Cloning and Characterization of a Novel Binding Factor (GMEB-2) of the Glucocorticoid Modulatory Element*

The 21-base pair glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene is the onlycis-acting element known to modulate the transcriptional activity of receptors bound to glucocorticoid response elements. Specifically, the GME increases the activity of complexes bound both by physiological concentrations of glucocorticoids, due to a left shift in the dose-response curve, and by saturating concentrations of anti-glucocorticoids. For this reason, the nuclear protein(s) that has been demonstrated to bind to the GME is of major interest as a possible transcription factor with hitherto undescribed properties. Subsequent studies indicated that not one but two proteins of 88 and 67 kDa (= GMEB-1 and -2, respectively) formed a heteromeric complex with double-stranded GME oligonucleotides in gel shift assays and participated in the expression of GME activity (Oshima, H., Szapary, D., and Simons, S. S., Jr. (1995) J. Biol. Chem.270, 21893–21910). Here, we report the use of polymerase chain reaction of degenerate oligonucleotides and 5′- and 3′-rapid amplification of cDNA ends to clone two cDNAs of 2.0 and 1.9 kilobase pairs that probably result from alternative splicing. Both cDNAs encoded open reading frames containing all four previously sequenced peptides. The longer 2.0-kilobase pair cDNA encoded an open reading frame for an acidic, 529-amino acid protein and afforded a major 67-kDa and a minor 58-kDa protein after in vitrotranscription/translation. Both proteins were recognized by a mono-epitopic antibody raised against a peptide of GMEB-2. The in vitro translated protein bound to GME DNA in gel shift assays. However, the binding to GME DNA increased markedly after mixing with authentic GMEB-1 to give a gel-shifted complex that was similar to that derived from HTC cell cytosol. GMEB-2 shares a unique domain (KDWKR) with proteins derived from diverse organisms as follows:Drosophila (DEAF-I), rat (Suppressin), andCaenorhabditis elegans (three unknown open reading frames). Collectively, these data suggest that the 67-kDa GMEB-2 not only is an important factor for the modulation of glucocorticoid receptor bound to glucocorticoid response elements but also may belong to a novel family of transcription factors.

Glucocorticoid induction of the rat liver tyrosine aminotrans-ferase (TAT) 1 gene has been a useful model for steroid-induced gene expression over the years for several reasons. It is a biologically relevant response. It was one of the first systems to show a correlation between the steroid binding of receptors and whole cell induction of a protein (1). TAT induction is a primary effect of the receptor-steroid complex in that the induction of enzyme requires mRNA synthesis and is down-regulated by the removal of steroid (2). Finally, the TAT gene was shown to contain specific DNA sequences, called glucocorticoid response elements (GREs), which are steroid-inducible enhancers. GREs are obligatory components in the formation of the ternary DNA-receptor-steroid complexes that, in turn, are believed to interact with the transcriptional machinery to increase the rate of TAT gene transcription (3,4).
Over the last few years, however, the picture has become much more complicated. Whereas the TAT GRE is still a "simple GRE" in that receptor-steroid complex binding to the isolated GRE is capable of inducing transcription without the help of other cis-acting elements or transcription factors (5), differences with other GREs have emerged, and many other TAT gene elements have been found to participate in the induction process. Thus, the TAT GREs are much further upstream from the start of transcription than for most other steroid hormone responsive elements (3,6), and the transcription factor binding to the second GRE appears to be HNF-3 as opposed to the glucocorticoid receptor (7,8). The region around the GREs of Ϫ2.6 to Ϫ2.3 kb has been found to be necessary for tissuespecific induction and has been called a glucocorticoid-responsive unit (6). In addition to the GRE, the glucocorticoid-responsive unit contains binding sites for the family of C/EBP proteins (9,10) and an Ets-related factor (11) and harbors an element for tissue-selective gene expression (12). Tissue-specific elements are located at Ϫ3.6 kb, which binds CREB (7,13,14), at Ϫ5.5 kb (6,9), at Ϫ11 kb (7,15), and possibly at Ϫ350 to ϩ1 bp (16,17). A tissue nonspecific element that affects the level of induced activity has been localized at Ϫ3.0 to Ϫ2.6 kb (12).
