Properties of the Glucocorticoid Modulatory Element Binding Proteins GMEB-1 and -2: Potential New Modifiers of Glucocorticoid Receptor Transactivation and Members of the Family of KDWK Proteins
Sunil Kaul,
John A. Blackford, Jr.,
Jun Chen,
Vasily V. Ogryzko and
S. Stoney Simons, Jr.
Steroid Hormones Section (S.K., J.A.B., J.C., S.S.S.) National
Institute of Diabetes and Digestive and Kidney Diseases/Laboratory of
Molecular and Cellular Biology, and The Eukaryotic Transcriptional
Regulation Section (V.V.O.) National Institute of Child Health and
Human Development/ Laboratory of Molecular Growth Regulation
National Institutes of Health Bethesda, Maryland 20892-0805
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ABSTRACT
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An important component of glucocorticoid steroid
induction of tyrosine aminotransferase (TAT) gene expression is the
glucocorticoid modulatory element (GME), which is located at -3.6 kb
of the rat TAT gene. The GME both mediates a greater sensitivity to
hormone, due to a left shift in the dose-response curve of agonists,
and increases the partial agonist activity of antiglucocorticoids.
These properties of the GME are intimately related to the binding of a
heteromeric complex of two proteins (GMEB-1 and -2). We previously
cloned the rat GMEB-2 as a 67-kDa protein. We now report the cloning of
the other member of the GME binding complex, the 88-kDa human GMEB-1,
and various properties of both proteins. GMEB-1 and -2 each possess an
intrinsic transactivation activity in mammalian one-hybrid assays,
consistent with our proposed model in which they modify glucocorticoid
receptor (GR)-regulated gene induction. This hypothesis is supported by
interactions between GR and both GMEB-1 and -2 in mammalian two-hybrid
and in pull-down assays. Furthermore, overexpression of GMEB-1 and -2,
either alone or in combination, results in a reversible right shift in
the dose-response curve, and decreased agonist activity of
antisteroids, as expected from the squelching of other limiting
factors. Additional mechanistic details that are compatible with the
model of GME action are suggested by the interactions in a two-hybrid
assay of both GMEBs with CREB-binding protein (CBP) and the absence of
histone acetyl transferase (HAT) activity in both proteins. GMEB-1 and
-2 share a sequence of 90 amino acids that is 80% identical. This
region also displays homology to several other proteins containing a
core sequence of KDWK. Thus, the GMEBs may be members of a new family
of factors with interesting transcriptional properties.
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INTRODUCTION
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The dose-response curve is a crucial characteristic of steroid
hormone action and a basic feature of pharmacology. It indicates which
agonists are active at physiological steroid concentrations. It also
reveals the dose at which the most dramatic changes in biological
activity occur with minimum differences in steroid concentration,
i.e. the EC50 of the steroid. Cellular
gene transcription mechanisms have evolved to capitalize on these
effects of subtle changes in hormone concentration to selectively
control the expression levels of key genes during development,
differentiation, and homeostasis. Finally, the dose-response curve
distinguishes between agonists and antagonists.
The dose-response curve has long been thought to be a property of each
individual steroid-receptor complex and independent of the cell or the
regulated gene. Thus, the determinants of the dose-response curve would
be among the various steps followed by all receptors in converting the
presence of steroidal ligand into the observed biological response (1 2 ). These steps include passive entry of the steroid into the cell,
binding to the intracellular receptor protein, which may or may not be
predominantly cytoplasmic as seen for the glucocorticoid receptor (GR)
(3 ), activation to a species with increased affinity for DNA, binding
of the receptor-steroid complex to hormone response elements (HREs),
and finally interaction with the transcriptional machinery and assorted
cofactors to modify the rates of gene transcription. The rate-limiting
step in this pathway has been concluded to be activation, which is
microscopically irreversible (4 ). Thus, the affinity of steroid binding
to receptor was envisaged to determine the ultimate dose-response curve
(4 5 6 ). Furthermore, the underlying assumption of all
structure-activity studies has been that one could generalize from the
results in one system to any other responsive gene in cells containing
the same receptor. Likewise, if a given steroid was an antagonist in
one system, it was presumed that it would be an antagonist in all
systems.
Accumulating data have begun to force a revision of this model.
Unexplained differences in the dose-response curves of different
estrogen-inducible genes in the same cells have been known for many
years (7 8 9 10 ). Similarly, we have documented variations in the
dose-response curves for glucocorticoid induction of tyrosine
aminotransferase (TAT) vs. glutamine synthetase, mouse
mammary tumor virus (MMTV), or transiently transfected TAT in
the same cell (11 12 13 ) and between the same TAT gene in differently
treated cells (14 15 ). Other longstanding conundrums have been why
most antisteroids display partial agonist activity and how the amount
of this residual activity can vary with the cell (16 17 18 ), the gene
(12 18 19 20 ), the promoter (16 17 18 21 ), and even the composition (18 22 ) or spacing (23 ) of the HRE. Again, the rat TAT gene has been a good
model system as it displays variations in the residual agonist activity
of antiglucocorticoids that parallel the observed shifts in the
dose-response curves of agonists (12 13 14 15 19 ). Further studies with the
rat TAT gene revealed a 21-bp cis-acting element that was
sufficient to reproduce all of the above effects (13 15 24 25 ).
Thus, at least one method of controlling the dose-response curve of
agonists, and the partial agonist activity of antagonists, was the
presence of the cis-acting element that we called a GME, or
glucocorticoid modulatory element.
A key component in the action of the GME is the binding of a
trans-acting factor because mutant oligonucleotides that did
not bind factor were biologically inactive (15 ). Consequently, the
proposed model for the mechanism of GME action consists of the
trans-acting factor binding to the GME and interacting with
both the HRE-bound GRs and unknown transcription factors to increase
the coupling efficiency of selected steps before the synthesis of the
induced gene transcript (15 26 27 ). A pivotal development in support
of this model was the finding that the trans-acting factor
was actually a heterooligomeric complex of two novel proteins (GMEB-1
and -2) with an aggregate size of about 550600 kDa (GMEB-1 and -2)
(28 ). We have recently cloned and characterized the rat GMEB-2 as a
67-kDa protein on SDS-polyacrylamide gels (29 ).
