post-transfection, respectively. GABP
or PRL protein levels were not
affected by transfection of the control lamin A/C siRNA at any time
point examined (Fig. 12, A and C). Because GABP
also regulates the transcription of mitochondrial genes, the reduction
of PRL expression by GABP
siRNA could be a nonspecific effect. To
ensure the reduction in PRL expression by GABP
is specific, Western
blots were probed with an antibody against
-tubulin. As shown in
Fig. 12B,
-tubulin protein levels are unaffected by any
siRNA condition tested, indicating that the reduction in PRL is
specific and is not a result of depressed mitochondrial function. In
conclusion, these data clearly show that knock down of GABP
/
1
results in a significant reduction of PRL protein levels, demonstrating
a critical role for GABP in the regulation of PRL gene expression in
GH3 pituitary cells.
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Fig. 12.
GABP is critical for PRL expression.
A, siRNA knock down of GABP reduces PRL protein. GH3
cells were transfected with the indicated siRNAs and harvested at the
times indicated. Whole cell lysates were subjected to Western blot
analysis and probed with antibodies against GABP or PRL.
B, equal loading of GH3 cell lysates. Western blots in A were stripped and re-probed for
-tubulin as described under "Experimental Procedures."
C and D, graphical representation of siRNA knock
down. The Western blots described in A and B were
subjected to densitometry, and the GABP
and PRL levels were
normalized to the
-tubulin levels. The percent of remaining GABP
and PRL was calculated based on the levels present in cells transfected
with the lamin A/C oligonucleotide. Percentages reflect the average of
three separate experiments, and error bars represent 1 S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
1 in the basal
regulation of the rPRL promoter via the EBS in the BTE. Specifically, we used a biochemical approach to purify and identify the Ets factor
that binds to the rPRL promoter BTE site, among the many Ets factors
that are expressed in GH3 cells and have been shown to bind to the BTE.
The data revealed that GABP is the selective and functionally relevant
Ets member that binds to the BTE. GABP activation of the
36 rPRL
promoter was dependent on the presence of transfected GABP, intact BTE
control elements upstream of the minimal promoter, and an intact EBS
within the BTE. Moreover, a dominant-negative GABP
1 construct almost
completely blocked basal rPRL promoter activity but did not affect the
basal activity of the evolutionarily related rGH promoter (Fig. 9 and
data not shown). Furthermore, although several Ets factors activate the proximal
425 rPRL promoter in GH3 cells, GABP
/
1 was the only Ets member that significantly activated a minimal
36rPRL promoter containing multiple copies of the BTE. Finally, knock down of GABP
using siRNA significantly and specifically reduced PRL protein levels.
(Fig. 7). Of note, the
sequence of rat GABP
has not yet been reported; however, the human
and mouse isoforms are highly homologous. Insufficient purified
material was obtained to identify the ~47-kDa protein by MALDI-TOF or
MS/MS sequencing, most likely due its wider range of elution (Fig.
5B). Nevertheless, we predicted the 47-kDa protein should be
GABP
1, because GABP
is the only known cofactor of GABP
, and
the predicted size of GABP
1 (48 kDa) is close to the calculated size
of the ~47-kDa purified protein (35, 36). The 47-kDa band was
confirmed to be GABP
by Western blot analysis (Fig. 8).
/GABP
1 is consistent with the
biochemical data presented in these and previous studies. GABP
/
is a ubiquitously expressed transcription factor (35, 36), and previous
gel shift analysis with the BTE probe showed that BTE binding activity
was present in both GH3 and HeLa nuclear extracts (17). These data
indicated that BTF is not a pituitary-specific factor and suggested
that BTF might be a ubiquitous protein (17). Additionally, previous
antibody supershift studies showed that antisera to GABP
, -
1, and
Ets-1 supershifted complexes containing BTF bound to the BTE probe
(17). It is possible that the Ets-1 antibody is not as specific as
previously thought and is able to recognize GABP
. Alternatively,
small amounts of Ets-1 may bind to the BTE probe, but the level of
binding could have been too weak to detect during the purification
process. Finally, UV cross-linking studies with purified fractions show
that only a protein of the predicted size of ~57 kDa specifically
binds to the BTE probe (Fig. 6) (17), which is consistent with the
ability of GABP
, but not GABP
1, to bind DNA (37).
/
1 is ubiquitously expressed, we have shown that
GABP is the Ets factor that binds to the BTE and contributes to the
regulation of the tissue-specific expression of the rPRL gene. Indeed,
GABP
/
1 has recently been shown to regulate the expression of a
variety of tissue-specific genes. The mechanism of this tissue-specific
regulation is unclear but is likely to involve co-regulation by other
factors at adjacent DNA elements. For example, a functional interaction
of GABP with C/EBP
is thought to contribute to the liver-specific
expression of factor IX (38). Additionally, the association of GABP
with c-Myb and C/EBP
is thought to regulate the expression of the
neutrophil elastase gene in myeloid cells (39). With regard to the
mechanism by which GABP regulates the pituitary-specific expression of
the rPRL gene, it is possible that GABP interacts with other
transcription factors that bind to adjacent and/or overlapping DNA
elements. For example, GABP might interact with F2F, an FPII DNA
binding activity that has yet to be identified (see Fig. 1) (7).
Alternatively, GABP might interact with C/EBP
, which stimulates
basal rPRL promoter activity and binds to DNA sequences overlapping the
BTE (16).
(40). Here we present biochemical, transcriptional, and siRNA
data that the BTE exhibits binding and functional selectivity for GABP. Taken together, these data suggest that the growth
factor-dependent regulation of the rPRL promoter via the
BTE is complex and may involve distinct Ets-factor binding to the BTE
that is modulated by specific signaling pathways. Nevertheless, the
studies presented here demonstrate a critical role for GABP in the
basal regulation of the rPRL promoter.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. David Gordon and James
Hagman for discussion and reading of the manuscript. We thank
Dr. Robert Sclafani for providing the FPLC and Dr.
Charles McHenry and Brad Glover for assistance with the
FPLC. We also thank Connie Mastalir for technical assistance.
We thank Drs. Tom Kristie and A. F. Parlow for kindly providing the GABP and PRL antibodies, respectively. These studies utilized the Tissue Culture Core Facility of the University of Colorado Comprehensive Cancer Center, supported by National Institutes of Health Grant NCI P30 CA46934 and the University of Colorado Health
Sciences Center DNA Sequencing Core.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK46868 (to A. G. H.) and AR39730 (to K. A. R.).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.
§ Present address: Dept. of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309.
To whom correspondence should be addressed: University of
Colorado Health Sciences Center, 4200 East Ninth Ave., Box B-151, Denver, CO 80262. Tel.: 303-315-8443; Fax: 303-315-4525; E-mail: a.gutierrez-hartmann@uchsc.edu.
Published, JBC Papers in Press, March 4, 2003, DOI 10.1074/jbc.M213063200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
rPRL, rat prolactin;
EBS, Ets-binding site;
FPLC, fast protein liquid chromatography;
BTE, basal transcription element;
EMSA, electrophoretic mobility shift
assays;
PRL, prolactin;
BTF, BTE binding factor;
MALDI-TOF, matrix-assisted laser desorption ionization/time of flight;
MS, mass
spectrometry;
siRNA, small interfering RNA;
FGF, fibroblast growth
factor;
GH, growth hormone;
C/EBP, CCAAT/enhancer-binding protein;
GH3NE, GH3 nuclear extract;
GABP, GA-binding protein.
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