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(Received for publication, April 30, 1995; and in revised form, June 21, 1995) From the
Glucocorticoid induction of the tyrosine aminotransferase gene
deviates from that of many glucocorticoid-responsive genes by having a
lower EC
For many years, the accepted model of steroid hormone action
predicted that the responses of all regulated genes were a property of
the steroid used. Thus, a gene is induced, or repressed, by agonists,
and the action of agonists is prevented by antisteroids. Furthermore,
the concentration of steroid required for half-maximal induction by an
agonist and the amount of agonist activity possessed by a given
antisteroid should be constant for each steroid and independent of the
gene examined (reviewed in (1) and (2) ). Recently,
this model has had to be modified as exceptions were defined. Thus, jun Other observations that
did not appear to fit with the conventional model of steroid hormone
action originated from studies on glucocorticoid induction of the
tyrosine aminotransferase (TAT) ( We
previously proposed that this modulation of TAT gene induction in rat
hepatoma cells occurred via the binding of a trans-acting
factor to a cis-acting element of the TAT gene(1) .
Stable (22) and transient (23) transfection assays
succeeded in identifying such a cis-acting element, at about
-3646 bp of the rat TAT gene, that conveyed all of the
glucocorticoid induction properties of the endogenous TAT gene to
heterologous genes and promoters. This cis-acting element was
called a glucocorticoid modulatory element (GME) and was found to bind
a trans-acting factor(s)(23) . The mechanism of action
of the GME, unlike that of the commonly discussed transcription factor
binding sites, does not involve synergism with the glucocorticoid
response element, or GRE(24) . This suggests that the GME-bound
factor(s) (GMEB) might be a novel protein. The binding site of the
GMEB has also been identified as a cyclic AMP-responsive element
(CRE)(25, 26, 27) , but several lines of
evidence indicate that two different sets of proteins are responsible
for GME and CRE activity. First, the biological activities mediated by
GMEB and the CRE binding protein (CREB) are quite dissimilar ((21, 22, 23) versus 25, 28).
Second, no additional element is needed for GME biological activity,
while a functional CRE requires a second TAT gene sequence, initially
called BIII (25) and more recently found to bind
HNF-4(27) . Third, both the GME (23) and the CRE (28) give a closely spaced, three-band pattern in gel shift
assays. However, the GMEB is responsible for slowest migrating of the
three bands (23) , which has been shown not to contain
CREB(26) . The GME contains the sequence CGTCA, which is a
common CRE element that binds homo- and heterodimers of CREB/ATF along
with other family members or unrelated proteins (29) such as
AP-1(30) . Thus, many known and unknown trans-acting
factors could bind to the GME/CRE site at -3646 bp of the TAT
gene. The purpose of this paper, therefore, was to characterize and
purify the binding protein(s) proposed to be responsible for the GME
activity of modulating glucocorticoid receptor function.
For size exclusion
chromatography, 200 µl of nuclear extract or cytosol was loaded
onto a Superose 6 HR 10/30 column (Pharmacia), which had been
equilibrated with buffer 150T at 4 °C. The column was run at 0.5
ml/min in a FPLC system (Pharmacia). Every 500-µl fraction was
collected and stored at -70 °C. For ion exchange
chromatography, HTC cytosol (about 30 ml) was loaded with a Superloop
in a FPLC system (Pharmacia) onto Mono Q HR 10/10 column that had been
equilibrated with buffer 20T at 1 ml/min. After washing the column with
the same buffer, the column was eluted with a linear gradient up to 40%
of buffer 1000T. Every 2-ml fraction was collected and stored at
-70 °C. For size fractionation by electrophoresis,
partially purified nuclear extract or cytosol was separated on 6 or 8%
SDS-PAGE gels (1.5 mm thick) (32) and the desired M
The presence of the GMEB in the above cell lines was
examined in gel shift assays. Normally, a three-band pattern was
obtained, of which the slowest migrating band corresponds to the
biologically relevant interaction of GMEB with the GME(23) .
Nuclear extracts from each cell line afforded somewhat different
patterns; but in each case, a complex of the same low mobility was
observed that was blocked only by excess non-labeled GME
oligonucleotide (Fig. 1A). In all cases, the
biologically inactive M2 oligonucleotide (23) was unable to
competitively inhibit the formation of this band (Fig. 1A). Thus, both liver and non-liver cells appear
to contain GMEB.
Figure 1:
Presence of GMEBs in
nuclear extracts of various cells (A) and nuclear extracts (Nuc. Extr.) versus cytosol solutions from HTC and
PC-12 cells (B). Nuclear extracts or cytosols from different
cell lines were analyzed in gel shift assays with
When
cytosols were prepared from HTC cells treated with saturating
concentrations of dexamethasone at 37 °C, conditions that cause the
nuclear translocation of most of the cytoplasmic glucocorticoid
receptors(37) , there was no appreciable decrease in the
intensity of the gel-shifted band with GMEB
bound to several polycationic columns. GMEB was eluted from
heparin-agarose at 0.3-0.8 M NaCl, from DEAE-Sepharose
CL-6B by 0.1-0.2 M NaCl, and from Mono Q columns with
0.2 M NaCl. Thus, GMEB would be predicted to contain at least
one surface containing a net abundance of anionic charges, as would be
expected for a factor involved in the modulation of transcription. GMEB
also bound to phenyl-Sepharose and a Mono S polyanionic column, but the
recovery was very low (data not shown). Several results indicate
that GMEB and CREB are different proteins. First, heating the nuclear
extracts to 65 °C (but not 37 °C) for 10 min eliminated the
formation of the GMEB-bound complex (data not shown). In contrast, CREB
is stable under these conditions (data not shown, (29) ).
