Properties of the Glucocorticoid Modulatory Element Binding Proteins GMEB-1 and -2: Potential New Modifiers of Glucocorticoid Receptor Transactivation and Members of the Family of KDWK Proteins

Sunil Kaul, John A. Blackford, Jr., Jun Chen, Vasily V. Ogryzko and S. Stoney Simons, Jr.

Steroid Hormones Section (S.K., J.A.B., J.C., S.S.S.) National Institute of Diabetes and Digestive and Kidney Diseases/Laboratory of Molecular and Cellular Biology, and The Eukaryotic Transcriptional Regulation Section (V.V.O.) National Institute of Child Health and Human Development/ Laboratory of Molecular Growth Regulation National Institutes of Health Bethesda, Maryland 20892-0805


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
An important component of glucocorticoid steroid induction of tyrosine aminotransferase (TAT) gene expression is the glucocorticoid modulatory element (GME), which is located at -3.6 kb of the rat TAT gene. The GME both mediates a greater sensitivity to hormone, due to a left shift in the dose-response curve of agonists, and increases the partial agonist activity of antiglucocorticoids. These properties of the GME are intimately related to the binding of a heteromeric complex of two proteins (GMEB-1 and -2). We previously cloned the rat GMEB-2 as a 67-kDa protein. We now report the cloning of the other member of the GME binding complex, the 88-kDa human GMEB-1, and various properties of both proteins. GMEB-1 and -2 each possess an intrinsic transactivation activity in mammalian one-hybrid assays, consistent with our proposed model in which they modify glucocorticoid receptor (GR)-regulated gene induction. This hypothesis is supported by interactions between GR and both GMEB-1 and -2 in mammalian two-hybrid and in pull-down assays. Furthermore, overexpression of GMEB-1 and -2, either alone or in combination, results in a reversible right shift in the dose-response curve, and decreased agonist activity of antisteroids, as expected from the squelching of other limiting factors. Additional mechanistic details that are compatible with the model of GME action are suggested by the interactions in a two-hybrid assay of both GMEBs with CREB-binding protein (CBP) and the absence of histone acetyl transferase (HAT) activity in both proteins. GMEB-1 and -2 share a sequence of 90 amino acids that is 80% identical. This region also displays homology to several other proteins containing a core sequence of KDWK. Thus, the GMEBs may be members of a new family of factors with interesting transcriptional properties.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The dose-response curve is a crucial characteristic of steroid hormone action and a basic feature of pharmacology. It indicates which agonists are active at physiological steroid concentrations. It also reveals the dose at which the most dramatic changes in biological activity occur with minimum differences in steroid concentration, i.e. the EC50 of the steroid. Cellular gene transcription mechanisms have evolved to capitalize on these effects of subtle changes in hormone concentration to selectively control the expression levels of key genes during development, differentiation, and homeostasis. Finally, the dose-response curve distinguishes between agonists and antagonists.

The dose-response curve has long been thought to be a property of each individual steroid-receptor complex and independent of the cell or the regulated gene. Thus, the determinants of the dose-response curve would be among the various steps followed by all receptors in converting the presence of steroidal ligand into the observed biological response (1 2 ). These steps include passive entry of the steroid into the cell, binding to the intracellular receptor protein, which may or may not be predominantly cytoplasmic as seen for the glucocorticoid receptor (GR) (3 ), activation to a species with increased affinity for DNA, binding of the receptor-steroid complex to hormone response elements (HREs), and finally interaction with the transcriptional machinery and assorted cofactors to modify the rates of gene transcription. The rate-limiting step in this pathway has been concluded to be activation, which is microscopically irreversible (4 ). Thus, the affinity of steroid binding to receptor was envisaged to determine the ultimate dose-response curve (4 5 6 ). Furthermore, the underlying assumption of all structure-activity studies has been that one could generalize from the results in one system to any other responsive gene in cells containing the same receptor. Likewise, if a given steroid was an antagonist in one system, it was presumed that it would be an antagonist in all systems.

Accumulating data have begun to force a revision of this model. Unexplained differences in the dose-response curves of different estrogen-inducible genes in the same cells have been known for many years (7 8 9 10 ). Similarly, we have documented variations in the dose-response curves for glucocorticoid induction of tyrosine aminotransferase (TAT) vs. glutamine synthetase, mouse mammary tumor virus (MMTV), or transiently transfected TAT in the same cell (11 12 13 ) and between the same TAT gene in differently treated cells (14 15 ). Other longstanding conundrums have been why most antisteroids display partial agonist activity and how the amount of this residual activity can vary with the cell (16 17 18 ), the gene (12 18 19 20 ), the promoter (16 17 18 21 ), and even the composition (18 22 ) or spacing (23 ) of the HRE. Again, the rat TAT gene has been a good model system as it displays variations in the residual agonist activity of antiglucocorticoids that parallel the observed shifts in the dose-response curves of agonists (12 13 14 15 19 ). Further studies with the rat TAT gene revealed a 21-bp cis-acting element that was sufficient to reproduce all of the above effects (13 15 24 25 ). Thus, at least one method of controlling the dose-response curve of agonists, and the partial agonist activity of antagonists, was the presence of the cis-acting element that we called a GME, or glucocorticoid modulatory element.

A key component in the action of the GME is the binding of a trans-acting factor because mutant oligonucleotides that did not bind factor were biologically inactive (15 ). Consequently, the proposed model for the mechanism of GME action consists of the trans-acting factor binding to the GME and interacting with both the HRE-bound GRs and unknown transcription factors to increase the coupling efficiency of selected steps before the synthesis of the induced gene transcript (15 26 27 ). A pivotal development in support of this model was the finding that the trans-acting factor was actually a heterooligomeric complex of two novel proteins (GMEB-1 and -2) with an aggregate size of about 550–600 kDa (GMEB-1 and -2) (28 ). We have recently cloned and characterized the rat GMEB-2 as a 67-kDa protein on SDS-polyacrylamide gels (29 ).