In the present study, we report the cloning of the second protein of
the heteromeric complex, i.e. the 88-kDa GMEB-1. Several
properties of GMEB-1, alone and in combination with GMEB-2, were
examined to determine whether they were compatible with the previously
determined biological properties of the GME. The biological activities
of GMEB-1 and -2 were assessed in a mammalian one-hybrid assay. We
examined the interactions of GMEB-1 and GMEB-2 with themselves, with
each other, with GR, and with other cofactors that are known to bind
steroid receptors (30 31 32 33 ). Possible associations with CREB, which is
known to bind to the same DNA sequence as the GMEBs (15 34 35 36 ), were
also investigated. Finally, each GMEB was overexpressed in whole cells
to examine its ability to modulate both the GR dose-response curve
and the partial agonist activity of antisteroids. Collectively, the
data indicate that the originally isolated GMEB-1 and -2 have now both
been cloned, that the GMEBs can modulate the transactivation properties
of GR-steroid complexes, and that GMEB-1 and -2 may be members of a new
class of transcription factors with potentially novel properties.
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RESULTS
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Cloning of GMEB-1
The amino acid sequence of three tryptic peptides of purified rat
GMEB1 was already known (28 ), and three new peptides (ESEEISENTLMF,
XQMI/FLQLQPVQQGXAE, and AVILETELR) were obtained by additional
sequencing. None of these peptide sequences exhibited any substantial
homology with any known protein or nucleic acid sequence in GenBank.
However, one tryptic peptide was highly homologous (16 of 18 amino
acids) with a human Expressed Sequence Tag (EST 129005) in the dbEST
database. This EST was resequenced and used to isolate clones from a
human heart library as described in Materials and Methods.
One 1.8-kb clone contained an open reading frame (ORF) of 563 amino
acids bounded on both sides by in-frame stop codons (Fig. 1A
).
Thus, this clone encoded the full-length protein. Of the 73 amino acids
in the six sequenced peptides of rat GMEB-1, 67 were identical in the
translated sequence from the human cDNA clone. Three nonidentical
residues were highly conserved (D vs. E, D vs. Q,
and V vs. M), one was similar (Q vs. H), and two
were different (Y vs. A and T vs. A). The
translation initiation codon was preceded by a minimal Kozak sequence,
and the cDNA had a respectable poly A tail at the 3'-end. Surprisingly,
there was substantial homology between GMEB-1 and its heterodimerizing
partner, GMEB-2 (29 37 ), which was greater at the cDNA level (55%)
than it was at the amino acid level (39%). Furthermore, the two
proteins are 80% identical over the 93 amino acids of positions
89181 of hGMEB-1 (Fig. 1B
).


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Figure 1. hGMEB-1
A, Nucleotide and deduced amino acid sequence of the 1.8-kb
GMEB-1 clone. In frame stop codons at either end of the ORF are
indicated by the asterisk under the codon. The two crucial
residues of the consensus Kozak sequence are marked by the double
dagger above the bases. The six tryptic peptides are
underlined. The new tryptic peptides are in bold
type. The differences from the rat sequences (28 ) are
italicized. B, Sequence homology between human (h)GMEB-1 and
rat (r)GMEB-2. The cross-hatched bar ( ) is
above the peptide of GMEB-2 that was used to prepare the
anti-GMEB antibody. The originally reported sequence of GMEB-2 (29 ) was
found by Hinrich Gronemeyer (Strasbourg, France), and confirmed by us,
to be in error at nucleotides 183186 (initial = GCCTGT;
revised = GCTGCTGCT), thus adding one amino acid to the protein
sequence at residue 62 (initial = AlaCysAla; revised =
AlaAlaAlaAla).
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Biochemical Properties of GMEB-1
The molecular mass predicted for hGMEB-1 from the cDNA
clone was much less (61 kDa) than that observed on gels for rat GMEB-1
protein (88 kDa) (28 ). However, cell-free translation of the 1.8-kb
clone with [35S]methionine yielded a major
product of 89 kDa, which migrated very closely with rat GMEB-1 on
SDS-PAGE gels (Fig. 2
). The minor
difference in migration was presumably due to the limited amino acid
differences between rat and human GMEB-1 (Fig. 1A
), as even single
amino acid differences can alter the gel mobility of proteins
(38 ). Western blotting with an antibody to an epitope shared by hGMEB-1
and rGMEB-2 (see Fig. 1
) revealed an approximately 88-kDa
protein both in the cell-free translation reaction with the appropriate
cDNA and in rat hepatoma tissue culture (HTC) cell cytosol (Fig. 2
).
Interestingly, both recombinant hGMEB-1 and native rGMEB-1 proteins
were less reactive to the anti-GMEB antibody than was rGMEB-2 (Fig. 2
and data not shown) even though 22 of 23 of the amino acids of the
epitope were conserved in hGMEB-1.

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Figure 2. SDS Gel Properties of in Vitro
Translated GMEB-1 cDNA
A, SDS gel migration of [35S]methionine-labeled protein.
The 1.8-kb GMEB-1 cDNA in pCMV-SPORT was in vitro
translated with [35S]methionine, separated on a 10%
SDS-polyacrylamide gel, and autoradiographed as described in
Materials and Methods. The open arrow
indicates the position of GMEB-1. The positions of the mol wt markers
are indicated. B, Detection of authentic and in vitro
translated GMEB-1 by Western blotting. Samples of HTC cell cytosol,
in vitro translated GMEB-1, or reticulocyte lysate that
had been incubated with luciferase cDNA as carrier DNA (CONTROL) were
separated on 10% SDS-polyacrylamide gels and transferred to
nitrocellulose for Western blotting with affinity-purified anti-GMEB
antibody. The open arrow indicates the position of
GMEB-1. The solid arrow indicates the position of
GMEB-2. Other bands in HTC cytosol are nonspecific, cross-reacting
species (29 ) that are more visible with the overexposure needed to
detect GMEB-1.
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DNA Binding of GMEB-1 and -2 in Gel Shift Assays
Two previously documented properties of rGMEB-1 are its binding to
GME oligonucleotides 1) weakly as a homooligomeric complex and 2)
synergistically with the 67-kDa rGMEB-2 to yield a slightly larger
heteromeric complex (28 29 ). We therefore examined the DNA binding
activities in gel shift assays of hGMEB-1 ± rGMEB-2 that were
in vitro translated from the corresponding cDNAs. The
recombinant hGMEB-1 protein alone bound to the GME somewhat more than
expected but in a highly specific manner (Fig. 3
). The binding was competed by a 25-fold
molar excess of cold GME. Those GME oligonucleotides with clustered
base substitutions that continued to display GME biological activity
(i.e. M1 and M3) also competed the binding of GMEB-1. In
contrast, an excess of the biologically inactive M2 oligonucleotide
(15 ) was unable to prevent the DNA binding of GMEB-1. When in
vitro translated hGMEB-1 and rGMEB-2 were mixed on ice overnight
to allow for efficient heterooligomer formation (28 ), a protein-GME
complex was formed that had virtually the same migration pattern as
seen with the authentic heterooligomer from HTC cell cytosol (Fig. 3
).