Second, an anti-CREB antibody supershifted some of the lower two bands
in gel shift assays but none of the more slowly migrating
GMEB-containing bands (data not shown). This confirms the earlier
report that only the lower two bands contain CREB(26) . Third,
a 0.5-h treatment with 10 µM of forskolin, which increases
the cellular cAMP (and activates CREB), had no effect on the amount of
GMEB in the gel shift assay (data not shown) or on the percent agonist
activity seen for 1 µM dexamethasone 21-mesylate with the
GREtkCAT reporter (30 versus 34%) and only slightly increased
the percent agonist for dexamethasone 21-mesylate with the GMEGREtkCAT
reporter (67 versus 57%). Fourth, a Two
factors that are closely related to CREB, which heterodimerize with
CREB, and can replace CREB in vivo are CREM and
ATF-1(39) . However, neither a broad spectrum antibody
(anti-CREM) nor a variety of mono-specific antibodies could supershift
the GMEB-containing band in gel shift assays. Similarly,
immunodepletion of the nuclear extracts with these antibodies did not
prevent the formation of the GMEB-containing band (data not shown).
These results argue that the GMEB is not ATF-1, -2, or -3, CREB-1 or
-2, CBP, or CREM. Similar experiments with anti-jun and
anti-fos antibodies ruled out jun and/or fos as being the GMEB (data not shown), even though AP-1 is active
with a highly homologous DNA sequence(40) .
Figure 2:
Methylation interference assay for GMEB
binding to GME. Nuclear extracts from Fu5-5 clone 27 cells were
incubated with partially methylated
When
compared to molecular weight markers in the gel shift
assay(41) , the size of the GMEB was calculated to be
Figure 3:
GMEB is a multimeric protein complex. A, size exclusion column chromatography of HTC cytosol. HTC
cytosol (200 µl) was separated on a Superose 6HR 10/30 column as
described under ``Materials and Methods.'' The amount of
protein in each fraction (0.5 ml) was monitored by the UV absorption at
280 nm. Molecular mass markers: T, thyroglobulin (670 kDa); G, bovine
Figure 4:
Reconstitution of GMEB activity requires
two separable species. HTC cell cytosol (about 37 µg of protein)
that had been partially purified by Mono Q column chromatography was
size fractionated on a 6% SDS-PAGE gel. Molecular weight ranges of HTC
cell proteins were isolated by cutting the sample lanes, as indicated
in the unshaded gel lane strip, relative to the migrations of
prestained markers (Life Technologies, Inc., Bio-Rad) in adjacent lanes
(note that gel was cut below the 71-kDa marker). Proteins in the
excised pieces of the gel were electroeluted, kept separate, or mixed
as indicated by the shadedgellanestrips, and then precipitated, denatured, and renatured
as described under ``Materials and Methods.'' About 1-2
µl of the 20 µl of renatured proteins was used in gel shift
assays with
Figure 5:
Purification and reconstitution of GMEB
activity. A, silver-stained SDS-PAGE gel of material during
various stages of purification. Unstained molecular weight markers, lanes1 and 8; about 100 ng each of HTC
cytosol, lane2; mono Q column purified cytosol, lane3; M2 DNA affinity column flow through, lane4; and the GME binding fractions from the first (lane5), second (lane6), and third (lane7) M3 DNA affinity column. The arrows indicate the three candidate GMEBs (labeled 1-3,
best seen in lanes6 and 7). B,
reconstitution of GMEB activity in gel shift assays. The three major
proteins in the eluant from the third M3 DNA affinity column (proteins
1-3 in A) were individually cut out from a
silver-stained gel such as in A, electroeluted, denatured, and
renatured as described under ``Materials and Methods.''
Aliquots (0.4 µl out of 17 µl) from the renatured sample were
analyzed in the gel shift assay. lanes1 and 9, purified GMEB from the third M3 DNA affinity column; lanes2-7, indicated individual proteins or
mixtures of proteins that were mixed, precipitated, denatured, and
renatured. C, specificity of binding activity of purified and
reconstituted GMEBs. Protein bands 1 and 2 from the SDS gel of A were isolated and reconstituted as in B and then analyzed
in the standard gel shift assay without (lane2) or
with a 100-fold molar excess of non-labeled specifically (GME) or
nonspecifically (M2) binding oligonucleotides. The complex formed with
GMEB from the third M3 affinity column, but not fractionated on SDS
gels, is shown in lane1.
About
130 µg of the two proteins GMEB1 (88 kDa) and GMEB2 (67 kDa) was
isolated from denaturing SDS-polyacrylamide gels and sent to the Kreck
Foundation (Yale University) for peptide sequencing. The procedure
involved in-gel trypsin digestion of each protein, HPLC separation of
the fragments, and laser desorption mass spectroscopy to determine
which fragments would be most amenable to micro-sequencing. The partial
sequences of the three peptides from each protein digest that were
sequenced are given in Table 3. The yield of most sequenced
peptides was 5-23%, which is the normal range. For peptide
2-73, a very high yield of 78% was obtained, which also proves
that this protein was not a mixture of two or more proteins when
isolated from the SDS-polyacrylamide gel. The high concentration of
acidic residues in some of these peptides (i.e. 1-109
and 2-145) is compatible with the retention of the proteins on
polycationic columns (see above). A TBLASTN search of numerous DNA and
protein data bases using the NCBI BLAST E-mail server (44) revealed no significant homology with any of the peptides
of Table 3. Therefore, the GMEB may be a heterooligomeric complex
of two novel proteins.
We have called the element at -3.6 kilobases of the rat
TAT gene a glucocorticoid modulatory element, or GME, because it
modulates the induction properties of both subsaturating concentrations
of agonists and saturating concentrations of antagonists(23) .