All of the above TAT gene elements have been defined in the context of how, and when, glucocorticoid receptor activates or represses gene transcription in the presence of saturating concentrations of steroid. Arguably even more important for the functioning of intact cells are the responses with subsaturating concentrations of glucocorticoids because the intact cell or organism rarely is exposed to micromolar concentrations of glucocorticoid. Whereas those mechanisms regulating the level of response to saturating concentrations of agonist steroids should persist at subsaturating concentrations, thereby leaving the EC 50 of the dose-response curve unaffected, the converse is not necessarily true. In fact, we have reported that the doseresponse curve for TAT gene induction in Fu5-5 cells is leftshifted (to give a lower EC 50 ) relative to the same gene in HTC cells (18 -21) or to a different gene in the same Fu5-5 cells (19,(21)(22)(23)(24). Thus, physiological concentrations of glucocorticoid elicited a greater percentage of the maximal induction of the TAT gene in Fu5-5 cells than of any other genes examined. Although the fold change in the percent of maximal activity seen with subsaturating concentrations of glucocorticoid may seem small (e.g. 60% for the TAT gene versus 30% for other glucocorticoid-regulated genes), it is more than sufficient to permit differential control of gene expression by the same subsaturating concentration of glucocorticoid that a cell would see during development, differentiation, and homeostasis.
Another relatively unexplored area of steroid hormone action concerns anti-steroids, which block the action of agonists and thus have clinical utility. In parallel with the above studies of TAT dose-response curves, we observed that the amount of agonist activity displayed by anti-glucocorticoids such as dexamethasone 21-mesylate was much greater for TAT gene induction in Fu5-5 cells than in HTC cells (19,21,22).
Both the left shift in the dose-response curve (18,19,(22)(23)(24)(25) and the increased agonist activity of anti-steroids (19,23,24,26) could be reproduced completely in the context of transiently transfected cells by a synthetic reporter gene (GREtkCAT) containing a 21-bp sequence (located at Ϫ3.6 kb of the TAT gene) that was positioned 5Ј of the GRE. Furthermore, synthetic reporter constructs containing the 21-bp TAT sequence mimicked the endogenous TAT gene in the two other properties that have been examined: control of expression at the level of correctly initiated transcripts (26,27) and response to changes in cell density (26 -28). Thus, the properties of the endogenous TAT gene were faithfully reproduced in synthetic reporter constructs containing a GRE and the 21-bp element. To reflect its activity, we have called this 21-bp sequence a glucocorticoid modulatory element, or GME (25)(26)(27)(28)(29).
The characteristics of this GME sequence appear to be unique among those elements previously documented to participate in the transcriptional activation by steroid receptors. A cis-acting element of the distal promoter of the rat progesterone receptor gene has been described to cause just the opposite effects of the GME, i.e. a right shift in the estrogen receptor induction of the progesterone receptor gene and decreased amounts of agonist activity with selected anti-estrogens (30). Thus, it is almost certain that different factors will be found to be responsible for the opposite effects of this progesterone receptor element and the GME.
Two proteins of 88 and 67 kDa that bind to the GME have been purified by oligo-affinity chromatography. The combination of these two proteins was sufficient to give a complex with the GME oligonucleotide in gel shift assays that was indistinguishable from that of the endogenous, cellular proteins (31). In both cases, biologically active, but not mutant inactive, GME oligonucleotide was able to inhibit the formation of a protein-DNA complex with 32 P-GME oligonucleotide (26,31). For these reasons, we have called the 88-and 67-kDa proteins glucocorticoid modulatory element-binding proteins (GMEB) 1 and 2, respectively. By several biochemical criteria, these GMEBs differed from many of the previously described CREB/CREM/ ATF family members, some of which also could bind to the the same GME DNA sequence (7,13,14,31). Furthermore, tryptic peptide fragments of the two GMEBs were unlike anything on GenBank TM , which additionally suggested that a novel process was being examined. Nevertheless, as most transcription factors are members of larger families with similar activities, it is probable that other proteins will be found that will be related to the GMEBs, either in structure or in function. In this report, we describe the cloning and characterization of GMEB-2 (the 67-kDa protein). Preliminary experiments indicate that other related proteins may exist which could be members of this putative new family of transcription factors. Cell Culture and Preparation of Cytosol-Rat hepatoma tissue culture cells (clone 27 of Fu5-5 cells) were grown at 37°C in a humidified incubator (5% CO 2 ) in Richter's improved minimum essential medium with zinc and supplemented with 0.03% glutamine and 10% heatinactivated fetal calf serum (Biofluids, Rockville, MD). Cytosols were prepared as usual by freeze-thaw lysis and ultracentrifugation (32).
