(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 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.
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, junfos 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 jun
jun 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) .
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) ()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.
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 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.
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 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.
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 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.
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 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).
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 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.
When
compared to molecular weight markers in the gel shift
assay(41) , the size of the GMEB was calculated to be 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.
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 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.
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 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.
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 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.
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.