The binding of a protein(s) to the GME was observed that was directly
related to the biological activity of the GME
oligonucleotide(23, 24) , which was different from
synergism(24) . We now report that the GMEB appears to be a
heterooligomer of two previously unsequenced proteins, GMEB1 and GMEB2,
of apparent molecular masses of 88 and 67 kDa, respectively. However,
conclusive identification must await the cloning of both proteins and a
demonstration of biological activity with the cloned proteins in cells
lacking GMEBs. Several properties of the GMEBs emerged during their
purification that pertain to the mechanism of GME action. First,
although the DNA sequence to which the GMEBs bind is very similar to
that for the CREB/CREM/ATF and the Jun/Fos/AP-1 superfamilies, and CREB
even binds to a non-consensus CRE at the same position as the GME at
-3646 bp of the TAT gene(25, 26, 27) ,
there is little similarity between the GMEBs and these other proteins.
The GMEBs are not related to CREB by the criteria of size, biological
activity(23, 24, 38) , antibody reactivity,
methylation interference patterns for protein binding (Fig. 2Aversus Fig. 7 of (28) ), or
amino acid sequence (Table 3). Some AP-1 sites contain the CGTC
of the GME, and AP-1 may bind to the GME/CRE, as it has recently been
reported that 12-O-tetradecanoylphorbol-13-acetate both
inhibited glucocorticoid (and cAMP) induction of TAT and caused a
decreased protein occupancy of the CRE at -3646 bp(45) .
However, again there was no similarity between the peptide sequences of
the GMEBs and AP-1, an anti-AP-1 antibody did not cause a supershift of
GMEB-GME complexes, and 12-O-tetradecanoylphorbol-13-acetate
alone did not elicit any response from GME-containing constructs (data
not shown). Thus, there is little physical or biological similarity
between the GMEBs and the other factors binding to the same DNA
sequence. This, then, is an additional example of different proteins
that bind to the same DNA region(46, 47) . Second,
we do not know if GMEB and CREB can both bind to the GME/CRE at the
same time. However, it seems that CREB is unable to block GMEB action.
The low levels of the protein kinase A regulatory subunit (Tse-1) in
liver cells are thought to result in high amounts of active CREB that
would bind to the GME/CRE(48) . Nevertheless, reporter
constructs containing either a single GME (GMEGREtkCAT) or other
elements that are needed for CRE activity, such as multiple tandem
repeats of the GME or the GME plus the downstream BIII
sequence(25) , show full GME activity in Fu5-5 rat
hepatoma cells(23) . Furthermore, conditions that elevate
protein kinase A activity, such as forskolin treatment, did not inhibit
GME activity. Thus, while CREB binds to the same DNA sequence as GMEB,
CREB does not appear to competitively inhibit GMEB binding in intact
cells. Third, GME activity is not limited to rat liver cells (Table 1), and the GMEBs are not tissue-specific proteins (Fig. 1A). Furthermore, the fact that the GME was
active with synthetic GREs and a variety of promoters, including a
minimum thymidine kinase promoter (23) , suggests that no
tissue-specific DNA binding factors are required for GME activity. Fourth, the GMEBs are clearly of nuclear origin but can be readily
extracted from nuclei under conditions where other factors, such as
CREB, stay in the nucleus. This is reminiscent of several other nuclear
proteins(49) , including the progesterone and estrogen
receptors, which are predominantly nuclear but appear in most cytosolic
preparations. The cytosolic appearance of the GMEBs could be indicative
of a dynamic equilibrium between the two cellular compartments, as
established for the progesterone
receptors(49, 50, 51) , or may simply reflect
a repartitioning of the GMEBs in the lysis buffer. Fifth, the mass
and stability of the GMEB complex are notable. The 550-600-kDa
size of the protein complex seen in both gel shift assays and size
exclusion chromatography (Fig. 3, A and B)
argue against a nonspecific aggregate. Involvement of the 265-kDa
protein CBP that binds phosphorylated CREB (52) was discounted
by the observed sizes of GMEB1 and -2 and their lack of
immunoreactivity with anti-CBP antibody. The most purified preparation
of GMEB appeared to contain about equal amounts of GMEB1 and -2 (Fig. 5A), which would require three or four molecules
of each protein in the final complex to achieve a molecular mass of
550-600 kDa. Such a massive complex is probably not too large to
be extracted intact from HTC cell nuclei because identically prepared
nuclei were found to be permeable to molecules as large as the 240-kDa
protein complex of phycoerythrin(53) . However, the GMEB
complex must be quite stable to retain specific binding to the GME
after extraction from the nucleus (Fig. 1), even in the presence
of up to 2.7 M guanidinium hydrochloride and after various
degrees of purification (Table 2). Despite the stability of the
GMEB complex with regard to dissociation, the rate of reassociation of
the separated components was relatively slow (Fig. 4). Finally, from the yield of purified GMEB1 and -2 in Table 3,
it can be calculated that there are about 40,000 molecules of each GMEB
per HTC cell. This is similar to the approximately 80,000 molecules of
glucocorticoid receptor that are present in an HTC cell(16) .
Considering the fact that most glucocorticoid-responsive genes contain
two GREs, each of which binds a dimer of the receptor, the ratio of
GME-bound GMEB complexes to GRE-bound receptors is about 1:2. Given the
facts that the GMEBs are not limited to rat liver cells and that
GME-like modulation has been observed with several other glucocorticoid
regulated genes(2) , it will be interesting to pursue the
possible role of a GME and its heteromeric binding complex in the
transcription of genes other than TAT. While CREB has been
identified as a component that also binds to the DNA sequence of the
GME/CRE(26) , it is not known if it is the only protein in the
complex. Several lines of evidence argue that the CREB-containing
complexes bound to the GME are also multimeric. Most obvious is the
size of the CREB-containing complexes, which were In
summary, we have found that a heteromeric complex of two potentially
new proteins binds to a cis-acting element of the TAT gene.