Antibody-A polyclonal rabbit antibody against the GMEB-2 sequence of ISPKEFVHLAGKSTLKDWKRAIR was prepared and affinity purified against the immunizing peptide by Zymed Laboratories (San Francisco, CA).
Cloning of GMEB-2 cDNAs-The rat GMEB-2 was cloned by preparing degenerate oligonucleotides of 20 bp in length for the three published tryptic fragments of GMEB-2 (31). For those amino acids such as leucine and serine, for each of which there are six possible codons, the degeneracy of the synthetic oligonucleotides was reduced by using only those codons that are most commonly used in rat. Each degenerate oligonucleotide contained the same last two 3Ј nucleotides to act as a "clamp" for hybridization, thereby increasing the frequency of productive elongation, with the cDNAs that were prepared by reverse transcription from poly(A)-enriched rat liver Fu5-5 cell, Clone 27, mRNA. Poly(A) ϩ RNA was isolated from total Fu5-5 RNA using either an Oligotex mRNA kit (Qiagen, Chatsworth, CA; for degenerate primer cloning) or two passes over oligo(dT)-cellulose spin columns (5 Prime/3 Prime; for RACE). cDNA was prepared from the poly(A) ϩ RNA using either avian myeloblastosis virus polymerase (for degenerate primer cloning) or Superscript or Superscript II reverse transcriptase (Life Technologies) with an oligo(dT) primer (for degenerate primer cloning), a GMEB2-specific primer (for 5Ј-RACE), or an anchored oligo(dT) primer (for 3Ј-RACE; used the CLONTECH (Palo Alto, CA) Amplifinder amplification primer sequence linked 5Ј to (dT) 17 ). After using all possible combinations of the primers for the three original tryptic fragments, and a second round of PCR using a second primer that was 3Ј of the first primer, a 278-bp cDNA was obtained which contained the appropriate portions of the peptides IMDSGELDFYQHDK and AGLL-DEVIQEFQQELEETMK at the 5Ј and 3Ј ends, respectively. This 278-bp cDNA was then used with the method of 5Ј-and 3Ј-rapid amplification of cDNA ends (RACE). Ligation-mediated 5Ј-RACE to generate a unique 5Ј sequence (0.8 kb) was performed essentially according to the manufacturer's recommendations using the CLONTECH 5Ј-RACE kit, cDNA prepared from poly(A) ϩ RNA (see above), proofreading DeepVent DNA polymerase (New England Biolabs), and two nested GMEB2-specific primers. Two 1.4-kb 3Ј sequences, which overlapped the 5Ј 0.8-kb sequence, were obtained using two nested GMEB2 genespecific primers and a primer complementary to the anchor portion of the oligo(dT) primer used for cDNA synthesis (see above) with the native, proofreading form of Vent DNA polymerase (New England Biolabs). The blunt-ended products of Vent and DeepVent synthesis were A-tailed with Taq DNA polymerase according the manufacturer's recommendations (New England Biolabs) and cloned onto the T/A cloning vector, pCR2.0 (Invitrogen, Carlsbad, CA). Three 5Ј-and 3Ј-RACE clones were completely sequenced on both strands. The full-length cDNAs were obtained by joining the 5Ј and 3Ј fragments together after ApaI cleavage in the overlapping region to give 2.0-and 1.9-kb clones.
To ensure that there were no PCR errors in this composite GMEB-2 sequence, an independent recloning of GMEB-2 was performed. A fulllength 2.0-kb cDNA was recloned directly from Fu5-5 cell (clone 27) mRNA by standard reverse transcription and PCR amplification methods using oligonucleotides corresponding to 5Ј-and 3Ј-untranslated sequences of the composite GMEB-2. The 2.0-kb GMEB-2 cDNA was ligated into pCR2.1 (Invitrogen). Dideoxy sequencing (Sequenase 2.0 from U. S. Biochemical Corp.) of this direct GMEB-2 cDNA revealed no differences within the GMEB-2 coding region of the original GMEB-2 clone.