This binding was synergistic as the total final complex was greater
than the sum of the components [1.9 ± 0.3
(SEM) fold higher, n = 5, P
= 0.04]. Again, the DNA binding of the recombinant heterooligomer was
specific as only the biologically active DNAs (GME, M1, and M3 but not
M2) were capable of inhibiting the formation of the protein-DNA
complex. No supershift with anti-GMEB antibody could be observed
because it recognizes only denatured protein (29 ).

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Figure 3. Binding of in Vitro Translated
GMEB-1 and -2 to GME Oligonucleotide in Gel Shift Assay
[32P]GME oligonucleotide was incubated with 1 U of
in vitro translated GMEB-1, 1 U of in
vitro translated GMEB-2, 1 U each of in vitro
translated GMEB-1 and -2, and HTC cell cytosol with a 25-fold excess of
the indicated nonradioactively labeled oligonucleotides. The bands were
visualized by autoradiography as described in Materials and
Methods (arrow marks position of the GMEB
heterocomplex as discussed in the text).
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The sequence, size, Western blotting, and specificity of DNA binding of
our cloned protein all support its identity as that of the human
homolog of the previously described rGMEB-1 (28 ). The DNA binding
properties of the recombinant hGMEB-1 and rGMEB-2 to biologically
active DNA sequences further strengthen this conclusion and demonstrate
that the hGMEB-1 is functionally equivalent to the rat homolog. These
data also reinforce our earlier determination that we had correctly
cloned GMEB-2 (29 ).
Transactivation and Oligomerization Properties of the GMEBs
Full-length GMEB-1 and -2 were fused to the C-termini of either
the GAL4 DNA-binding domain (GAL4-DBD) or the VP16 activation domain
(VP16-AD) and examined for their ability to induce a GAL4-regulated
reporter gene containing five tandem copies of the GAL4 binding element
or upstream activating sequence (UAS). GMEB-1 and -2 each displayed
intrinsic transactivation activity (Fig. 4A
, inset). These results
support our model that GMEB-1 and -2 are key components in the
modulation of GR transcriptional activity by interacting with the
transcriptional machinery (15, 26).

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Figure 4. Activity and Interactions of GMEBs
A, Activity of GMEB chimeras in mammalian one- and two-hybrid assays.
Triplicate dishes of Cos-7 cells containing the indicated GAL4-GMEB
fusions without (inset) and with (main
figure) VP16-GMEB chimeras were cotransfected with the
GAL4-regulated Luciferase reporter. The induced levels of Luciferase
were determined as described in Materials and Methods.
The error bars represent the SD of the
triplicate plates for each sample in the assay. Similar results were
obtained in two other independent experiments. B, Binding of GMEB-1 and
-2 to immobilized GMEBs in pull-down assay.
[35S]methionine-labeled, in vitro
translated GMEB-1 and -2 were added to M2-Agarose columns containing
previously immobilized FLAG-tagged GMEB-1 or -2 (f:GMEB-1 or f:GMEB-2)
that had been overexpressed from Baculovirus vectors in insect cells.
The proteins remaining after washing were eluted, separated on
SDS-polyacrylamide gels, and visualized as described in
Materials and Methods. In vitro
translated Luciferase and CREB were used to assay nonspecific
retention. The column labeled "10% input" displays one tenth of
the indicated in vitro translated, full-length proteins
that was loaded to the Agarose columns. The column labeled "M2
Agarose" shows the amount of each protein that was retained in the
absence of GMEBs. The columns labeled "f:GMEB-1 or -2/M2 Agarose"
depict the amount of each protein that was retained by the immobilized
GMEBs. Similar results were obtained in a second experiment.
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When GMEB-1 and -2 chimeras were cotransfected with the GAL4-regulated
reporter, a synergistic increase in transactivation was observed (Fig. 4A
). This suggested an ability of each protein to both homo- and
heterooligomerize, supporting the earlier and above results. It should
be noted that the net activity of heterodimerization was very sensitive
to the orientation of the two chimeras: GAL-B2/VP16-B1 was much more
active than GAL-B1/VP16-B2. Thus, the interactions between GMEB-1 and
-2 may be affected by the nature and/or position of the fused protein
of the chimera.
More evidence for interactions of GMEB-1 and -2 with themselves and
each other was obtained from pull-down assays using FLAG-tagged GMEB-1
and -2 that were overexpressed from Baculovirus vectors in insect
cells. As shown in Fig. 4B
, column-purified recombinant GMEB-1 and
GMEB-2 formed not only homooligomers but also heterooligomers with
35S-labeled, in vitro translated
GMEB-1 and -2. These interactions were selective, as evidenced by the
absence of interactions on columns lacking immobilized GMEBs and a
greatly reduced retention of the control proteins Luciferase and CREB
(see also below). Thus, it appears that direct interactions occur
between two molecules of GMEB-1, of -2, and between GMEB-1 and -2.
Interaction of GMEBs with GR
As an interaction of the heteromeric GMEB protein complex with GR
is a central postulate in the mechanism of GME action (15 26 27 ), we
sought supporting evidence for this. Only a very weak association was
observed in mammalian two-hybrid assays between GAL4-GR and VP16-GMEB-1
or -2 either in the absence or presence of the glucocorticoid
dexamethasone (Dex) (data not shown). Interestingly, Gal4-GR and
VP16-GR also did not appear to interact, either with or without Dex;
the response with both chimeras was about 40% less than the sum of the
individual activities (data not shown; see also below). However, a
significant steroid-dependent interaction between each of the GMEBs and
GR was observed when the experiment was performed with the opposite
pair of chimeras (GAL4-GMEB-1 or -2 and VP16 vs. VP16-GR)
(Fig. 5A
). Little consistent increase was
noted when the components were mixed in the absence of steroid (data
not shown).

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Figure 5. GMEB-GR Interactions
A, Interaction of GMEBs and GR in mammalian two-hybrid assays.
Triplicate dishes of Cos-7 cells containing the indicated GAL4 and VP16
chimeras were cotransfected with the GAL4-regulated Luciferase reporter
and then treated with EtOH ± 1 µM Dex. The induced
levels of Luciferase were determined as described in Materials
and Methods and expressed as fold increase over the
steroid-free values. The bar graph gives the average
values (±SD) of 1415 experiments (P
<0.0001 for GMEB-1/GR, P = 0.0073 for GMEB-2/GR).
B, Binding of GR to immobilized GMEBs in pull-down assay.
[35S]methionine-labeled, in vitro
translated GR was incubated with buffer (untreated), EtOH, or 1
µM of the indicated steroids in EtOH to form the
receptor-steroid complexes. These complexes were then added to
M2-Agarose columns containing previously immobilized FLAG-tagged GMEB-1
or -2 (f:GMEB-1 or -2/M2 Agarose) that had been overexpressed from
Baculovirus vectors in insect cells. The proteins remaining after
washing were eluted, separated on SDS-polyacrylamide gels, and
visualized as described in Materials and Methods. In
vitro translated CREB was used to assay nonspecific retention.