These two proteins, GMEB1 and GMEB2, are associated with changes in the
transcriptional activity of antiglucocorticoids and low concentrations
of glucocorticoids. These are phenomena that have not been previously
described for steroid receptors and thus are of considerable
mechanistic interest. It remains to be seen whether the GMEBs interact
with glucocorticoid receptors and the transcriptional machinery in the
manner that we have proposed(2, 23) . The cloning of
GMEB1 and GMEB2 and the production of specific antibodies will be of
major assistance in understanding the mechanistic details of this
interesting system.
Volume 270,
Number 37,
Issue of September 15, pp. 21893-21901, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and displaying more agonist activity with a given
antiglucocorticoid. A cis-acting element, located 3646 base
pairs upstream of the start of tyrosine aminotransferase gene
transcription, has been found to be sufficient to reproduce these
variations with heterologous genes and promoters (Oshima, H., and
Simons, S. S., Jr.(1992) Mol. Endocrinol. 6, 416-428).
This element has been called a glucocorticoid modulatory element, or
GME. Others have called this sequence a cyclic AMP-responsive element
(CRE) due to the binding of the cyclic AMP response element binding
protein (CREB). We now report the partial purification and
characterization of two new proteins (GMEB1 and -2) of 88 and 67 kDa
that bind to the GME/CRE as a heteromeric complex. This purification
was followed by the formation of a previously characterized,
biologically relevant band in gel shift assays. By several biochemical
criteria, the GMEBs differed from many of the previously described
CREB/CREM/ATF family members. Partial peptide sequencing revealed that
the sequences of these two proteins have not yet been described. Size
exclusion chromatography and molecular weight measurements of the
gel-shifted band demonstrated that the GMEBs bound to the GME as a
macromolecular complex of about 550 kDa that could be dissociated by
deoxycholate. Similar experiments showed that CREB bound to the GME as
heteromeric complexes of about 310 and 360 kDa. As determined from gel
shift assays, GMEB1 and -2 are not restricted to rat liver cells but
appear to be ubiquitous. Thus, these novel GMEBs may participate in a
similar modulation of other glucocorticoid-inducible genes in a variety
of cells.
fos heterodimers (AP-1), and AP-1 inducers
such as phorbol esters block steroid induction (3, 4) in what can be a cell-specific
manner(5, 6) , while junjun homodimers augment glucocorticoid induction(4) . Cyclic
AMP, via protein kinase A, can often (but not
always(7, 8, 9) ) cause greater induction by
agonists (10, 11) and increased percentages of agonist
activity for antisteroids(11, 12) . Heat shock, or
chemical shock, afforded a synergistic increase in glucocorticoid
inducibility(13) , while the immunosuppressive agent FK506
augmented the activity of subsaturating concentrations of
glucocorticoids(14) . Finally, dopamine can cause
ligand-independent gene activation of some receptors(15) . None
of the above agents effected any shift in the dose-response curve for
agonists except for FK506, which was postulated to increase the nuclear
binding of activated complexes(14) .
)gene in rat hepatoma
tissue culture (HTC) cells, which had become a paradigm for
steroid-inducible genes. We found that the dose-response curve for
dexamethasone induction of TAT gene expression in the related
Fu5-5 rat hepatoma cell line was left shifted compared to that in
HTC cells(16) . Similarly, TAT enzyme activity was induced at
lower cAMP concentrations in Fu5-5 cells than in HTC
cells(7) . Furthermore, all antiglucocorticoids examined
displayed a higher percentage of agonist activity for TAT gene
expression in Fu5-5 than in HTC
cells(16, 17, 18) . This left shift in the
TAT dose-response curve, and increased agonist activity with
antisteroids, was not a general response of all
glucocorticoid-inducible genes in Fu5-5 cells (19) and
occurred at the level of correctly initiated
transcripts(7, 19) . Surprisingly, the magnitudes both
of the left shift in the dose-response curve and of the increased
amount of agonist activity were not constant but varied slowly over
time (17, 20) in a manner that was eventually found to
be related to the density of the cells in culture(21) .
Therefore, it appeared that some event downstream of steroid binding to
the glucocorticoid receptor selectively modulated the properties of TAT
gene induction by glucocorticoid agonists and antagonists.
Chemicals
The following chemicals were
obtained from the indicated sources: [P]dCTP and
dATP (3000Ci/mmol), DuPont NEN; deoxycholate,
dimethyl sulfate, and Nonidet P-40, Sigma; p-aminoethylbenzenesulfonyl fluoride, ICN (Cleveland, OH);
acrylamide, bisacrylamide, and molecular weight markers for Superose 6
HR columns and silver staining kit, Bio-Rad; prestained molecular
weight markers, Bio-Rad and Life Technologies, Inc.; native molecular
weight markers for gel shift assays and SDS-polyacrylamide gels,
Pharmacia Biotech Inc.
Antibodies
The listed antibodies were
gifts of, or purchased from, the sources in parentheses: anti-CREB (Dr.
G. Schütz), anti-fos (PC05) and
anti-jun (PC07) (Oncogene Science), rabbit polyclonal anti-CBP
(
HCBP3, Dr. R. Goodman), rabbit polyclonal anti-CREM (Dr. J.