Isolation of Authentic GMEB-2-HTC cytosol was separated on 8% SDS-polyacrylamide gels, and the molecular mass range of 67 kDa, based on the migration of prestained molecular weight markers in adjacent lanes, was cut out of the gel and electroeluted in 1ϫ 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 90 min at r.t. After elution, the protein was precipitated with 4 volumes of acetone (Ϫ60°C). The pellets were redissolved in LSB buffer and reprecipitated with acetone as before. After the second precipitation, the recovered samples were denatured and renatured by redissolving in 100 l of LSB buffer containing 6 M guanidinium hydrochloride and dialyzing against LSB buffer for 16 h. After dialysis, the renatured samples were concentrated to about 40 l with a Microcon 10 concentrator and stored at Ϫ70°C.
In Vitro Transcription and Translation-GMEB-2 protein was expressed in vitro from the clone in pCR2.1 with TNT T7-coupled reticulocyte lysate system (Promega) following the manufacturer's suggestions.
Gel Shift Assays and Quantitation of Bands-The oligonucleotides 5Ј-CTTCTGTATGAGCGCCAGTAT-3Ј and 3Ј-GAAGACATACTCGCG-GTCATA-5Ј, which correspond to the GME of the rat TAT sequence at Ϫ3654 to Ϫ3634 bp from the start of transcription, were annealed and 32 P-end-labeled by Lofstrand Laboratories (Gaithersburg, MD). Gel shift experiments were performed as described (26). Briefly, cytosol (0.5-2.0 l) and the in vitro transcription/translation product (0.5-4.0 l) were incubated with 20,000 cpm of the 32 P-end-labeled GME (0.6 fmol) in a total volume of 20 l for 20 min with sheared, non-denatured herring sperm DNA (0.3 g) as a nonspecific competitor. For immunoinhibition experiments, the antibody was added to the 20-l reaction after the initial 20 min at 0°C, and the incubation was continued for an additional 15 min at r.t. After electrophoresis in a 5% non-denaturing polyacrylamide gel at 150 V in 0.4ϫ TBE, the dried gels were autoradiographed for 12-24 h at r.t. with Kodak X-Omat XAR-5 film. Alternatively, the gels were exposed to the phosphorimaging screen for the Molecular Dynamics Image-Quant system for 16 -48 h at r.t. The amount of each specific band was calculated as the intensity of that band (calculated by the Molecular Dynamics software) minus the constant background value of the same area from an unrelated region of the gel.
SDS-Polyacrylamide Gels and Western Blotting-Samples diluted 1:2 in 2ϫ SDS buffer were analyzed on constant percentage acrylamide gels (between 7 and 14% with a 1:37.5 ratio of bisacrylamide to acrylamide) run in a water-cooled (15°C) Protean II slab gel apparatus (Bio-Rad) at 35 mA/gel. Gels were fixed, stained, dried without fluorescence additives, marked at the positions of the molecular weight markers with a fluorescent paint, and autoradiographed for 10 -24 h as described (33). The gels were equilibrated in transfer buffer for 2 min at r.t. prior to electrophoretic transfer of proteins to nitrocellulose membranes in a Bio-Rad Transblot Apparatus (100 mA overnight followed by 250 mA for 2 h). The nitrocellulose was stained in Ponceau S (0.02% Ponceau S and 0.04% glacial acetic acid in water) to localize molecular weight markers, incubated with 10% Carnation nonfat dry milk in TBS for 45 min, and washed three times with TBS containing 0.1% Tween (0.1 TTBS) for 5 min. Primary antibody was diluted in 0.1 TTBS (1:1000) and added to the nitrocellulose for 2 h at r.t. Biotinylated anti-rabbit secondary antibody and ABC reagents (each diluted 1:1000; Vector Laboratories, Burlingame, CA) were each added for sequential 30-min incubations at r.t. After the incubation periods with primary antibody, secondary antibody, and ABC reagents, the nitrocellulose was washed three times for 5 min each with 0.1 TTBS, and an additional three washes with TBS containing 0.3% Tween immediately after incubation with the ABC reagents. Detection of signal was performed by enhanced chemiluminescence using the recommended protocol of the supplier (Amersham Pharmacia Biotech). The positions of the molecular weight markers (Amersham Pharmacia Biotech) were indicated by overlaying with a fluorescent paint marker.