In the present study, we report the cloning of the second protein of the heteromeric complex, i.e. the 88-kDa GMEB-1. Several properties of GMEB-1, alone and in combination with GMEB-2, were examined to determine whether they were compatible with the previously determined biological properties of the GME. The biological activities of GMEB-1 and -2 were assessed in a mammalian one-hybrid assay. We examined the interactions of GMEB-1 and GMEB-2 with themselves, with each other, with GR, and with other cofactors that are known to bind steroid receptors (30 31 32 33 ). Possible associations with CREB, which is known to bind to the same DNA sequence as the GMEBs (15 34 35 36 ), were also investigated. Finally, each GMEB was overexpressed in whole cells to examine its ability to modulate both the GR dose-response curve and the partial agonist activity of antisteroids. Collectively, the data indicate that the originally isolated GMEB-1 and -2 have now both been cloned, that the GMEBs can modulate the transactivation properties of GR-steroid complexes, and that GMEB-1 and -2 may be members of a new class of transcription factors with potentially novel properties.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of GMEB-1
The amino acid sequence of three tryptic peptides of purified rat GMEB1 was already known (28 ), and three new peptides (ESEEISENTLMF, XQMI/FLQLQPVQQGXAE, and AVILETELR) were obtained by additional sequencing. None of these peptide sequences exhibited any substantial homology with any known protein or nucleic acid sequence in GenBank. However, one tryptic peptide was highly homologous (16 of 18 amino acids) with a human Expressed Sequence Tag (EST 129005) in the dbEST database. This EST was resequenced and used to isolate clones from a human heart library as described in Materials and Methods. One 1.8-kb clone contained an open reading frame (ORF) of 563 amino acids bounded on both sides by in-frame stop codons (Fig. 1AGo). Thus, this clone encoded the full-length protein. Of the 73 amino acids in the six sequenced peptides of rat GMEB-1, 67 were identical in the translated sequence from the human cDNA clone. Three nonidentical residues were highly conserved (D vs. E, D vs. Q, and V vs. M), one was similar (Q vs. H), and two were different (Y vs. A and T vs. A). The translation initiation codon was preceded by a minimal Kozak sequence, and the cDNA had a respectable poly A tail at the 3'-end. Surprisingly, there was substantial homology between GMEB-1 and its heterodimerizing partner, GMEB-2 (29 37 ), which was greater at the cDNA level (55%) than it was at the amino acid level (39%). Furthermore, the two proteins are 80% identical over the 93 amino acids of positions 89–181 of hGMEB-1 (Fig. 1BGo).




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Figure 1. hGMEB-1

A, Nucleotide and deduced amino acid sequence of the 1.8-kb GMEB-1 clone. In frame stop codons at either end of the ORF are indicated by the asterisk under the codon. The two crucial residues of the consensus Kozak sequence are marked by the double dagger above the bases. The six tryptic peptides are underlined. The new tryptic peptides are in bold type. The differences from the rat sequences (28 ) are italicized. B, Sequence homology between human (h)GMEB-1 and rat (r)GMEB-2. The cross-hatched bar ({boxtimes}) is above the peptide of GMEB-2 that was used to prepare the anti-GMEB antibody. The originally reported sequence of GMEB-2 (29 ) was found by Hinrich Gronemeyer (Strasbourg, France), and confirmed by us, to be in error at nucleotides 183–186 (initial = GCCTGT; revised = GCTGCTGCT), thus adding one amino acid to the protein sequence at residue 62 (initial = AlaCysAla; revised = AlaAlaAlaAla).

 
Biochemical Properties of GMEB-1
The molecular mass predicted for hGMEB-1 from the cDNA clone was much less (61 kDa) than that observed on gels for rat GMEB-1 protein (88 kDa) (28 ). However, cell-free translation of the 1.8-kb clone with [35S]methionine yielded a major product of 89 kDa, which migrated very closely with rat GMEB-1 on SDS-PAGE gels (Fig. 2Go). The minor difference in migration was presumably due to the limited amino acid differences between rat and human GMEB-1 (Fig. 1AGo), as even single amino acid differences can alter the gel mobility of proteins (38 ). Western blotting with an antibody to an epitope shared by hGMEB-1 and rGMEB-2 (see Fig. 1Go) revealed an approximately 88-kDa protein both in the cell-free translation reaction with the appropriate cDNA and in rat hepatoma tissue culture (HTC) cell cytosol (Fig. 2Go). Interestingly, both recombinant hGMEB-1 and native rGMEB-1 proteins were less reactive to the anti-GMEB antibody than was rGMEB-2 (Fig. 2Go and data not shown) even though 22 of 23 of the amino acids of the epitope were conserved in hGMEB-1.



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Figure 2. SDS Gel Properties of in Vitro Translated GMEB-1 cDNA

A, SDS gel migration of [35S]methionine-labeled protein. The 1.8-kb GMEB-1 cDNA in pCMV-SPORT was in vitro translated with [35S]methionine, separated on a 10% SDS-polyacrylamide gel, and autoradiographed as described in Materials and Methods. The open arrow indicates the position of GMEB-1. The positions of the mol wt markers are indicated. B, Detection of authentic and in vitro translated GMEB-1 by Western blotting. Samples of HTC cell cytosol, in vitro translated GMEB-1, or reticulocyte lysate that had been incubated with luciferase cDNA as carrier DNA (CONTROL) were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose for Western blotting with affinity-purified anti-GMEB antibody. The open arrow indicates the position of GMEB-1. The solid arrow indicates the position of GMEB-2. Other bands in HTC cytosol are nonspecific, cross-reacting species (29 ) that are more visible with the overexposure needed to detect GMEB-1.

 
DNA Binding of GMEB-1 and -2 in Gel Shift Assays
Two previously documented properties of rGMEB-1 are its binding to GME oligonucleotides 1) weakly as a homooligomeric complex and 2) synergistically with the 67-kDa rGMEB-2 to yield a slightly larger heteromeric complex (28 29 ). We therefore examined the DNA binding activities in gel shift assays of hGMEB-1 ± rGMEB-2 that were in vitro translated from the corresponding cDNAs. The recombinant hGMEB-1 protein alone bound to the GME somewhat more than expected but in a highly specific manner (Fig. 3Go). The binding was competed by a 25-fold molar excess of cold GME. Those GME oligonucleotides with clustered base substitutions that continued to display GME biological activity (i.e. M1 and M3) also competed the binding of GMEB-1. In contrast, an excess of the biologically inactive M2 oligonucleotide (15 ) was unable to prevent the DNA binding of GMEB-1. When in vitro translated hGMEB-1 and rGMEB-2 were mixed on ice overnight to allow for efficient heterooligomer formation (28 ), a protein-GME complex was formed that had virtually the same migration pattern as seen with the authentic heterooligomer from HTC cell cytosol (Fig. 3Go). This binding was synergistic as the total final complex was greater than the sum of the components [1.9 ± 0.3 (SEM) fold higher, n = 5, P = 0.04]. Again, the DNA binding of the recombinant heterooligomer was specific as only the biologically active DNAs (GME, M1, and M3 but not M2) were capable of inhibiting the formation of the protein-DNA complex. No supershift with anti-GMEB antibody could be observed because it recognizes only denatured protein (29 ).