The row labeled "M2 Agarose" indicates the amount of receptor
± indicated steroid, or CREB, that was retained in the absence of
immobilized GMEBs. The row labeled "10% input" displays one tenth
of the in vitro translated proteins that were loaded to
the Agarose columns. Similar results were obtained in a second
experiment.
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Pull-down assays substantiated the above apparent interactions of GR
with the GMEBs. As seen in Fig. 5B
, either GMEB-1 or GMEB-2 bound to
M2-Agarose immobilized significant amounts of
[35S]methionine-labeled GR but not CREB
(see also below). Interestingly, the amount of GR that was retained did
not change appreciably whether the receptor was steroid-free or bound
by agonist (Dex) or antagonist [Dex-Mes or progesterone (Prog)]. The
amount of GR retained on GMEB-1 and GMEB-2 columns ranged from 18 to
28% and 16 to 28%, respectively, of the input GR, indicating an
efficient binding. For these reasons, coupled with their intrinsic
transactivation activity, the GMEBs may be potential new
transcriptional cofactors for GR.
Whole-Cell Activity of Overexpressed GMEBs
Given the complexity of the proposed model of GME action, in which
the two proteins GMEB-1 and -2 combine to bind to the GME and then
modulate GR transcriptional activity (15 26 27 ), the effect of GMEB
overexpression could not be easily predicted. The simplest
result would be that increased amounts of either protein caused
a further left shift in the dose-response curve and increased agonist
activity of antisteroids. Alternatively, decreased activities might
result from squelching, caused by the sequestering of a limiting
component (39 ). In CV-1 cells, overexpression of either GMEB-1 or -2 in
CV-1 cells gave both a right shift in the Dex dose-response curve and
decreased agonist activity with the antiglucocorticoid Dex-Mes (Fig. 6A
).
Cotransfection of GMEB-1 and -2 gave only marginally more right shift,
or reduced partial agonist activity (Fig. 6A
), suggesting that the
effects of squelching by the GMEBs are saturable. This activity of the
GMEBs was found to be dependent upon the concentration of both the
GMEBs and GR. Thus, the ability of the GMEBs to effect a right shift in
the dose-response curve (as shown by lower activities of 1
nM Dex) and decreased partial agonist activity of 1
µM Dex-Mes was more pronounced with increased
concentrations of either GMEB (Fig. 6B
). Conversely, the repression
seen with each GMEB could be reversed simply by adding more GR (Fig. 6C
). These effects appear to be selective as equal amounts of truncated
GMEB-1 and -2 proteins are inactive (J. Chen, S. Kaul, and S.
S. Simons, Jr., in preparation). Therefore, GMEB-1 and -2 have the
properties expected of proteins involved in GME action, namely the
ability to modulate, in a reversible fashion, the GR induction
parameters in intact cells.

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Figure 6. Whole-Cell Biological Activity of
GMEBs
A, Overexpressed GMEB-1 and -2 cause a right shift in the Dex
dose-response curve and decreased partial agonist activity of Dex-Mes.
Triplicate dishes of CV-1 cells containing 40 ng of pSVLGR plus 180 ng
of GMEB-1, or 70 ng of GMEB-2, were cotransfected with 1 µg of
GMEGREtkLuc reporter and then treated with EtOH ± the indicated
concentrations of Dex or 1 µM Dex-Mes. The induced levels
of Luciferase were determined as described in Materials and
Methods. The induction above basal activity with each steroid
concentration was determined and plotted as percent of the maximal
induction by Dex. The activity of 1 µM Dex-Mes is given
by the bars. The error bars represent the
SD of triplicate plates. B, Inhibition of GR activities by
added GMEB-1 or -2. Triplicate dishes of CV-1 cells containing 80 ng of
pSVLGR plus the indicated amounts of GMEB-1 or -2 were cotransfected
with 1 µg of GMEGREtkLuc reporter and then treated with EtOH ±
1 nM Dex, 1 µM Dex, or 1 µM
Dex-Mes. Empty vector DNA was used to keep the concentration of GMEB
plasmid vector constant in all plates. The induced levels of Luciferase
were determined as described in Materials and Methods.
The induction above basal activity with each steroid concentration was
determined and plotted as percent of the maximal induction by Dex. The
error bars represent the SD of triplicate
plates. Similar results were obtained in two other independent
experiments. C, Reversal of GMEB inhibition of GR activities by added
GR. Triplicate dishes of CV-1 cells containing 200 ng of GMEB-1, or 100
ng of GMEB-2, plus the indicated amount of pSVLGR were cotransfected
with 1 µg of GMEGREtkLuc reporter and then treated with EtOH ±
1 nM Dex, 1 µM Dex, or 1 µM
Dex-Mes. Empty vector DNA was used to keep the concentration of GMEB
plasmid vector constant in all plates. The induced levels of Luciferase
were determined as described in Materials and Methods.
The induction above basal activity with each steroid concentration was
determined and plotted as percent of the maximal induction by Dex. The
error bars represent the SD of triplicate
plates. Similar results were obtained in at least two other independent
experiments.
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GMEBs Do Not Possess Histone Acetyltransferase (HAT) Activity
A commonly proposed mechanism for the action of the
transcriptional cofactors of steroid/nuclear receptors is nucleosome
reorganization after histone acetylation (40 41 42 ). In fact, several
cofactors have been found to possess histone acetyl transferase (HAT)
activity (32 33 43 44 45 ). Therefore, we inquired whether the above
biological activity of the GMEBs might require HAT activity.
Interestingly, recombinant GMEB-1 and GMEB-2 with a FLAG tag at the N
terminus that were expressed from Baculovirus vectors in SF-9 cells, or
their mixtures, did not show any HAT activity (43 ) while the p300
positive control displayed a strong reaction in the same gel (data not
shown).
Interactions of GMEBs with Other Proteins
The proposed mechanism of GMEB action involves interactions with
GR (Fig. 5
) and other unidentified proteins (15 26 27 ). Attractive
candidates are the cointegrators p300, CREB-binding protein (CBP), and
p300/CBP-associated factor (PCAF) (30 31 32 43 45 ). Mammalian-two
hybrid assays suggest an interaction with CBP but not p300 or PCAF
(Fig. 7
). Whether this results from a
functional difference between CBP and p300 (46 47 48 ) is not known. No
binding of either GMEB with the corepressors SMRT (silencing mediator
for retinoid and thyroid hormone receptors) and or NCoR (nuclear
receptor corepressor) could be detected in the mammalian two-hybrid
assay (data not shown).