Statistics-Analyses were performed with the unpaired two-tailed Student's t test using the program InStat 2.03 for Macintosh (GraphPad Software, Inc., San Diego, CA).

Cloning of GMEB-2-
The rat GMEB-2 was cloned using the approach of PCR, with the primers being the degenerate oligonucleotides derived from the three tryptic fragments of GMEB-2 (31). After using all possible combinations of the three tryptic fragment primers, and a second round of PCR using internal nested primers, a 278-bp cDNA was obtained. 5Ј-and 3Ј-RACE were used to generate a unique 5Ј sequence (0.8 kb) with minor differences upstream of the open reading frame (see below), which overlapped with two 1.4-kb 3Ј sequences containing more major differences at the 3Ј ends (Fig. 1). Fusions of the two sets of fragments yielded two cDNA clones of 2.0 and 1.9 kb. Both clones encoded open reading frames (529 and 485 for the 2.0-and 1.9-kb clones, respectively) bounded by in frame stop codons and contained the three sequenced GMEB-2 peptides (31) (Fig. 2). These results argue that both clones represent full-length clones and that the smaller 1.9-kb clone originated from alternative splicing of the precursor of the longer 2.0-kb mRNA transcript. A fourth tryptic peptide of the original 67-kDa protein was sequenced (Keck Foundation, Yale University) in hopes of obtaining a fragment that was unique to For this reason, the bulk of the intervening sequences have been omitted. The divergence after position 1520 is not due simply to inserts, deletions, or a few mutations but corresponds to an entirely different sequence.
one of the two 3Ј sequences, thereby permitting a direct identification of the cDNA for the isolated protein. Unfortunately, the fourth peptide (XXVLLNNIVONFGMLDLVK) was also common to both 3Ј clones, and no other tryptic fragment of the GMEB-2 protein (31) was suitable for further sequencing.
To ensure that there were no PCR errors in the originally obtained GMEB-2 sequence, an independent recloning of GMEB-2 was carried out. Based on the known 5Ј-and 3Јnontranslated sequences of GMEB-2, the full-length 2.0-kb cDNA was recloned directly from Fu5-5 clone 27 cellular mRNA by the reverse transcription and PCR amplification method. No sequence disagreements within the GMEB-2 coding region were observed between the above two independent 2.0-kb GMEB-2 clones as determined by DNA sequencing with Sequenase 2.0. Thus, this appears to be the correct sequence for the rat protein. This protein is predicted to be an acidic protein with a pI of 5.0. This protein appears to be a novel protein. Routine BLAST searches of GenBank TM have yet to reveal any other protein that is identical or even homologous to that encoded by either clone (last search was 2/11/98).

Biochemical Properties of GMEB-2-
The predicted molecular weight of both suspected GMEB-2 clones was much less than that expected. The 2.0-kb cDNA encoded protein has a calculated molecular mass of 56,535 Da versus the expected 67 kDa, whereas the calculated size for the 1.9-kb cDNA product is 52,174 Da. However, cell-free translation with [ 35 S]methionine of the 2.0-kb clone yielded a major product, which migrated with the expected 67-kDa molecular mass on SDS gels (Fig. 3), along with a smaller product that could result from a downstream start of translation at Met-52 (Fig. 2). In contrast, cell-free translation of a luciferase cDNA clone gave neither of these species (data not shown). Thus, the 56.5-kDa protein encoded by the 2.0-kb cDNA migrated on SDS gels much slower than would be expected.
Western blotting with an antibody to an epitope of both cDNA clones revealed a 67-kDa protein both in the cell-free translation reaction with the appropriate cDNA and in HTC cell cytosols (Fig. 4). The inability to detect the same proteins in the presence of excess immunogenic peptide indicated that the 67-kDa species (indicated by arrows in Fig. 4) is the only species that is specifically recognized by this antibody. It should be noted that this antibody readily recognized the denatured GMEB-2 on Western blots but was unable to immunoprecipitate GMEB-2 in solution, whether present as just GMEB-2 from the in vitro translation reactions or as the GMEB-1⅐-2 complex from HTC cell cytosol (data not shown). This suggests that the immunogenic region of GMEB-2 is occluded in solution, possibly due to homo-and hetero-oligomerization.