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Figure 3. Binding of in Vitro Translated GMEB-1 and -2 to GME Oligonucleotide in Gel Shift Assay

[32P]GME oligonucleotide was incubated with 1 U of in vitro translated GMEB-1, 1 U of in vitro translated GMEB-2, 1 U each of in vitro translated GMEB-1 and -2, and HTC cell cytosol with a 25-fold excess of the indicated nonradioactively labeled oligonucleotides. The bands were visualized by autoradiography as described in Materials and Methods (arrow marks position of the GMEB heterocomplex as discussed in the text).

 
The sequence, size, Western blotting, and specificity of DNA binding of our cloned protein all support its identity as that of the human homolog of the previously described rGMEB-1 (28 ). The DNA binding properties of the recombinant hGMEB-1 and rGMEB-2 to biologically active DNA sequences further strengthen this conclusion and demonstrate that the hGMEB-1 is functionally equivalent to the rat homolog. These data also reinforce our earlier determination that we had correctly cloned GMEB-2 (29 ).

Transactivation and Oligomerization Properties of the GMEBs
Full-length GMEB-1 and -2 were fused to the C-termini of either the GAL4 DNA-binding domain (GAL4-DBD) or the VP16 activation domain (VP16-AD) and examined for their ability to induce a GAL4-regulated reporter gene containing five tandem copies of the GAL4 binding element or upstream activating sequence (UAS). GMEB-1 and -2 each displayed intrinsic transactivation activity (Fig. 4AGo, inset). These results support our model that GMEB-1 and -2 are key components in the modulation of GR transcriptional activity by interacting with the transcriptional machinery (15, 26).



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Figure 4. Activity and Interactions of GMEBs

A, Activity of GMEB chimeras in mammalian one- and two-hybrid assays. Triplicate dishes of Cos-7 cells containing the indicated GAL4-GMEB fusions without (inset) and with (main figure) VP16-GMEB chimeras were cotransfected with the GAL4-regulated Luciferase reporter. The induced levels of Luciferase were determined as described in Materials and Methods. The error bars represent the SD of the triplicate plates for each sample in the assay. Similar results were obtained in two other independent experiments. B, Binding of GMEB-1 and -2 to immobilized GMEBs in pull-down assay. [35S]methionine-labeled, in vitro translated GMEB-1 and -2 were added to M2-Agarose columns containing previously immobilized FLAG-tagged GMEB-1 or -2 (f:GMEB-1 or f:GMEB-2) that had been overexpressed from Baculovirus vectors in insect cells. The proteins remaining after washing were eluted, separated on SDS-polyacrylamide gels, and visualized as described in Materials and Methods. In vitro translated Luciferase and CREB were used to assay nonspecific retention. The column labeled "10% input" displays one tenth of the indicated in vitro translated, full-length proteins that was loaded to the Agarose columns. The column labeled "M2 Agarose" shows the amount of each protein that was retained in the absence of GMEBs. The columns labeled "f:GMEB-1 or -2/M2 Agarose" depict the amount of each protein that was retained by the immobilized GMEBs. Similar results were obtained in a second experiment.

 
When GMEB-1 and -2 chimeras were cotransfected with the GAL4-regulated reporter, a synergistic increase in transactivation was observed (Fig. 4AGo). This suggested an ability of each protein to both homo- and heterooligomerize, supporting the earlier and above results. It should be noted that the net activity of heterodimerization was very sensitive to the orientation of the two chimeras: GAL-B2/VP16-B1 was much more active than GAL-B1/VP16-B2. Thus, the interactions between GMEB-1 and -2 may be affected by the nature and/or position of the fused protein of the chimera.

More evidence for interactions of GMEB-1 and -2 with themselves and each other was obtained from pull-down assays using FLAG-tagged GMEB-1 and -2 that were overexpressed from Baculovirus vectors in insect cells. As shown in Fig. 4BGo, column-purified recombinant GMEB-1 and GMEB-2 formed not only homooligomers but also heterooligomers with 35S-labeled, in vitro translated GMEB-1 and -2. These interactions were selective, as evidenced by the absence of interactions on columns lacking immobilized GMEBs and a greatly reduced retention of the control proteins Luciferase and CREB (see also below). Thus, it appears that direct interactions occur between two molecules of GMEB-1, of -2, and between GMEB-1 and -2.

Interaction of GMEBs with GR
As an interaction of the heteromeric GMEB protein complex with GR is a central postulate in the mechanism of GME action (15 26 27 ), we sought supporting evidence for this. Only a very weak association was observed in mammalian two-hybrid assays between GAL4-GR and VP16-GMEB-1 or -2 either in the absence or presence of the glucocorticoid dexamethasone (Dex) (data not shown). Interestingly, Gal4-GR and VP16-GR also did not appear to interact, either with or without Dex; the response with both chimeras was about 40% less than the sum of the individual activities (data not shown; see also below). However, a significant steroid-dependent interaction between each of the GMEBs and GR was observed when the experiment was performed with the opposite pair of chimeras (GAL4-GMEB-1 or -2 and VP16 vs. VP16-GR) (Fig. 5AGo). Little consistent increase was noted when the components were mixed in the absence of steroid (data not shown).



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Figure 5. GMEB-GR Interactions

A, Interaction of GMEBs and GR in mammalian two-hybrid assays. Triplicate dishes of Cos-7 cells containing the indicated GAL4 and VP16 chimeras were cotransfected with the GAL4-regulated Luciferase reporter and then treated with EtOH ± 1 µM Dex. The induced levels of Luciferase were determined as described in Materials and Methods and expressed as fold increase over the steroid-free values. The bar graph gives the average values (±SD) of 14–15 experiments (P <0.0001 for GMEB-1/GR, P = 0.0073 for GMEB-2/GR). B, Binding of GR to immobilized GMEBs in pull-down assay. [35S]methionine-labeled, in vitro translated GR was incubated with buffer (untreated), EtOH, or 1 µM of the indicated steroids in EtOH to form the receptor-steroid complexes. These complexes were then added to M2-Agarose columns containing previously immobilized FLAG-tagged GMEB-1 or -2 (f:GMEB-1 or -2/M2 Agarose) that had been overexpressed from Baculovirus vectors in insect cells. The proteins remaining after washing were eluted, separated on SDS-polyacrylamide gels, and visualized as described in Materials and Methods. In vitro translated CREB was used to assay nonspecific retention. The row labeled "M2 Agarose" indicates the amount of receptor ± indicated steroid, or CREB, that was retained in the absence of immobilized GMEBs. The row labeled "10% input" displays one tenth of the in vitro translated proteins that were loaded to the Agarose columns. Similar results were obtained in a second experiment.