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Figure 7. Interaction of GMEBs with Transcriptional Cofactors
in Mammalian Two-Hybrid Assays
Triplicate dishes containing the indicated GAL4 chimeras and equimolar
amounts of either VP16 chimeras or VP16 were cotransfected with the
GAL4-regulated Luciferase reporter. The induced levels of Luciferase
were determined as described in Materials and Methods.
The error bars represent the SD of the
triplicate plates for each sample in the assay. Similar results were
obtained in four other independent experiments.
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Another candidate binder of the GMEBs is CREB. Both Oshima and Simons
(15 ) and Schutz et al. (34 35 36 ) have reported that CREB also
binds to the GME. Furthermore, as CREB and the GMEBs both form
complexes with GME oligonucleotides in gel shift assays that are of
very similar size and much larger than expected for the binding of
monomers, and as the GMEBs are known to bind to the GME as a
heterooligomer (28 29 ), it seemed possible that the large
complexes found to contain CREB might also contain one or more GMEBs.
Overexpression of CREB did not hinder the expression of GME activity
(27 ). Similarly, no interaction was observed between either of the
GMEBs and CREB in the pull-down assay (Figs. 4B
and 5B
) or in the
mammalian two-hybrid assay with both orientations of the GAL4-DBD and
VP16 activation domain chimeras (data not shown). Therefore, there is
currently no evidence for interactions between CREB and the GMEBs.
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DISCUSSION
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The glucocorticoid modulator element (GME) has the unique property
of modulating the glucocorticoid induction properties of the rat TAT
and other target genes (13 14 15 24 25 ). This whole-cell activity is
associated with the binding of a heteromeric complex of two proteins,
GMEB-1 and -2 (15 28 ), the latter of which we have recently cloned
(29 ). Both GMEB-1 and -2 are novel proteins that were not present on
GenBank, which is consistent with no other proteins being reported to
possess similar activities. This paper describes the cloning of the
second member of the biologically active heterooligomer, the human
equivalent of rat GMEB-1, as a 563-amino acid protein. The assignment
of the cloned protein as GMEB-1 was based on the more than 91%
identity of the predicted amino acid sequence of the human cDNA clone
with the six sequenced tryptic fragments distributed throughout the rat
GMEB-1, the nearly identical size of the in vitro translated
protein with rat GMEB-1, and detection by Western blotting with an
antibody that was raised against a peptide that was 96% identical.
Furthermore, the newly cloned material acted just like authentic GMEB-1
(28 29 ) in its ability to homo- and heterooligomerize with
itself and GMEB-2 and to bind to GME oligonucleotides as a homo- and
heterooligomer in gel shift assays. A crucial property of these
proteins is that they be able to modify the dose-response curve of
agonist steroids, and the partial agonist activity of antagonists, in
intact cells. GMEB-1 and -2 modulate these activities of GR whether
overexpressed individually or together. Therefore, both rat GMEB-2, and
now the human equivalent of rat GMEB-1, appear to have been cloned.
Several properties of the GMEBs made us question whether we had
identified the correct proteins. First, the size of GMEB-1 predicted
from the cDNA clone was too small. However, the protein encoded by the
1.8-kb hGMEB-1 cDNA clone migrated on SDS-PAGE (Fig. 2
) as a much
larger protein than expected from its sequence (88 vs. 62
kDa). This behavior was similar to that of the rGMEB-2 (29 ) and might
be due to posttranslational modifications. GMEB-1 and GMEB-2 both
contain S/T-rich regions and have several potential phosphorylation
sites for casein kinase and protein kinase. GMEB-1 also contains some
cAMP- and cGMP-dependent protein kinase sites.
Second, after our biochemical characterization of hGMEB-1 was completed
(49 ), the cloning of human GMEB-1 as a protein that binds heat shock
protein 27 in a yeast two-hybrid screen was reported (50 ).
Interestingly, this clone of hGMEB-1 contains a 10-amino acid insert
(LFIDGHFYNR) after amino acid 43 that was not present either in the
originally isolated rat GMEB-1, as determined from the new sequenced
peptide reported here that corresponds to amino acids 3246, or in our
human clone (Fig. 1
). This GMEB-1 of Theriault et al. (50 )
appears to be a GMEB-1 isoform (37 ). It is currently not possible to
determine the relative abundance of these two forms of GMEB-1 as the
antibodies used in the two studies can not discriminate between the two
proteins. Whether the longer form of hGMEB-1 (50 ) has the same
activities as seen for our clone and for the isolated GMEB-1 remains to
be established.
Third, another report of the cloning of human GMEB-1, and of human
GMEB-2, appeared while this paper was being revised. This report
described the GMEBs as HeLa cell factors essential for parvovirus
replication (51 ). While it is not unusual for a given protein to have
two biological activities, the ability of GMEB-1 to bind to heat shock
protein 27 and the essential role of the GMEBs in parvovirus
replication made it imperative for us to determine, first, that we had
cloned the correct molecules and, second, whether GMEB-1 and -2 were in
fact relevant for the GME action. The identification of three
additional sequenced peptides from the originally purified rat GMEB-1
protein (28 ) in our human GMEB-1 clone strongly argues that we have
cloned the correct protein (Fig. 1A
). All of the biochemical properties
of the overexpressed proteins from our GMEB-1 and -2 (29 ) clones (size,
antibody reaction, DNA binding as homo- and heterooligomers, and
specificity of DNA binding) are also consistent with those of the
endogenous proteins (Figs. 2
and 3
). Finally, the overexpressed GMEB-1
and -2 proteins can modulate GR activity (Fig. 6
). Thus, we conclude
that we have cloned two proteins that are biologically relevant for GME
activity. The fact that these proteins also have other cellular
functions promises to make future investigations particularly
interesting.
The effect of overexpressed GMEBs to cause a right shift in the
dose-response curve, and a decrease in the partial agonist activity of
antisteroids, as opposed to the left shift and increased partial
agonist activity seen with the GME, was not completely unexpected. Our
proposed model of GME action involves a multiprotein complex (15 26 27 ). Unless the GMEBs are the limiting factor for the expression of the
GR transactivational properties, overexpression of either GMEB could
sequester other required factors to cause squelching (39 ) and a
decrease in GR activities. For example, while CBP is a coactivator and
generally increases the total transactivation by steroid receptors,
overexpression of CBP in cells in which CBP is abundant causes a
decrease in GR transactivation (48 ). In our systems, GR appears to be a
limiting reagent (39 52 ). Furthermore, when GR concentrations are
increased so that they are not limiting, the GME is no longer active
(S. Chen, N. Sarlis, and S. S. Simons, Jr., in preparation). This
squelching is not due simply to a large imbalance in the ratio of
GMEB-1 to GMEB-2 because overexpression of both GMEBs does not reverse
the behavior. Instead, the effects of GMEB-1 and -2 are slightly more
than that of GMEB-2 alone (Fig. 6A
). This suggests the existence of a
lower limit for the squelching by elevated GMEBs, consistent with the
diminishing changes with higher levels of GMEB-2 alone (Fig. 6B
). While
it is possible that a left shift in the dose-response curve, and
increased agonist activity of antiglucocorticoids, may be achieved with
some other ratio of transfected GMEBs, we suspect that this behavior
will be seen only in cells where the GMEBs are limiting. We have not
yet found such a cell line (28 ). Nevertheless, both GMEBs do modulate
GR activity (Fig. 6
), thus fulfilling a necessary requirement for these
proteins to be considered as potential cofactors in the mechanism of
GME action.