Habener), mouse monoclonal anti-ATF-1 (C41-5.1), anti-ATF-2
(F2BR-1), anti-CREB-1 (24H4B), rabbit polyclonal anti-ATF-3 (C-19), and
anti-CREB-2 (C-20) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).Buffers
Buffer 20T contained 20 mM Tris (pH 8.0 at room temperature), 2 mM MgCl
,
0.5 mM dithiothreitol, 50 µMp-aminoethylbenzenesulfonyl fluoride, 10% glycerol, 0.2 mM EDTA, and 20 mM NaCl. Buffers 150T and 1000T were the
same as buffer 20T except that the NaCl concentrations were 150 and
1000 mM, respectively. Buffer TN was the same as buffer 20T
with 0.05% Nonidet P-40 but without NaCl. TBE buffer contained 50
mM Tris base, 50 mM boric acid, and 1 mM EDTA.Cell Culture, Transfections, and Preparation of
Cellular Fractions
Rat hepatoma tissue culture cells
(uncloned and clone 27 Fu5-5 cells (19) and uncloned and
clone 28 HTC cells(20) ) were grown at 37 °C in a
humidified incubator (5% CO) in Richter's improved
minimum essential medium with zinc and supplemented with 0.03%
glutamine and 10% heat-inactivated fetal calf serum (Biofluids,
Rockville, MD) as described. HeLa (G. Hager, NIH), L
(tk; G. Schütz, Heidelberg), and
PC12 cells (M. Iadarola, NIH) were grown in Dulbecco's modified
Eagle's medium (low glucose with 0.4% glutamine and 110 mg/ml
sodium pyruvate) with 10% heat-inactivated fetal calf serum. Transient
transfections of cells were achieved with calcium phosphate (22) or Lipofectin (Life Technologies, Inc.) (23) and
analyzed as described. Nuclear extracts (23) and cytosols (31) were prepared as usual.
Gel Shift Assays
The following
double-stranded GME oligonucleotide, 5`-tcgaCTTCTGCGTCAGCGCCAGTATg-3`,
3`-GAAGACGCAGTCGCGGTCATAcagct-5` (capitalized letters correspond to the
rat TAT sequence from -3654 to -3634 bp from the start of
transcription; lower case letters are for added nucleotides to make SalI cohesive ends), was used for gel shift assays, after
filling in the single-stranded DNA with Klenow enzyme and labeling with
[P]dCTP at room temperature. Gel shift
experiments were performed as described (23) with some
modifications. In brief, nuclear extracts (3 µg) or cytosol
preparations (7 µg) were incubated with 20,000 cpm of the
P-end-labeled probe (0.6 fmol) in a total volume of 10
µl for 15 min at 0 °C with sheered, non-denatured herring sperm
DNA (0.15 µg) as a nonspecific competitor. After electrophoresis at
4 °C in a 5% non-denaturing polyacrylamide gel at 150 V in 0.4
TBE, the dried gels were autoradiographed for 12-24 h at
room temperature with Kodak X-Omat XAR-5 film or were exposed to the
phosphorimagizing screen for the Molecular Dynamics ImageQuant system
(Molecular Dynamics) for 16-72 h at room temperature. For
supershift experiments, the antibodies (0.8 µl) were preincubated
with the nuclear extracts or cytosol for 60 min at 0 °C before
adding spermidine, herring sperm DNA, and P-labeled GME.
The upper strand sequences of blunt-ended, double strand
oligonucleotides used for competition experiments were as follows: GME,
5`-CTTCTGCGTCAGCGCCAGTAT-3`; M1, 5`-TGCTGACGTCAGCGCCAGTAT-3`; M2,
5`-CTTCTGTATGAGCGCCAGTAT-3`; M3, 5`-CTTCTGCGTCAGTATGCGTAT-3`; M4,
5`-CTTCTGCGTCAGCGCCATGCG-3`; AP-1, 5`-TTCCGGCTGACTCATCAAGCG-3`; CRE,
5`-AGAGATTGCCTGACGTCAGAGAGCTAG-3` ( (23) and (24) and
references therein).
Immunodepletion
Nuclear extracts (6
µg) were incubated with antibodies (2 µl) in 20 µl of 10
mM HEPES (pH 7.9 at room temperature), 25 mM KCl, 5
mM MgCl, 0.1 mM EDTA, 0.25 mM dithiothreitol, 5% glycerol, 50 µMp-aminoethylbenzenesulfonyl fluoride for 1 h at 0 °C.
Protein A/G-agarose (Pierce) (2 µl of a 50% slurry in 25 mM Tris (pH 8.0), 0.5 mM EDTA, 1 mg/ml bovine serum albumin)
was added and incubated an additional 1-16 h at 0 °C. The
mixture was then centrifuged at 14,000 g for 2 min at
0 °C, and the supernatant (8.45 µl) was used for gel shift
assays.Fractionation Procedures for GMEB
Nuclear
extracts (50 µl) in the absence or presence of 1 or 2.7 M guanidine hydrochloride were loaded onto a Microcon 100 (molecular
cut-off, 100 kDa) (Amicon) and centrifuged at 2500 g for 20-25 min until 50% of the initial volume had passed
through the membrane. The retentates and pass-through fractions
containing guanidine hydrochloride were dialyzed against buffer 20T at
4 °C for 16 h and stored at -70 °C.