 
Pull-down assays substantiated the above apparent interactions of GR with the GMEBs. As seen in Fig. 5BGo, either GMEB-1 or GMEB-2 bound to M2-Agarose immobilized significant amounts of [35S]methionine-labeled GR but not CREB (see also below). Interestingly, the amount of GR that was retained did not change appreciably whether the receptor was steroid-free or bound by agonist (Dex) or antagonist [Dex-Mes or progesterone (Prog)]. The amount of GR retained on GMEB-1 and GMEB-2 columns ranged from 18 to 28% and 16 to 28%, respectively, of the input GR, indicating an efficient binding. For these reasons, coupled with their intrinsic transactivation activity, the GMEBs may be potential new transcriptional cofactors for GR.

Whole-Cell Activity of Overexpressed GMEBs
Given the complexity of the proposed model of GME action, in which the two proteins GMEB-1 and -2 combine to bind to the GME and then modulate GR transcriptional activity (15 26 27 ), the effect of GMEB overexpression could not be easily predicted. The simplest result would be that increased amounts of either protein caused a further left shift in the dose-response curve and increased agonist activity of antisteroids. Alternatively, decreased activities might result from squelching, caused by the sequestering of a limiting component (39 ). In CV-1 cells, overexpression of either GMEB-1 or -2 in CV-1 cells gave both a right shift in the Dex dose-response curve and decreased agonist activity with the antiglucocorticoid Dex-Mes (Fig. 6AGo). Cotransfection of GMEB-1 and -2 gave only marginally more right shift, or reduced partial agonist activity (Fig. 6AGo), suggesting that the effects of squelching by the GMEBs are saturable. This activity of the GMEBs was found to be dependent upon the concentration of both the GMEBs and GR. Thus, the ability of the GMEBs to effect a right shift in the dose-response curve (as shown by lower activities of 1 nM Dex) and decreased partial agonist activity of 1 µM Dex-Mes was more pronounced with increased concentrations of either GMEB (Fig. 6BGo). Conversely, the repression seen with each GMEB could be reversed simply by adding more GR (Fig. 6CGo). These effects appear to be selective as equal amounts of truncated GMEB-1 and -2 proteins are inactive (J. Chen, S. Kaul, and S. S. Simons, Jr., in preparation). Therefore, GMEB-1 and -2 have the properties expected of proteins involved in GME action, namely the ability to modulate, in a reversible fashion, the GR induction parameters in intact cells.



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Figure 6. Whole-Cell Biological Activity of GMEBs

A, Overexpressed GMEB-1 and -2 cause a right shift in the Dex dose-response curve and decreased partial agonist activity of Dex-Mes. Triplicate dishes of CV-1 cells containing 40 ng of pSVLGR plus 180 ng of GMEB-1, or 70 ng of GMEB-2, were cotransfected with 1 µg of GMEGREtkLuc reporter and then treated with EtOH ± the indicated concentrations of Dex or 1 µM Dex-Mes. The induced levels of Luciferase were determined as described in Materials and Methods. The induction above basal activity with each steroid concentration was determined and plotted as percent of the maximal induction by Dex. The activity of 1 µM Dex-Mes is given by the bars. The error bars represent the SD of triplicate plates. B, Inhibition of GR activities by added GMEB-1 or -2. Triplicate dishes of CV-1 cells containing 80 ng of pSVLGR plus the indicated amounts of GMEB-1 or -2 were cotransfected with 1 µg of GMEGREtkLuc reporter and then treated with EtOH ± 1 nM Dex, 1 µM Dex, or 1 µM Dex-Mes. Empty vector DNA was used to keep the concentration of GMEB plasmid vector constant in all plates. The induced levels of Luciferase were determined as described in Materials and Methods. The induction above basal activity with each steroid concentration was determined and plotted as percent of the maximal induction by Dex. The error bars represent the SD of triplicate plates. Similar results were obtained in two other independent experiments. C, Reversal of GMEB inhibition of GR activities by added GR. Triplicate dishes of CV-1 cells containing 200 ng of GMEB-1, or 100 ng of GMEB-2, plus the indicated amount of pSVLGR were cotransfected with 1 µg of GMEGREtkLuc reporter and then treated with EtOH ± 1 nM Dex, 1 µM Dex, or 1 µM Dex-Mes. Empty vector DNA was used to keep the concentration of GMEB plasmid vector constant in all plates. The induced levels of Luciferase were determined as described in Materials and Methods. The induction above basal activity with each steroid concentration was determined and plotted as percent of the maximal induction by Dex. The error bars represent the SD of triplicate plates. Similar results were obtained in at least two other independent experiments.

 
GMEBs Do Not Possess Histone Acetyltransferase (HAT) Activity
A commonly proposed mechanism for the action of the transcriptional cofactors of steroid/nuclear receptors is nucleosome reorganization after histone acetylation (40 41 42 ). In fact, several cofactors have been found to possess histone acetyl transferase (HAT) activity (32 33 43 44 45 ). Therefore, we inquired whether the above biological activity of the GMEBs might require HAT activity. Interestingly, recombinant GMEB-1 and GMEB-2 with a FLAG tag at the N terminus that were expressed from Baculovirus vectors in SF-9 cells, or their mixtures, did not show any HAT activity (43 ) while the p300 positive control displayed a strong reaction in the same gel (data not shown).

Interactions of GMEBs with Other Proteins
The proposed mechanism of GMEB action involves interactions with GR (Fig. 5Go) and other unidentified proteins (15 26 27 ). Attractive candidates are the cointegrators p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF) (30 31 32 43 45 ). Mammalian-two hybrid assays suggest an interaction with CBP but not p300 or PCAF (Fig. 7Go). Whether this results from a functional difference between CBP and p300 (46 47 48 ) is not known. No binding of either GMEB with the corepressors SMRT (silencing mediator for retinoid and thyroid hormone receptors) and or NCoR (nuclear receptor corepressor) could be detected in the mammalian two-hybrid assay (data not shown).