Numerous similarities between our GMEB-1 and -2 clones suggest that
they are closely related proteins. GMEB-1 and -2 are 55% identical at
the cDNA level and 39% identical on the basis of amino acid sequence
and contain a region of 90 amino acids that is 80% identical.
Interestingly, the anti-GMEB antibody only weakly recognizes GMEB-1
even though the antigenic site in GMEB-1 is virtually identical to that
of GMEB-2 (22 of 23 amino acids). Both GMEBs possess intrinsic
transactivation activity in the mammalian one-hybrid assay. The
mammalian two-hybrid assay indicates that each protein forms
homooligomers and heterooligomers with the other protein in solution
(Fig. 4
). GMEB-1 and -2 also bind specifically to the same DNA sequence
both as a homooligomer and as a heterooligomer with each other (Fig. 3
). Quantitative comparisons of the homo- and heterooligomeric
interactions of GMEB-1 and -2 in the two-hybrid assay are complicated
by the observation that the activities of the GMEB chimeras are
orientation selective and most effective for GAL4-B2 interacting with
VP16-B1 (Fig. 4A
). Similar orientation specificities in the two-hybrid
assay have been observed by others (53 54 ).
Based on the biological activities of GME oligonucleotides that do, and
do not, bind the GMEBs (15 ), the mechanism of action of the GMEBs has
been proposed to involve their binding to the GME and subsequent
interaction with both GRE-bound GRs and the transcriptional machinery
(15 26 27 ). In fact, in mammalian two-hybrid assays, both GMEB-1 and
-2 display a modest steroid-dependent interaction with GR (Fig. 5A
).
The interaction of GR with GMEBs is particularly notable as the
chimeras GAL4-GR and VP16-GR exhibit no synergistic response, even in
the presence of steroid. This is not due to an inactivity of VP16-GR,
as witnessed from the responses with the GAL4-GMEBs, but may be due to
an attenuated activity of GAL4-GR. This could follow from either a
misfolding of the GAL-GR chimera or the formation of GAL-GR dimers,
thereby excluding the binding of VP16-GR chimeras. Alternatively, these
results are consistent with the hypothesis that the dimerization of
many DNA-bound receptors (55 ) occurs not in solution (56 57 ) but only
after a DNA binding-induced conformational change (57 58 59 60 61 ). Similarly,
the binding of cofactors to receptors has been found to be modified by
the DNA binding of receptors (62 63 ). In this case, the interactions
of the GMEBs might be stronger with GRE-bound GRs.
The strength of the associations of GAL4-GMEBs with VP16-GR in the
two-hybrid assays appears to be less than those of the FLAG-GMEBs and
wild-type GR in pull-down assays where there was no noticeable
difference in the presence or absence of either agonist or antagonist
steroid (Fig. 5
and data not shown). The binding of the GMEBs to
antagonist-bound receptors was anticipated from the ability of the GME
to augment the residual agonist activity of several different
GR-antisteroid complexes (13 15 24 25 64 ). A similar
ligand-independent interaction with GR, but ligand-dependent increases
in GR transactivation, has been seen with TIF1ß (65 ). The fact that
the GMEBs and steroid-free GR can bind may not be physiologically
relevant, however, as the predominantly cytoplasmic location of
steroid-free GR (3 ) may effectively prevent association with the
nuclear GMEBs (15 ). This could explain the steroid dependency for
GMEB/GR interactions in the two-hybrid assays but not in the pull-down
assays.
GMEB-1 or -2 binds to GR but neither contain a LxxLL motif, which has
been found to be intimately involved in cofactor binding to the steroid
receptors (66 67 ). However, the receptor binding proteins, TIF1ß
(65 ) and SUG1, or ARA70 (68 ), also have no LxxLL motifs. Furthermore,
not all LxxLL motifs interact with receptors (67 69 70 ). Another
difference between the GMEBs and many other coactivators of the
steroid/nuclear receptors is the absence of HAT activity (32 33 43 44 45 ), which is proposed to disrupt the nucleosomes and initiate
chromatin remodeling to facilitate the access of other transcription
factors (40 41 42 ). However, TIF2, a well established coactivator for
steroid receptors, is like the GMEBs and does not possess HAT activity
(70 ). Furthermore, recent data suggest that HAT activity may not be
required in all instances of steroid receptor-activated gene
transcription (71 72 73 ) and that chromatin architecture is not a
controlling factor in GR induction of TAT gene expression (71 74 ).
These observations are consistent with reports that the modulation of
TAT induction occurs just as well for the endogenous gene as for stably
and transiently transfected genes containing the GME (13 15 64 75 ),
conditions under which ordered nucleosome structures are not formed
(76 77 ). Thus, the modulation of GR gene induction activity by the
GME, which does not alter the fold induction by glucocorticoids (64 ),
may not require HAT activity, which has been seen most commonly in
factors that increase the fold induction of steroid receptors (32 33 43 44 45 ).
A second component of the proposed mechanism of GME action is the
association of the GMEBs with a transcription factor(s) (15 26 27 ).
Thus, the functional interaction of both GMEBs with CBP (Fig. 7
), a
common transcription integrating factor (30 31 78 ), is intriguing.
This whole cell interaction occurred with the C-terminal third of CBP,
which is missing both the HAT activity domain (79 ) and the receptor
interaction domain of CBP (30 31 ). We have recently reported that
increased concentrations of CBP cause a left shift in the dose-response
curve, and increased agonist activity of antiglucocorticoids, in the
absence of a GME (52 ). If the GMEBs bind to GR and CBP via different
domains, one attractive mechanism for a left shift by the GME is thus
to increase the net affinity of CBP and GR for each other, thereby
increasing the concentration of GR-CBP complexes without changing the
concentration of GR or CBP. Further experiments are needed to test this
hypothesis.
GMEB-2 displays some homology to several proteins including
DEAF-1 and Suppressin (29 ), which appears to be the short form of a
protein called NUDR (80 ). With the addition of GMEB-1, two human
proteins (nuclear phosphoprotein and lymphoid-specific SP100 homolog),
and two mouse ESTs, the size of the potential family of related
proteins has expanded, and the putative consensus sequence of this
family (29 ) can be refined (Fig. 8
).