Gel shift assays were performed to confirm the identity of the putative rat GMEB-2 cDNA clone. Two previously documented properties (31) were examined as follows: the ability to bind to the GME oligonucleotide 1) as a homo-oligomeric complex and 2) synergistically with the 88-kDa GMEB-1 to yield a slightly larger heteromeric complex. In gel shift assays with GME oligonucleotides, unprogrammed reticulocyte lysate did afford a weak gel-shifted band, possibly due to low concentrations of rabbit GMEB-2 in the lysate. However, programmed lysate containing the GMEB-2 cDNA gave much more of a complex with the same mobility,which, as previously reported (31), migrated slightly faster than the complex with GMEB-1 and -2 ( Fig. 5A). This binding was competed by excess GME oligonucleotide but not by the biologically inactive oligonucleotide M2 (26), thus showing that the binding of the in vitro translated GMEB-2 is sequence-specific. 2 Authentic GMEB-1 and -2 were then isolated from HTC cell cytosol. As shown in Fig. 5B, the gel-shifted complex with recombinant GMEB-2 migrated slightly faster than that of both the endogenous GMEB-1⅐-2 complex from HTC cell cytosol (lanes 6 versus 7) and the reconstituted complex with recombinant GMEB-2 and gel-purified GMEB-1 (lanes 6 versus 5). Furthermore, the combination of recombinant GMEB-2 and authentic GMEB-1 synergized to give more gel-shifted complex than the sum of the two individual components (lanes 5 versus  2 and 6).
Finally, anti-GMEB-2 antibody could reduce the amount of gel-shifted complex formed between the GME oligonucleotide and GMEB-2 (compare lane 5 versus lane 4 in Fig. 6, GMEB-2 complex is indicated by the arrow). However, a 15-min incubation at r.t. was required to see the immuno-disruption of the GME⅐GMEB-2 complexes, consistent with the inability of anti-GMEB-2 antibody to immunoprecipitate GMEB-2 from solution at 0°C. It is of interest that the same antibody treatment 2 S. Kaul and S. S. Simons, manuscript in preparation.

FIG. 4. Detection of authentic and in vitro translated GMEB-2 by Western blotting.
Samples of HTC cell cytosol, in vitro translated GMEB-2 (programed lysate), or reticulocyte lysate that had been incubated with luciferase cDNA as carrier DNA (unprogramed lysate) were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose for Western blotting with affinity purified anti-GMEB-2 antibody in the absence (w/o Peptide) or presence (Peptide) of antigenic peptide (4 g/ml). The arrow indicates the position of GMEB-2. The ECL background in the presence of the antigenic peptide was very high, presumably due to a high binding affinity of the peptide to the nitrocellulose filter. In order to block the nonspecific binding of peptide to filter, 10% non-fat dried milk (Carnation) was included in all solutions from the addition of peptide to the addition of secondary antibody.
FIG. 5. Binding of in vitro translated GMEB-2 to GME oligonucleotide in gel shift assay. 32 P-GME oligonucleotide was incubated with HTC cell cytosol, unprogramed lysate, or various combinations of programed lysate or gel purified GMEB-1 or -2 from HTC cell cytosol. A, comparison of complex formation with GME of in vitro translated GMEB-2 (3), unprogramed lysate, and HTC cell cytosol containing GMEB-1 and 2. B, synergistic binding of authentic, or in vitro translated, GMEB-2 and authentic GMEB-1 to GME oligonucleotide. Authentic GMEB-1 and -2 were separated on SDS-polyacrylamide gels, extracted, and individually renatured overnight as described (31). The various combinations of proteins were incubated with labeled DNA overnight and then analyzed as described under "Experimental Procedures." The position of the authentic complex of GMEB-1 and -2 with 32 P-GME oligonucleotide is indicated by the arrow. at r.t. was unable to reduce the amount of complex formed with the GMEB-1/-2 heteromer (lanes 3 versus 2), perhaps indicating a tighter association of the heteromeric complex, which is reflected in the approximately equal amount of heteromeric complex that was formed from HTC cell cytosols and in vitro translated GMEB-2 (lanes 2 and 4 of Fig. 6) even though HTC cell cytosol contains less GMEB-2 (see Fig. 4). In three separate experiments, the immuno-depletion of the GMEB-2 complexes (final ϭ 65 Ϯ 10%, S.D.) was significantly greater than that of the GMEB-1⅐-2 complexes from HTC cells (final ϭ 108 Ϯ 7%, S.D., p Ͻ 0.0013). Therefore, by several criteria, the 2.0-kb clone that we have isolated encodes a novel protein that corresponds to the 67-kDa GMEB-2.