ranges, based on the migration of pre-stained
molecular weight markers (Life Technologies, Inc., Bio-Rad) in adjacent
lanes, were cut out of the gel and electroeluted in 1 SDS
running buffer (25 mM Tris, 0.19 M glycine, 0.1% SDS)
at 125 V in a model 1750 electroeluter (ISCO, Lincoln, NE) for 60 min
at room temperature. After elution, the protein was precipitated with 4
volumes of acetone (-60 °C), and the pellets were washed with
80% acetone, 20% buffer 20T (4 °C) as described (33) . The
pellets were redissolved in buffer 20T and reprecipitated with acetone
as before. After the second precipitation, the recovered samples were
denatured and renatured by redissolving in 60 µl of buffer 20T
containing 6 M guanidine-hydrochloride and dialyzing against
buffer 20T at 4 °C for 16 h. After dialysis, the renatured samples
were concentrated to about 20 µl with a Microcon 10 concentrator
(12,000 rpm for 15 min at 4 °C) (Amicon) and stored at -70
°C.Methylation Interference Assay
The
sequence of the double-stranded oligonucleotide used was as follows
(capitalized letters correspond to the rat TAT sequence from
-3654 to -3634 bp from the start of transcription):
5`-tcgaCTTCTGCGTCAGCGCCAGTATg-3`, 3`-gctGAAGACGCAGTCGCGGTCATAcg-5`. The
probe was filled-in by Klenow fragment with
[P]dCTP or [
P]dATP for
labeling specifically the upper or lower strand, respectively. The
labeled probes were partially methylated with dimethyl sulfate as
described by Maniatis et al.(34) . The binding
reaction and gel electrophoresis were performed as for the gel shift
assay, except that the binding reaction was scaled up 8-fold and wider
sample wells (5 cm width) were used. After electrophoresis, the gel was
electroblotted onto DEAE filters (NA45; Schleicher & Schuell) in
0.5
TBE buffer at 30 V at 4 °C for 16 h. The filter was
exposed to Kodak X-Omat XAR-5 film at 4 °C for 120 min. The
retarded and free bands were identified from the x-ray film and cut out
from the filter. The DNA was extracted from the excised filter in
elution buffer (1 M NaCl, 0.1 mM EDTA, 20 mM Tris (pH 8.0 at room temperature)) for 30 min at 60 °C,
precipitated by ethanol with 5 µg of native, sheered herring sperm
DNA, and subjected to piperidine treatments(35) . Equivalent
amounts of radioactivity obtained from the retarded and free gel shift
bands were loaded onto 15% sequencing gels. After electrophoresis, the
gel was dried and exposed to Kodak X-Omat XAR-5 film at -70
°C for 3-5 days.In Vitro Transcription and
Translation
Mouse CREB, mouse c-Jun, and rat c-Fos proteins
were expressed in vitro with TNT SP6 or TNT T7-coupled
reticulocyte lysate system (Promega) from pET-3/m
CREB (Dr. G.
Schütz), c-Jun (Dr. I. Verma), and pc-Fos (rat)-1
(Dr. T. Curran) plasmids, respectively.DNA Affinity
Chromatography
Oligonucleotides for the M3 sequence
(5`-tcgaCTTCTGCGTCAGTATGCGTATg-3`,
3`-GAAGACGCAGTCATACGCATAcagct-3`) or M2 sequence
(5`-tcgaCTTCTGTATGAGCGCCAGTATg-3`,
3`-GAAGACATACTCGCGGTCATAcagct-3`) (capitalized letters
correspond to the rat TAT sequence from -3654 to -3634 bp
from the start of transcription, bold letters indicate mutations of
native TAT sequence, and lower case letters are for added nucleotides
to make SalI cohesive ends) were synthesized and purified by
HPLC. Sequence-specific DNA affinity columns were prepared as described (36) except that 500 µg of oligonucleotides were coupled
to 1 ml (bed volume) of CNBr-activated Sepharose CL-4B (Pharmacia).
GMEB activity throughout the chromatography was monitored by gel shift
assays with P-labeled GME. Mono Q fractions containing
GMEB activity (42.5 ml) were pooled, diluted with an equal volume of
buffer TN to reduce the NaCl concentration to about 100 mM,
mixed with sheered herring sperm DNA (final concentration of 5
µg/ml) and spermidine (final concentration of 2 mM; U. S.
Biochemical Corp.), and incubated on ice for 10 min before being spun
at 12,000
g for 10 min at 4 °C. The supernatant
(20 ml) was applied with gravity onto the M2 DNA affinity column
(400-µl bed volume) equilibrated with buffer TN plus 100 mM NaCl. The column was then washed with 4 2 ml of buffer TN
containing 100 mM NaCl followed by 550 µl of buffer TN
with 200, 300, 400, or 800 mM NaCl in stepwise fashion. The
flow-through fraction that contained GMEB activity was loaded onto a
400-µl bed volume M3 DNA affinity column that was washed as for the
M2 DNA affinity column and eluted with buffer TN containing 200, 300,
400, 500, 600, or 800 mM NaCl. The fractions (300-500
mM NaCl) containing GMEB activity were pooled and diluted with
buffer TN to a final NaCl concentration of 100 mM and reloaded
on the M3 column. A total of three sequential M3 DNA affinity column
purifications were performed. The final M3 DNA affinity column had a
250-µl bed volume, and the GMEB activity was eluted in the
300-500 mM NaCl fraction. All samples were aliquoted,
quickly frozen in a dry ice-methanol bath, and stored at -70
°C.
GMEB Is Present and Active in Non-hepatic
Cells
The GME was initially defined in the context of the
cloned rat hepatoma tissue culture cells Fu5-5 and HTC. There,
the percentage of maximal agonist activity obtained from transiently
transfected GREtkCAT reporter constructs with either subsaturating
concentrations of agonists or saturating concentrations of
antiglucocorticoids was increased by the presence of the
GME(23) . As shown in Table 1with the agonist
dexamethasone and the antagonist dexamethasone 21-mesylate, GME
activity was not restricted to the originally investigated cloned
hepatoma cells. The GME caused increased percentages of agonist
activity in uncloned Fu5-5 cells as well as transformed mouse
fibroblasts (L cells) and human cervical adenocarcinoma cells (HeLa
cells) in a manner that required the crucial CGTC sequence of the GME,
which was not present in the inactive M2 mutation of the
GME(23) . Thus, the capacity to express GME activity, which
presumably reflects the existence of the requisite GMEB factor(s), is
not restricted to hepatic cells but crosses species lines. The
inactivity of the GME in rat adrenal pheochromocytoma cells (PC-12)
suggests, though, that there is some cellular selectivity for GME
action.