GMEB-1 is highly homologous to, but is not a splicing variant of,
GMEB-2 (37 ) and displays a greater homology with GMEB-2 than any other
protein yet described. Therefore, GMEB-1 and -2 are the most closely
related proteins in this putative new family of related proteins. Of
those proteins that have been characterized, no function has yet been
demonstrated for the consensus sequence that includes the sequence
KDWK, although it has been proposed to be involved in DNA binding (81 ).
The GMEBs (Fig. 3
) (28 29 ), DEAF-1 (81 ), Suppressin (S. Kaul,
J. A. Blackford, Jr., and S. S. Simons, Jr., in preparation),
and NUDR (80 ) have all been found to bind DNA. The human
lymphoid-specific SP100 homolog is a nuclear protein that appears as
nuclear dots, some of which colocalized with PML nuclear bodies
(82 83 ). The GMEBs, DEAF-1, Suppressin/NUDR, and Sp100 (84 ) each
have characteristics of transcription factors, and the GMEBs and NUDR
(80 ) possess intrinsic transactivation activity in transfected cells.
The human nuclear phosphoprotein may also be involved in signal
transduction (85 ). Given the unusual activities that have already been
associated with these proteins, further investigations of this emerging
new family of proteins promises to uncover new details of cellular
transcriptional control mechanisms.

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|
Figure 8. Possible GMEB Family Members
Alignment of sequences of the high homology region of GMEB-1 and -2
with rat and human Suppressin (NUDR is not included as it appears to be
an extended form of Suppressin), Drosophila DEAF-1, the
ORFs of four Caenorhabditis elegans cosmids, two human
proteins (nuclear phosphoprotein and lymphoid-specific SP100 homolog),
and two mouse ESTs. The alignment was performed by SeqApp, which
ascribes different colors to various amino acids that are unrelated to
the homology. The region of highest homology for all proteins is
underlined.
|
|
 |
MATERIALS AND METHODS
|
---|
Unless otherwise indicated, all operations were performed
at 0 C.
Steroids
Dex was purchased from Sigma (St. Louis, MO).
Des-Mes was synthesized as described previously (86 ).
Antibody
The antibody used to detect both native and
recombinant GMEB1 was raised against a GMEB-2 peptide
(ISPKEFVHLAGKSTLKDWKRAIR) that was highly homologous to a
region of GMEB-1 (29 ).
Cloning of GMEB-1
In addition to the sequences of the three reported tryptic
peptides of GMEB-1 (28 ), three more peptides were sequenced at the Keck
Foundation (Yale University, New Haven, CT). The sequences of these
peptides and the yields (in parentheses) were ESEEISENTLMF (16.4%),
with a minor species of XQMI/FLQLQPVQQGXAE, and AVILETELR (44.2%). The
amino acid sequences of all GMEB-1 peptides were blasted against the NR
database (in all six frames) of GenBank (87 ). No homology was found
with any known proteins or nucleic acid. However, one of the tryptic
peptides displayed substantial homology (16 of 18 amino acids) with a
human EST (129005) in the dbEST database of GenBank. This EST was then
used to identify related ESTs in dbEST database. Two ESTs (129796 and
hbc1096) showed high homology to particular areas of EST 129005. Based
on sequence of the above three ESTs, SeqApp 1.9 (D. Gilbert, Indiana
University, Indianapolis, IN) was used to generate a 1-kb continuous
sequence. EST 129005 was obtained from ATCC (Manassas, VA) and
resequenced. There were several errors in the published one-pass
sequence of EST129005 but no errors in the region encoding the sequence
that was homologous with the tryptic peptide. Two oligonucleotides
(5'-CATCCAGGCTCCCCTTCCAAG-3' and 5'-CCAGGAACCAAGTAGAGCAGGG-3') were
designed based on the sequence of the above 1-kb sequence. Both
oligonucleotides were biotinylated and used to screen a cDNA library
from human heart (Life Technologies, Inc., Gaithersburg,
MD) using the solution hybridization method of Gene Trapper cDNA
positive selection system (Life Technologies, Inc.)
according to manufacturers instructions. The captured cDNA for each
oligonucleotide was electroporated into electromax DH10B cells
(Life Technologies, Inc.). Colony PCR was performed on 120
clones of 200 generated and 8 of them had inserts. Most of the positive
clones had an insert size of around 2 kb. One large clone, GM106
(GMEB-1/pCMV-SPORT), was sequenced by MacConnell Research Corp. (San
Diego, CA) on a Licor machine from both ends.
Plasmids
pRBalI17 used for in vitro synthesis of GR
protein and pSVLGR used to transiently transfect GR into CV-1
cells were obtained from Keith Yamamoto (University of California, San
Francisco, CA). GALp300, with the entire p300, was obtained from A.
Giordano (Thomas Jefferson University) while GAL4CBP, containing amino
acids 16782441 of CBP, was obtained from Richard Goodman (Vollum
Institute). pCi.GAL4pCAF was prepared by modifying pCI (Promega Corp., Madison, WI) to pCI.FlagGal4(94) (sequence is available
upon request to V.V.O.). PCAF was then inserted into the
MluI and NotI sites to give the final construct.
GMEGREtkLuc reporter was described previously (27 ).
GAL/GMEB-2 [GMEB-pM] and VP16/GMEB-2, consisting of the GAL4-DBD or
the VP16 transactivation domain fused to the amino terminus of
full-length GMEB-2, were constructed by cloning a 2-kb blunt ended
NcoI-HindIII fragment of GMEB-2/pCR2.1 (29 ) into
the EcoRV site of pcDNA3.1/His A (Invitrogen,
San Diego, CA). Clones were digested with BamHI to ensure
the proper orientation of cDNA. A 2-kb BamHI fragment from
GMEB-2 pcDNA3.1/His A was then cloned into BamHI site of pM
(GAL4-DBD) and pVP16 (VP16 activation domain) (CLONTECH Laboratories, Inc. Palo Alto, CA).
GMEB-1/pCM-SPORT was mutated with the Quick Change Site-Directed
Mutagenesis kit (Stratagene, La Jolla, CA) just upstream
of the initiation codon to remove the upstream stop codon and create an
EcoRI site. The 2-kb EcoRI and XbaI
fragment from the above mutated clone of GMEB-1/pCM-SPORT (clone 101)
was cloned in the same sites of pM, pVP16, and pcDNA3.1/His A to
generate GAL/GMEB-1, VP16/GMEB-1, and GMEB-1/pcDNA3.1/His A.