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Figure 7. Interaction of GMEBs with Transcriptional Cofactors in Mammalian Two-Hybrid Assays

Triplicate dishes containing the indicated GAL4 chimeras and equimolar amounts of either VP16 chimeras or VP16 were cotransfected with the GAL4-regulated Luciferase reporter. The induced levels of Luciferase were determined as described in Materials and Methods. The error bars represent the SD of the triplicate plates for each sample in the assay. Similar results were obtained in four other independent experiments.

 
Another candidate binder of the GMEBs is CREB. Both Oshima and Simons (15 ) and Schutz et al. (34 35 36 ) have reported that CREB also binds to the GME. Furthermore, as CREB and the GMEBs both form complexes with GME oligonucleotides in gel shift assays that are of very similar size and much larger than expected for the binding of monomers, and as the GMEBs are known to bind to the GME as a heterooligomer (28 29 ), it seemed possible that the large complexes found to contain CREB might also contain one or more GMEBs. Overexpression of CREB did not hinder the expression of GME activity (27 ). Similarly, no interaction was observed between either of the GMEBs and CREB in the pull-down assay (Figs. 4BGo and 5BGo) or in the mammalian two-hybrid assay with both orientations of the GAL4-DBD and VP16 activation domain chimeras (data not shown). Therefore, there is currently no evidence for interactions between CREB and the GMEBs.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glucocorticoid modulator element (GME) has the unique property of modulating the glucocorticoid induction properties of the rat TAT and other target genes (13 14 15 24 25 ). This whole-cell activity is associated with the binding of a heteromeric complex of two proteins, GMEB-1 and -2 (15 28 ), the latter of which we have recently cloned (29 ). Both GMEB-1 and -2 are novel proteins that were not present on GenBank, which is consistent with no other proteins being reported to possess similar activities. This paper describes the cloning of the second member of the biologically active heterooligomer, the human equivalent of rat GMEB-1, as a 563-amino acid protein. The assignment of the cloned protein as GMEB-1 was based on the more than 91% identity of the predicted amino acid sequence of the human cDNA clone with the six sequenced tryptic fragments distributed throughout the rat GMEB-1, the nearly identical size of the in vitro translated protein with rat GMEB-1, and detection by Western blotting with an antibody that was raised against a peptide that was 96% identical. Furthermore, the newly cloned material acted just like authentic GMEB-1 (28 29 ) in its ability to homo- and heterooligomerize with itself and GMEB-2 and to bind to GME oligonucleotides as a homo- and heterooligomer in gel shift assays. A crucial property of these proteins is that they be able to modify the dose-response curve of agonist steroids, and the partial agonist activity of antagonists, in intact cells. GMEB-1 and -2 modulate these activities of GR whether overexpressed individually or together. Therefore, both rat GMEB-2, and now the human equivalent of rat GMEB-1, appear to have been cloned.

Several properties of the GMEBs made us question whether we had identified the correct proteins. First, the size of GMEB-1 predicted from the cDNA clone was too small. However, the protein encoded by the 1.8-kb hGMEB-1 cDNA clone migrated on SDS-PAGE (Fig. 2Go) as a much larger protein than expected from its sequence (88 vs. 62 kDa). This behavior was similar to that of the rGMEB-2 (29 ) and might be due to posttranslational modifications. GMEB-1 and GMEB-2 both contain S/T-rich regions and have several potential phosphorylation sites for casein kinase and protein kinase. GMEB-1 also contains some cAMP- and cGMP-dependent protein kinase sites.

Second, after our biochemical characterization of hGMEB-1 was completed (49 ), the cloning of human GMEB-1 as a protein that binds heat shock protein 27 in a yeast two-hybrid screen was reported (50 ). Interestingly, this clone of hGMEB-1 contains a 10-amino acid insert (LFIDGHFYNR) after amino acid 43 that was not present either in the originally isolated rat GMEB-1, as determined from the new sequenced peptide reported here that corresponds to amino acids 32–46, or in our human clone (Fig. 1Go). This GMEB-1 of Theriault et al. (50 ) appears to be a GMEB-1 isoform (37 ). It is currently not possible to determine the relative abundance of these two forms of GMEB-1 as the antibodies used in the two studies can not discriminate between the two proteins. Whether the longer form of hGMEB-1 (50 ) has the same activities as seen for our clone and for the isolated GMEB-1 remains to be established.

Third, another report of the cloning of human GMEB-1, and of human GMEB-2, appeared while this paper was being revised. This report described the GMEBs as HeLa cell factors essential for parvovirus replication (51 ). While it is not unusual for a given protein to have two biological activities, the ability of GMEB-1 to bind to heat shock protein 27 and the essential role of the GMEBs in parvovirus replication made it imperative for us to determine, first, that we had cloned the correct molecules and, second, whether GMEB-1 and -2 were in fact relevant for the GME action. The identification of three additional sequenced peptides from the originally purified rat GMEB-1 protein (28 ) in our human GMEB-1 clone strongly argues that we have cloned the correct protein (Fig. 1AGo). All of the biochemical properties of the overexpressed proteins from our GMEB-1 and -2 (29 ) clones (size, antibody reaction, DNA binding as homo- and heterooligomers, and specificity of DNA binding) are also consistent with those of the endogenous proteins (Figs. 2Go and 3Go). Finally, the overexpressed GMEB-1 and -2 proteins can modulate GR activity (Fig. 6Go). Thus, we conclude that we have cloned two proteins that are biologically relevant for GME activity. The fact that these proteins also have other cellular functions promises to make future investigations particularly interesting.