It should be noted that preimmune serum had no effect on the level of complex containing GMEB-1 and -2 (lanes 2 versus 3). However, both preimmune and immune sera contained an uncharacterized species that afforded a much more slowly migrating complex (lanes 2 versus 3 and lanes 4 versus 5). The lower levels of this unknown species in the immune serum was probably a result of the partial purification of the anti-GMEB antibody. DISCUSSION We had previously reported that the 67-kDa GMEB-2 is part of a heteromeric complex of two new proteins that bind to a cis-acting GME element of the TAT gene (31) in a manner that appears to modulate the activity of glucocorticoid receptors bound to a GRE (23)(24)(25)(26)29). In view of the unusual properties of the GME, which causes a left shift in the dose-response curve of GR-agonist complexes and increased amounts of agonist activity for GR-antagonist complexes, the identification of the two proteins binding to the GME was of considerable interest. We now report the cloning and characterization of the GMEB-2.
PCR from degenerate oligonucleotides, followed by 5Ј-and 3Ј-RACE, yielded a 2.0-kb cDNA with an open reading frame containing all four sequenced peptides that were isolated from tryptic digestion of the purified protein. This open reading frame was bounded by in frame stop codons. Therefore, we are confident that we have isolated the complete gene. The calculated molecular mass of 56.5 kDa for GMEB-2 was much less than that expected from the migration of the purified protein on SDS gels (31). However, the protein that was obtained from in vitro translation of the cDNA clone migrated as a 67-kDa protein on SDS gels. Furthermore, Western blotting with an antibody raised against a non-sequenced peptide of the GMEB-2 indicated that the synthetic protein co-migrated with a 67-kDa protein in HTC cell cytosol, which is known to contain GMEB-2 (31). This Western blotting of endogenous and in vitro translated GMEB-2 was selectively blocked by the presence of excess antigenic peptide. Finally, the recombinant GMEB-2 protein formed a sequence-specific gel-shifted complex with a GME oligonucleotide that could be inhibited by anti-GMEB-2 antibody and displayed the same gel shift properties as did authentic GMEB-2. Thus, we conclude that the cDNA that we have cloned does encode the rat GMEB-2 protein.
The difference between predicted size of GMEB-2 and that observed on SDS gels was unusually large. However, the discrepancy does not appear to result from posttranslational modifications as the DNA binding and oligomerization of material prepared by in vitro translation are the same as for the GMEB-2 from cells. Although further studies are required to confirm this, we suspect that the aberrant migration on SDS gels is due to the presence of some sequence, just as has been identified for GR, which migrates as a protein that is about 10 kDa larger than its predicted size (34).
A demonstration of the biological activity, and relevance, of GMEB-2 awaits the cloning and characterization of the heterooligomeric partner, GMEB-1 (31). However, GMEB-2 binding to the GME oligonucleotide is prevented only by biologically active mutant oligonucleotides (26), and GMEB-2 does have some intrinsic transcriptional activity in mammalian cells. 2 The entire genome of several prokaryotic organisms has recently been cloned (Ref. 35 and references therein). The fact that none of these genomes contain sequences that are highly homologous to GMEB-2, as determined by a tBLASTN search of GenBank TM (36), suggests that GMEB-2 is an evolutionarily recent protein. This conclusion is in keeping with the apparent role of GMEB-2 in modulating the activity of glucocorticoid receptor-regulated gene expression (18 -22, 25-29).