GR/pcDNA3.1/HisC was generated by ligating three fragments: 1) a 718-bp
SspI and BglII fragment obtained by digesting
pSVLGR with the same restriction enzymes; 2) a 2-kb fragment of pSVLGR
digested with BglII and XbaI; and 3) vector
pcDNA3.1/HisC that had been digested with EcorV and XbaI.
GAL/GR and VP16/GR were again constructed by ligating three DNA
fragments: 1) a 0.7-kb fragment obtained by digesting GR-pcDNA3.1/HisC
with EcoRI and BglII; 2) a 2-kb fragment obtained
after digesting GR-pcDNA3.1/HisC with BglII and
XbaI; and 3) vectors pM and pVP16 digested with
EcoRI and XbaI.
The baculovirus expressed GMEBs contained a Flag tag for use in
immunoadsorption assays. GMEB-1/Flag p/FastBac1 was constructed by
cloning a 2-kb EcoRI and XbaI fragment from
GMEB-1/pcDNA3.1/HisA into Flag/pFastBac1 (Life Technologies, Inc.). GMEB-2 was first cloned into the BamHI site of
pSP73 (Promega Corp.) to clone GMEB-2 cDNA into the
correct frame of Flag/pFastBac1. Finally, a 2-kb XbaI and
SacI fragment from GMEB-2/pSP73 was cloned in
Flag/pFastBac1.
CREB-pCR2.1 was constructed by cloning a EcoRI fragment from
pSV-CREB (William Walker, University of Pennsylvania, Philadelphia, PA)
into pCR2.1. CREB was cloned in frame in pM and pVP16 by first
amplifying by PCR the CREB ORF, and creating BamHI (at
5'-end) and HindIII (at 3'-end) sites, using pSV-CREB as a
template with high-fidelity Pfu polymerase (Stratagene).
The PCR product was digested with BamHI and
HindIII and cloned into the same sites of pM and pVP16.
Cell Culture and Transient Transfection
Cos-7 or CV-1 cells were grown in DMEM (Life Technologies, Inc.) supplemented with 5%, or 10%, respectively, of FCS
(Biofluids Inc., Rockville, MD) at 37 C in a humidified
incubator (5% CO2). The transient transfections
were done using Lipofectamine Reagent (Life Technologies, Inc.) in the presence of media without serum (OPTI-MEM I;
Life Technologies, Inc.). Cells were incubated with
plasmid DNA, OPTI-MEM I, and lipofectamine reagent for 4 or 16 h
and then placed in the normal media (5 or 10% FCS, DMEM) for 16 h
before being induced with the appropriate steroid for 24 h.
Transfected cells were lysed and assayed for reporter gene using
Luciferase Assay Reagent according to manufacturers instruction
(Promega Corp.). Luciferase activity was measured in an
EG&G Berthhold Luminometer (Microlumat LB 96 P).
Expression of GMEB-1 and GMEB-2 in Baculovirus
GMEB-1 and GMEB-2 proteins with the FLAG tag at the N terminus
were expressed as a Baculovirus protein in SF9 cells using BAC to BAC
Baculovirus Expression System according to the manufacturers
instruction (Life Technologies, Inc.).
In Vitro Transcription and Translation
GMEB-1 (SP6), GMEB-2 (T7), CREB (T7), and GR (SP6) were
expressed from clones in pCMV-SPORT, PCR 2.1, pSP73, and pBAL117,
respectively, using either SP6 or T7 RNA polymerase (as indicated in
parentheses) in TNT rabbit reticulocyte lysate system (Promega Corp.) according to manufacturers instructions.
Gel Shift Assay
Gel shift assays to detect the binding of GMEB-1, GMEB-2, CREB
(single proteins or their mixtures), or HTC cytosol to
32P-GME oligonucleotides were performed as
published (29 ). The mixtures of two or more proteins were incubated on
ice for 1216 h for the formation of protein complexes (28 ). The
sequence of GME and mutant oligonucleotides M1, M2, and M3 have been
described (15 ). The specificity of DNA binding was determined from the
competition by 25-fold molar excess of cold biologically active GME,
M1, and M3 oligonucleotides and the biologically inactive M2
oligonucleotide.
Pull-Down Assay
M2 agarose beads [anti-FLAG monoclonal antibody conjugated to
agarose (Sigma)] were recharged by treating them with 100
mM glycine.HCl buffer, pH 2.5, (10
vol.) for 23 min and then neutralized with 1 M
Tris.HCl, pH 8.0, (10 vol.). The beads were
washed once with PBS, pH 7.4 (BioWhittaker, Inc.,
Walkersville, MD) and once with High Salt Buffer (10% glycerol, 20
mM Tris.HCl, 0.4 mM EDTA,
0.2% Tween, 10 mM mercaptoethanol, and 500 mM
KCl). Baculovirus-infected cell extracts containing overexpressed
GMEB-1 or GMEB-2 (100 µl) were incubated with the above prepared M2
agarose for 23 h at room temperature. The beads were then washed five
times with High Salt Buffer for 10 min each. The beads were washed once
with Low Salt Buffer (10% glycerol, 20 mM
Tris.HCl, 0.4 mM EDTA, 0.2% Tween,
10 mM mercaptoethanol, and 250 mM KCl). The
in vitro translated proteins (labeled with
[35S]methionine) were added separately to the
M2 beads bound with either Flag GMEB-1 or -2 and incubated for 1 h
at room temperature and then 1216 h at 4 C. The beads were then
washed four to five times with Low Salt Buffer (0.5 ml) for 10 min at
room temperature. Finally the beads were resuspended in 20 µl of Low
Salt Buffer and treated with 20 µl of 2x SDS-PAGE buffer for 15 min
at 37 C. The supernatant proteins were loaded onto a SDS-PAGE gel
(10%) and visualized by fluorography.
Data Analysis
Gel shift bands were quantitated on a Macintosh Power G3
computer using the public domain NIH Image program (developed at the
NIH and available on the Internet at
http://rsb.info.nih.gov/nih-image/). All statistical analyses were by
two-tailed Students t test using the program InStat 2.03
for Macintosh (GraphPad Software, Inc., San Diego,
CA).
 |
ACKNOWLEDGMENTS
|
---|
We thank A. Giordano, Richard Goodman, William Walker, and Keith
Yamamoto for generously providing plasmids, Hinrich Gronemeyer for
alerting us to the sequencing error of GMEB-2, Yoshihiro Nakatani
(NICHD, NIH) for support and helpful discussion of this work, and Alan
Wolffe (NICHD, NIH) for critical review of this paper.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. S. Stoney Simons, Jr., Building 8, Room B2A-07, NIDDK/LMCB, NIH, Bethesda, Maryland 20892.
Received for publication August 8, 1999.
Revision received February 18, 2000. Revision received April 5, 2000.
Accepted for publication April 7, 2000.
 |
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