The effect of overexpressed GMEBs to cause a right shift in the dose-response curve, and a decrease in the partial agonist activity of antisteroids, as opposed to the left shift and increased partial agonist activity seen with the GME, was not completely unexpected. Our proposed model of GME action involves a multiprotein complex (15 26 27 ). Unless the GMEBs are the limiting factor for the expression of the GR transactivational properties, overexpression of either GMEB could sequester other required factors to cause squelching (39 ) and a decrease in GR activities. For example, while CBP is a coactivator and generally increases the total transactivation by steroid receptors, overexpression of CBP in cells in which CBP is abundant causes a decrease in GR transactivation (48 ). In our systems, GR appears to be a limiting reagent (39 52 ). Furthermore, when GR concentrations are increased so that they are not limiting, the GME is no longer active (S. Chen, N. Sarlis, and S. S. Simons, Jr., in preparation). This squelching is not due simply to a large imbalance in the ratio of GMEB-1 to GMEB-2 because overexpression of both GMEBs does not reverse the behavior. Instead, the effects of GMEB-1 and -2 are slightly more than that of GMEB-2 alone (Fig. 6AGo). This suggests the existence of a lower limit for the squelching by elevated GMEBs, consistent with the diminishing changes with higher levels of GMEB-2 alone (Fig. 6BGo). While it is possible that a left shift in the dose-response curve, and increased agonist activity of antiglucocorticoids, may be achieved with some other ratio of transfected GMEBs, we suspect that this behavior will be seen only in cells where the GMEBs are limiting. We have not yet found such a cell line (28 ). Nevertheless, both GMEBs do modulate GR activity (Fig. 6Go), thus fulfilling a necessary requirement for these proteins to be considered as potential cofactors in the mechanism of GME action.

Numerous similarities between our GMEB-1 and -2 clones suggest that they are closely related proteins. GMEB-1 and -2 are 55% identical at the cDNA level and 39% identical on the basis of amino acid sequence and contain a region of 90 amino acids that is 80% identical. Interestingly, the anti-GMEB antibody only weakly recognizes GMEB-1 even though the antigenic site in GMEB-1 is virtually identical to that of GMEB-2 (22 of 23 amino acids). Both GMEBs possess intrinsic transactivation activity in the mammalian one-hybrid assay. The mammalian two-hybrid assay indicates that each protein forms homooligomers and heterooligomers with the other protein in solution (Fig. 4Go). GMEB-1 and -2 also bind specifically to the same DNA sequence both as a homooligomer and as a heterooligomer with each other (Fig. 3Go). Quantitative comparisons of the homo- and heterooligomeric interactions of GMEB-1 and -2 in the two-hybrid assay are complicated by the observation that the activities of the GMEB chimeras are orientation selective and most effective for GAL4-B2 interacting with VP16-B1 (Fig. 4AGo). Similar orientation specificities in the two-hybrid assay have been observed by others (53 54 ).

Based on the biological activities of GME oligonucleotides that do, and do not, bind the GMEBs (15 ), the mechanism of action of the GMEBs has been proposed to involve their binding to the GME and subsequent interaction with both GRE-bound GRs and the transcriptional machinery (15 26 27 ). In fact, in mammalian two-hybrid assays, both GMEB-1 and -2 display a modest steroid-dependent interaction with GR (Fig. 5AGo). The interaction of GR with GMEBs is particularly notable as the chimeras GAL4-GR and VP16-GR exhibit no synergistic response, even in the presence of steroid. This is not due to an inactivity of VP16-GR, as witnessed from the responses with the GAL4-GMEBs, but may be due to an attenuated activity of GAL4-GR. This could follow from either a misfolding of the GAL-GR chimera or the formation of GAL-GR dimers, thereby excluding the binding of VP16-GR chimeras. Alternatively, these results are consistent with the hypothesis that the dimerization of many DNA-bound receptors (55 ) occurs not in solution (56 57 ) but only after a DNA binding-induced conformational change (57 58 59 60 61 ). Similarly, the binding of cofactors to receptors has been found to be modified by the DNA binding of receptors (62 63 ). In this case, the interactions of the GMEBs might be stronger with GRE-bound GRs.

The strength of the associations of GAL4-GMEBs with VP16-GR in the two-hybrid assays appears to be less than those of the FLAG-GMEBs and wild-type GR in pull-down assays where there was no noticeable difference in the presence or absence of either agonist or antagonist steroid (Fig. 5Go and data not shown). The binding of the GMEBs to antagonist-bound receptors was anticipated from the ability of the GME to augment the residual agonist activity of several different GR-antisteroid complexes (13 15 24 25 64 ). A similar ligand-independent interaction with GR, but ligand-dependent increases in GR transactivation, has been seen with TIF1ß (65 ). The fact that the GMEBs and steroid-free GR can bind may not be physiologically relevant, however, as the predominantly cytoplasmic location of steroid-free GR (3 ) may effectively prevent association with the nuclear GMEBs (15 ). This could explain the steroid dependency for GMEB/GR interactions in the two-hybrid assays but not in the pull-down assays.

GMEB-1 or -2 binds to GR but neither contain a LxxLL motif, which has been found to be intimately involved in cofactor binding to the steroid receptors (66 67 ). However, the receptor binding proteins, TIF1ß (65 ) and SUG1, or ARA70 (68 ), also have no LxxLL motifs. Furthermore, not all LxxLL motifs interact with receptors (67 69 70 ). Another difference between the GMEBs and many other coactivators of the steroid/nuclear receptors is the absence of HAT activity (32 33 43 44 45 ), which is proposed to disrupt the nucleosomes and initiate chromatin remodeling to facilitate the access of other transcription factors (40 41 42 ). However, TIF2, a well established coactivator for steroid receptors, is like the GMEBs and does not possess HAT activity (70 ). Furthermore, recent data suggest that HAT activity may not be required in all instances of steroid receptor-activated gene transcription (71 72 73 ) and that chromatin architecture is not a controlling factor in GR induction of TAT gene expression (71 74 ). These observations are consistent with reports that the modulation of TAT induction occurs just as well for the endogenous gene as for stably and transiently transfected genes containing the GME (13 15 64 75 ), conditions under which ordered nucleosome structures are not formed (76 77 ). Thus, the modulation of GR gene induction activity by the GME, which does not alter the fold induction by glucocorticoids (64 ), may not require HAT activity, which has been seen most commonly in factors that increase the fold induction of steroid receptors (32 33 43 44 45 ).

A second component of the proposed mechanism of GME action is the association of the GMEBs with a transcription factor(s) (15 26 27 ). Thus, the functional interaction of both GMEBs with CBP (Fig. 7Go), a common transcription integrating factor (30 31 78 ), is intriguing. This whole cell interaction occurred with the C-terminal third of CBP, which is missing both the HAT activity domain (79 ) and the receptor interaction domain of CBP (30 31 ). We have recently reported that increased concentrations of CBP cause a left shift in the dose-response curve, and increased agonist activity of antiglucocorticoids, in the absence of a GME (52 ). If the GMEBs bind to GR and CBP via different domains, one attractive mechanism for a left shift by the GME is thus to increase the net affinity of CBP and GR for each other, thereby increasing the concentration of GR-CBP complexes without changing the concentration of GR or CBP. Further experiments are needed to test this hypothesis.