P GME
probe as described under ``Materials and Methods.'' Bands
were visualized by autoradiography (A and HTC cell data of B) or by phosphorimagizer. Unlabeled oligonucleotides were
added in 100-fold molar excess. The filledarrow indicates the position of the GMEB-containing band, the two
CREB-containing bands are just below, and the openarrow is at the position of nonspecifically bound
species.
Properties of GMEB
The GMEB-DNA complex
observed in gel shift assays migrated only slightly slower than several
other complexes (Fig. 1A). As expected, cytosol
solutions prepared by lysis of cells with hypotonic buffer or by the
freeze-thaw techniques used to obtain crude glucocorticoid receptors (31) contained very few DNA binding species. However, each
cytosol solution still appeared to include the same GME binding species
that was observed in nuclear extracts (Fig. 1B). This
cytosolic binding protein (Fig. 1B) was further
identified as the GMEB because the only DNAs that competitively
inhibited the formation of the gel-shifted complex were the known
biologically active oligonucleotides of GME, M1, M3, and
M4(23) . This procedure thus provided a simple method for
separating the GMEB from most of the other GME binding proteins. The
presence of the GMEB in PC-12 cells, where the GME is inactive (Table 1), suggests either that other factors besides the GMEB
and glucocorticoid receptors are required for activity or that the GMEB
is present in PC-12 cells in a biologically inactive form.P GME (data not
shown). Similarly, cytosolic preparations of receptors treated with
saturating concentrations of dexamethasone and then heated to activate
the complexes to the DNA binding form did not display an increased
amount of the GMEB complex in gel shift assays (data not shown).
Therefore, we conclude that this GMEB-containing band does not involve
any interaction of the glucocorticoid receptor with the GME.
P
oligonucleotide containing the consensus CRE (38) of the
somatostatin gene (SOM/CRE) (25, 26) differs from the
GME in 9 out of 19 nucleotides (see below; identical nucleotides are
underlined, and lower case letters indicate flanking DNA in the
reporter plasmids) and did not afford the slower migrating,
GMEB-containing band in gel shift assays (GME,
5`-tcgaCTTCTGCGTCAGCGCCAGTATtcga-3`; SOM/CRE,
5`-gatccCTCTCTGACGTCAGCCAAGGAgatc-3`). Also, non-labeled SOM/CRE
oligonucleotides only weakly competed for the formation of this band
with the
P GME oligonucleotide (data not shown).
GMEB Is a Multimeric Protein
Methylation
interference experiments revealed that all of the guanines in a 10-bp
region of the GME are important for binding in the gel shift assay (Fig. 2). An even larger region of 26 bp is protected from DNase
I digestion (data not shown). Under most circumstances, more than one
protein would be required to cover such a large stretch of DNA.
P GME probe and
fractionated on a 5% non-denaturing PAGE gel. The top and bottomstrands of free and GMEB-bound probe DNA were
processed for sequencing and autoradiographed as described under
``Materials and Methods.'' The DNA sequence of the top and bottomstrands is shown at the left and right, respectively, of the autoradiograph. B, bound probe; F, free probe; arrows mark
those guanosine residues that must remain unmethylated for complex
formation with GMEB to occur.
550
kDa; the sizes of the CREB-containing bands were about 310 and 360 kDa
(data not shown). A similar very large size of 600 kDa for the GMEB was
observed by gel shift assays of the peak binding activity after
fractionation by size exclusion chromatography on Superose 6 HR (Fig. 3, A and B) and Sepharose S-3000. To
determine whether this 550-600-kDa species was a monomeric or
oligomeric protein, we made use of the report that deoxycholate
dissociates protein-protein complexes, but not protein-DNA complexes,
in a manner that can be reversed by added Nonidet P-40
detergent(42, 43) . As shown in Fig. 3C, the appearance of the GMEB-containing band
(and the lower CREB-containing bands) was blocked by deoxycholate and
restored by added Nonidet P-40. However, these GMEB and CREB protein
complexes are relatively resistant to dissociation by salt. All three
bands were seen in gel shift assays of material retained by a Microcon
100 filter in the presence of 1 M guanidinium hydrochloride
while no band was observed in the flow through, which would contain
species
100 kDa (Fig. 3D). Raising the guanidinium
hydrochloride concentration to 2.7 M still did not allow any
GMEB to appear in the flow-through of the Microcon 100 filter, as
evidenced by the formation of the appropriate gel shift bands (data not
shown). Collectively, these data suggest that GMEB and the 42-kDa
CREB either can exist as a multimeric complex that resists dissociation
in salt or requires other proteins larger than 100 kDa to form the
observed DNA complexes on gels.
globulin (158 kDa); O, chicken
ovalbumin (44 kDa); M, equine myoglobulin (17 kDa); V, vitamin B
(1.35 kDa). B, gel shift
properties of size-fractionated HTC cytosol. Unfractionated cytosol (lanes1 and 24) and aliquots (3 µl) of
the indicated column fractions were assayed in the gel shift assay as
in Fig. 1and visualized by phosphorimagizer. The arrow marks the position of the GMEB-containing band. C,
effects of deoxycholate and Nonidet P-40 on the binding of GMEB and
CREB to GME. Fu5-5 nuclear extract was incubated with
P GME oligonucleotide in the presence of the indicated
percentage of deoxycholate ± 1% Nonidet P-40. Complexes were
separated on 5% non-denaturing polyacrylamide gel as described under
``Materials and Methods'' and visualized by autoradiography.
The filledarrow indicates the position of the
GMEB-containing band, the two CREB-containing bands are just below, and the openarrow is at the position
of nonspecifically bound species. D, size fractionation of
GMEB in 1 M guanidine hydrochloride. Fu5-5 nuclear
extracts ± 1 M guanidine hydrochloride were centrifuged
through a membrane (Microcon 100) whose molecular cutoff was 100 kDa.