Many transcription factors, such as the steroid receptors (37), NF-B/Rel (38), and Jun/Fos/CREB (39), are members of a larger superfamily of related proteins. We were therefore surprised to find that there were no large regions of GMEB-2 that were homologous to anything in GenBank TM . A family of proteins that interacts with steroid receptors has recently been found to be composed of SRC-1, TIF2, pCIP, ACTR, RAC3, and AIB1 (40), each of which contains a variety of shared motifs such as basic helix-loop-helix/PAS, serine/threonine-rich, glu-  6. Inhibition of GMEB-2 binding to GME oligonucleotide by anti-GMEB-2 antibody. 32 P-GME oligonucleotide was incubated with GMEB-1/-2 from HTC cell cytosol, followed by buffer, preimmune serum, or non-purified anti-GMEB antibody. Similarly, 32 P-GME oligonucleotide was incubated with in vitro translated GMEB-2, followed by preimmune serum or non-purified anti-GMEB antibody. The migration of the GMEB-2 complex is indicated by the arrow.
tamine-rich, and CBP-interacting domains (41)(42)(43). These proteins also interact with the receptors via domains that contain the small sequences of LXXLL (40,41,44). GMEB-2 does contain both serine/threonine-rich (32 and 26% in sequences 171-232 and 336 -516, respectively) and glutamine-rich (17% among amino acids 250 -321) domains, but their functional significance is not yet known. No basic helix-loop-helix/PAS, CBP-interacting, or receptor-interacting domain with homology to those of SRC-1, TIF2, pCIP, ACTR, RAC3, and AIB1 were found (data not shown). However, one local homology was noted with another selection of proteins. GMEB-2 contains a KDWKR sequence which has recently been reported in the DEAF-1 protein from Drosophila (45), rat Suppressin (46,47), three human EST clones, and a Caenorhabditis elegans cosmid. We have found essentially the same sequence in rat and human. 3 Suppressin, DEAF-1, and three other C. elegans cosmids (Fig. 7). Both Suppressin and DEAF-1 are transcription factors with interesting properties. Suppressin is a 63-kDa protein that has all the characteristics of a global negative regulator of cell proliferation and especially the immune system. Suppressin arrests lymphocytes in the G 0 /G 1 phase of the cell cycle after reduction of their RNA, protein, and DNA synthesis, suggesting that Suppressin inhibits the processes required for G 0 transition to G 1 . DEAF-1, an 85-kDa protein, binds to a specific DNA region of the 120-bp homeotic response element that is regulated specifically by the Deformed gene product in Drosophila embryos. DEAF-1 and its element are required for the functional activity of the 120-bp Deformed response element and thus is functionally a cofactor. Among these known and putative proteins, a consensus sequence of (S/T)P-(E/ Q)F--(K/R)-KDWK-I(R/K) has emerged that is slightly different than that noted by others (45). These data suggest that the novel 529-amino acid protein GMEB-2 is a potentially novel transcription factor which might belong to a new transcription factor family that includes DEAF-1 and Suppressin. It is interesting to note that one tryptic peptide of GMEB-1, FVHLAGK (31), is identical to the sequence of GMEB-2 within this consensus sequence and thus may be another member of this potential larger family.
The function of the consensus sequence of (S/T)P-(E/Q)F-----(K/R)---KDWK--I(R/K) is not known. However, the fact that an antibody raised against this sequence could not immunoprecipitate GMEB-2 but was very sensitive in recognizing GMEB-2 on Western blots suggests that this region is involved in protein-protein interactions. Further experiments should clarify the possible function of this sequence.
We suspect that the smaller, 1.9-kb cDNA clone (GMEB2Ј in Fig. 1) results from alternative splicing, as the DNA sequence of the open reading frame is identical up to nucleotide 1520. A potential splice site of AAG/GT exists at nucleotide 1515, just upstream of the divergence. Genomic sequencing will be required to ascertain the existence of a splice site here and the origin of the GMEB2Ј clone.
In conclusion, we have succeeded in cloning one of the two proteins that appear to be associated with changes in the transcriptional activity of anti-glucocorticoids and low concentrations of glucocorticoids (26,29). The fact that this protein had not been previously described is consistent with it having unique modulatory properties. Equally intriguing is the probability that the GMEBs are members of a new class of transacting factors. Experiments are currently in progress to test this provocative hypothesis in greater detail.