GMEB-2 displays some homology to several proteins including DEAF-1 and Suppressin (29 ), which appears to be the short form of a protein called NUDR (80 ). With the addition of GMEB-1, two human proteins (nuclear phosphoprotein and lymphoid-specific SP100 homolog), and two mouse ESTs, the size of the potential family of related proteins has expanded, and the putative consensus sequence of this family (29 ) can be refined (Fig. 8Go). GMEB-1 is highly homologous to, but is not a splicing variant of, GMEB-2 (37 ) and displays a greater homology with GMEB-2 than any other protein yet described. Therefore, GMEB-1 and -2 are the most closely related proteins in this putative new family of related proteins. Of those proteins that have been characterized, no function has yet been demonstrated for the consensus sequence that includes the sequence KDWK, although it has been proposed to be involved in DNA binding (81 ). The GMEBs (Fig. 3Go) (28 29 ), DEAF-1 (81 ), Suppressin (S. Kaul, J. A. Blackford, Jr., and S. S. Simons, Jr., in preparation), and NUDR (80 ) have all been found to bind DNA. The human lymphoid-specific SP100 homolog is a nuclear protein that appears as nuclear dots, some of which colocalized with PML nuclear bodies (82 83 ). The GMEBs, DEAF-1, Suppressin/NUDR, and Sp100 (84 ) each have characteristics of transcription factors, and the GMEBs and NUDR (80 ) possess intrinsic transactivation activity in transfected cells. The human nuclear phosphoprotein may also be involved in signal transduction (85 ). Given the unusual activities that have already been associated with these proteins, further investigations of this emerging new family of proteins promises to uncover new details of cellular transcriptional control mechanisms.



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Figure 8. Possible GMEB Family Members

Alignment of sequences of the high homology region of GMEB-1 and -2 with rat and human Suppressin (NUDR is not included as it appears to be an extended form of Suppressin), Drosophila DEAF-1, the ORFs of four Caenorhabditis elegans cosmids, two human proteins (nuclear phosphoprotein and lymphoid-specific SP100 homolog), and two mouse ESTs. The alignment was performed by SeqApp, which ascribes different colors to various amino acids that are unrelated to the homology. The region of highest homology for all proteins is underlined.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Unless otherwise indicated, all operations were performed at 0 C.

Steroids
Dex was purchased from Sigma (St. Louis, MO). Des-Mes was synthesized as described previously (86 ).

Antibody
The antibody used to detect both native and recombinant GMEB1 was raised against a GMEB-2 peptide (ISPKEFVHLAGKSTLKDWKRAIR) that was highly homologous to a region of GMEB-1 (29 ).

Cloning of GMEB-1
In addition to the sequences of the three reported tryptic peptides of GMEB-1 (28 ), three more peptides were sequenced at the Keck Foundation (Yale University, New Haven, CT). The sequences of these peptides and the yields (in parentheses) were ESEEISENTLMF (16.4%), with a minor species of XQMI/FLQLQPVQQGXAE, and AVILETELR (44.2%). The amino acid sequences of all GMEB-1 peptides were blasted against the NR database (in all six frames) of GenBank (87 ). No homology was found with any known proteins or nucleic acid. However, one of the tryptic peptides displayed substantial homology (16 of 18 amino acids) with a human EST (129005) in the dbEST database of GenBank. This EST was then used to identify related ESTs in dbEST database. Two ESTs (129796 and hbc1096) showed high homology to particular areas of EST 129005. Based on sequence of the above three ESTs, SeqApp 1.9 (D. Gilbert, Indiana University, Indianapolis, IN) was used to generate a 1-kb continuous sequence. EST 129005 was obtained from ATCC (Manassas, VA) and resequenced. There were several errors in the published one-pass sequence of EST129005 but no errors in the region encoding the sequence that was homologous with the tryptic peptide. Two oligonucleotides (5'-CATCCAGGCTCCCCTTCCAAG-3' and 5'-CCAGGAACCAAGTAGAGCAGGG-3') were designed based on the sequence of the above 1-kb sequence. Both oligonucleotides were biotinylated and used to screen a cDNA library from human heart (Life Technologies, Inc., Gaithersburg, MD) using the solution hybridization method of Gene Trapper cDNA positive selection system (Life Technologies, Inc.) according to manufacturer’s instructions. The captured cDNA for each oligonucleotide was electroporated into electromax DH10B cells (Life Technologies, Inc.). Colony PCR was performed on 120 clones of 200 generated and 8 of them had inserts. Most of the positive clones had an insert size of around 2 kb. One large clone, GM106 (GMEB-1/pCMV-SPORT), was sequenced by MacConnell Research Corp. (San Diego, CA) on a Licor machine from both ends.

Plasmids
pRBalI17 used for in vitro synthesis of GR protein and pSVLGR used to transiently transfect GR into CV-1 cells were obtained from Keith Yamamoto (University of California, San Francisco, CA). GALp300, with the entire p300, was obtained from A. Giordano (Thomas Jefferson University) while GAL4CBP, containing amino acids 1678–2441 of CBP, was obtained from Richard Goodman (Vollum Institute). pCi.GAL4pCAF was prepared by modifying pCI (Promega Corp., Madison, WI) to pCI.FlagGal4(94) (sequence is available upon request to V.V.O.). PCAF was then inserted into the MluI and NotI sites to give the final construct. GMEGREtkLuc reporter was described previously (27 ).

GAL/GMEB-2 [GMEB-pM] and VP16/GMEB-2, consisting of the GAL4-DBD or the VP16 transactivation domain fused to the amino terminus of full-length GMEB-2, were constructed by cloning a 2-kb blunt ended NcoI-HindIII fragment of GMEB-2/pCR2.1 (29 ) into the EcoRV site of pcDNA3.1/His A (Invitrogen, San Diego, CA). Clones were digested with BamHI to ensure the proper orientation of cDNA. A 2-kb BamHI fragment from GMEB-2 pcDNA3.1/His A was then cloned into BamHI site of pM (GAL4-DBD) and pVP16 (VP16 activation domain) (CLONTECH Laboratories, Inc. Palo Alto, CA).