After centrifugation, the filtrate (Filt.) and retentate (Ret.) fractions were dialyzed and analyzed individually or in
combination in gel shift assays with
P GME probe as
described under ``Materials and Methods.'' The filledarrow indicates the position of the GMEB-containing
band.
GMEB Is a Heterooligomer
Southwestern
blotting of proteins that had been separated on SDS-polyacrylamide gels
and then renatured prior to being probed with P GME
oligonucleotide was performed to determine the size of the monomeric
GMEB. No signal was seen with either crude or partially purified (by
FPLC on a Mono Q column) GMEB under conditions where CREB was readily
visualized at
42 kDa (data not shown). This implies that the GMEB
may be comprised of proteins of different molecular weights. Direct
evidence for this conclusion came from gel shift assays with Mono Q
purified material that was separated on, and then extracted from,
SDS-polyacrylamide gels. No one size of fractionated proteins yielded
the original band in gel shift assays, even though samples containing a
broad size range of proteins retained the ability to form the
gel-shifted band both after exposure to SDS (Fig. 4, lane2) and after extraction from an SDS gel (Fig. 4, lane3). However, when two fractions encompassing
species of 101-80 kDa and 80-62 kDa were mixed, a strong
gel shift band was obtained (Fig. 4, lanes5, 13, and 20). Each fraction by itself bound DNA weakly
and afforded different complexes. The reassociation of both components
occurred more efficiently when they were mixed before, as opposed to
after, renaturation (Fig. 4, lanes20versus22). When the components were mixed after
renaturation, increased incubation time favored complex formation (Fig. 4, lanes20-22).
P-GME probe. Lane1, mono
Q-purified cytosol; lane2, mono Q-purified HTC
cytosol mixed with the SDS loading buffer followed by precipitation,
denaturation, and renaturation; lane3, proteins
eluted from the entire molecular weight range of the SDS-PAGE gel; lanes7-10, 14-17, and 20, protein fractions were mixed before denaturation; lanes21 and 22, proteins were renatured
separately and then mixed for indicated time on ice just before the gel
shift assay. The position of the GMEB-containing band is indicated by
the arrow.
Purification and Peptide Sequencing of GMEB1 and
-2
The above data indicated that the GMEB is composed of
two proteins. To confirm this conclusion, the GMEBs were purified from
about 130 g of HTC cells (Table 2). Each stage of the
purification scheme was monitored for its ability to give the
appropriate gel-shifted band. Thus, the GMEBs in crude cytosol devoid
of CREB were fractionated first on a preparative Mono Q HR 10/10 column
and then on a column containing tandem repeats (>10) of the
biologically inactive GME mutant oligonucleotide M2 (23) to
remove nonspecific binding proteins. The flow-through from this column
was loaded onto a column with tandem repeats (>10) of the GME mutant
oligonucleotide M3 to bind GMEB. The mutant oligonucleotide M3 was used
because M3 had a higher apparent affinity for GMEB than did the native
GME, as determined from competitive gel shift experiments (Fig. 1B) and affinity chromatography with multiple
repeats of DNA oligonucleotides (data not shown). After three rounds of
DNA affinity chromatography, the GMEBs had been purified about
5000-fold (Table 2). As with crude GMEB, the gel-shifted band
obtained with purified GMEB was inhibited only by added biologically
active oligonucleotides (i.e. GME, M1, M3, M4 but not M2,
AP-1, or CRE) ( (23) and data not shown). Analysis of this
purified material on denaturing SDS-polyacrylamide gels followed by
staining with copper, Coomassie Blue, or silver (Fig. 5A) revealed three major species. However, only
the combination of the highest and lowest molecular mass proteins at 88
and 67 kDa could reconstitute the GMEB band in gel shift assays after
the elution of each band from SDS-polyacrylamide gels (Fig. 5B). Confirmation that this was the authentic
gel-shifted band came from the ability of only the biologically active
GME and not the mutant M2 oligonucleotide (23) to competitively
inhibit the formation of this band (Fig. 5C).
310-360
kDa in gel shift assays and 400 kDa on size exclusion columns (Fig. 3A and data not shown). Deoxycholate blocked the
formation of the CREB-containing bands in gel shift assays, just as was
observed for GMEB (Fig. 3B). Given the fact that CREB
is relatively small (
42 kDa), it would seem that the CREB
complexes must contain either multiple copies of CREB or other
proteins. CREB will bind to the GME-containing oligonucleotide in
Southwestern blots (see ``Results'') and will afford
gel-shifted bands with a CRE-containing oligonucleotide (26) so
that homooligomeric complexes of CREB may form. However, the
gel-shifted band that the somatostatin CRE formed with purified CREB
exhibited a faster migration than that with crude nuclear
extracts(26) . Thus, the CREB-containing complex from nuclear
extracts probably is not the same as that formed just from CREB and
would contain some other protein(s). Further experiments are required
to determine whether the suspected additional proteins are CBP (52) , other members of the CREB/CREM/ATF superfamily that can
heterodimerize with CREB(39) , or even GMEB1 or -2.
)
We thank Mari Oshima and Yoko Hirata (NICHD, National
Institutes of Health (NIH)) for technical suggestions with the FPLC,
David Jackson (NIDDK, NIH) for helpful comments, Mark Reitman (NIDDK,
NIH) for critical review of the paper, Tom Curran (Roche Institute of
Molecular Biology) for pc-Fos-1, Richard H. Goodman (Vollum Institute)
for anti-CBP, Joel Habener (Massachusetts General Hospital) for
anti-CREM, Gordon Hager (NCI, NIH) for HeLa cells, Michael J. Iadarola
(NIDR, NIH) for PC-12 cells, Günther
Schütz (Heidelberg, Germany) for L cells and
anti-CREB and pET-3/mCREB antibodies, and Inder Verma (Salk
Institute) for a c-Jun plasmid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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