GMEB-1/pCM-SPORT was mutated with the Quick Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) just upstream of the initiation codon to remove the upstream stop codon and create an EcoRI site. The 2-kb EcoRI and XbaI fragment from the above mutated clone of GMEB-1/pCM-SPORT (clone 101) was cloned in the same sites of pM, pVP16, and pcDNA3.1/His A to generate GAL/GMEB-1, VP16/GMEB-1, and GMEB-1/pcDNA3.1/His A. GR/pcDNA3.1/HisC was generated by ligating three fragments: 1) a 718-bp SspI and BglII fragment obtained by digesting pSVLGR with the same restriction enzymes; 2) a 2-kb fragment of pSVLGR digested with BglII and XbaI; and 3) vector pcDNA3.1/HisC that had been digested with EcorV and XbaI. GAL/GR and VP16/GR were again constructed by ligating three DNA fragments: 1) a 0.7-kb fragment obtained by digesting GR-pcDNA3.1/HisC with EcoRI and BglII; 2) a 2-kb fragment obtained after digesting GR-pcDNA3.1/HisC with BglII and XbaI; and 3) vectors pM and pVP16 digested with EcoRI and XbaI.

The baculovirus expressed GMEBs contained a Flag tag for use in immunoadsorption assays. GMEB-1/Flag p/FastBac1 was constructed by cloning a 2-kb EcoRI and XbaI fragment from GMEB-1/pcDNA3.1/HisA into Flag/pFastBac1 (Life Technologies, Inc.). GMEB-2 was first cloned into the BamHI site of pSP73 (Promega Corp.) to clone GMEB-2 cDNA into the correct frame of Flag/pFastBac1. Finally, a 2-kb XbaI and SacI fragment from GMEB-2/pSP73 was cloned in Flag/pFastBac1.

CREB-pCR2.1 was constructed by cloning a EcoRI fragment from pSV-CREB (William Walker, University of Pennsylvania, Philadelphia, PA) into pCR2.1. CREB was cloned in frame in pM and pVP16 by first amplifying by PCR the CREB ORF, and creating BamHI (at 5'-end) and HindIII (at 3'-end) sites, using pSV-CREB as a template with high-fidelity Pfu polymerase (Stratagene). The PCR product was digested with BamHI and HindIII and cloned into the same sites of pM and pVP16.

Cell Culture and Transient Transfection
Cos-7 or CV-1 cells were grown in DMEM (Life Technologies, Inc.) supplemented with 5%, or 10%, respectively, of FCS (Biofluids Inc., Rockville, MD) at 37 C in a humidified incubator (5% CO2). The transient transfections were done using Lipofectamine Reagent (Life Technologies, Inc.) in the presence of media without serum (OPTI-MEM I; Life Technologies, Inc.). Cells were incubated with plasmid DNA, OPTI-MEM I, and lipofectamine reagent for 4 or 16 h and then placed in the normal media (5 or 10% FCS, DMEM) for 16 h before being induced with the appropriate steroid for 24 h. Transfected cells were lysed and assayed for reporter gene using Luciferase Assay Reagent according to manufacturer’s instruction (Promega Corp.). Luciferase activity was measured in an EG&G Berthhold Luminometer (Microlumat LB 96 P).

Expression of GMEB-1 and GMEB-2 in Baculovirus
GMEB-1 and GMEB-2 proteins with the FLAG tag at the N terminus were expressed as a Baculovirus protein in SF9 cells using BAC to BAC Baculovirus Expression System according to the manufacturer’s instruction (Life Technologies, Inc.).

In Vitro Transcription and Translation
GMEB-1 (SP6), GMEB-2 (T7), CREB (T7), and GR (SP6) were expressed from clones in pCMV-SPORT, PCR 2.1, pSP73, and pBAL117, respectively, using either SP6 or T7 RNA polymerase (as indicated in parentheses) in TNT rabbit reticulocyte lysate system (Promega Corp.) according to manufacturer’s instructions.

Gel Shift Assay
Gel shift assays to detect the binding of GMEB-1, GMEB-2, CREB (single proteins or their mixtures), or HTC cytosol to 32P-GME oligonucleotides were performed as published (29 ). The mixtures of two or more proteins were incubated on ice for 12–16 h for the formation of protein complexes (28 ). The sequence of GME and mutant oligonucleotides M1, M2, and M3 have been described (15 ). The specificity of DNA binding was determined from the competition by 25-fold molar excess of cold biologically active GME, M1, and M3 oligonucleotides and the biologically inactive M2 oligonucleotide.

Pull-Down Assay
M2 agarose beads [anti-FLAG monoclonal antibody conjugated to agarose (Sigma)] were recharged by treating them with 100 mM glycine.HCl buffer, pH 2.5, (10 vol.) for 2–3 min and then neutralized with 1 M Tris.HCl, pH 8.0, (10 vol.). The beads were washed once with PBS, pH 7.4 (BioWhittaker, Inc., Walkersville, MD) and once with High Salt Buffer (10% glycerol, 20 mM Tris.HCl, 0.4 mM EDTA, 0.2% Tween, 10 mM mercaptoethanol, and 500 mM KCl). Baculovirus-infected cell extracts containing overexpressed GMEB-1 or GMEB-2 (100 µl) were incubated with the above prepared M2 agarose for 2–3 h at room temperature. The beads were then washed five times with High Salt Buffer for 10 min each. The beads were washed once with Low Salt Buffer (10% glycerol, 20 mM Tris.HCl, 0.4 mM EDTA, 0.2% Tween, 10 mM mercaptoethanol, and 250 mM KCl). The in vitro translated proteins (labeled with [35S]methionine) were added separately to the M2 beads bound with either Flag GMEB-1 or -2 and incubated for 1 h at room temperature and then 12–16 h at 4 C. The beads were then washed four to five times with Low Salt Buffer (0.5 ml) for 10 min at room temperature. Finally the beads were resuspended in 20 µl of Low Salt Buffer and treated with 20 µl of 2x SDS-PAGE buffer for 15 min at 37 C. The supernatant proteins were loaded onto a SDS-PAGE gel (10%) and visualized by fluorography.

Data Analysis
Gel shift bands were quantitated on a Macintosh Power G3 computer using the public domain NIH Image program (developed at the NIH and available on the Internet at http://rsb.info.nih.gov/nih-image/). All statistical analyses were by two-tailed Student’s t test using the program InStat 2.03 for Macintosh (GraphPad Software, Inc., San Diego, CA).


    ACKNOWLEDGMENTS
 
We thank A. Giordano, Richard Goodman, William Walker, and Keith Yamamoto for generously providing plasmids, Hinrich Gronemeyer for alerting us to the sequencing error of GMEB-2, Yoshihiro Nakatani (NICHD, NIH) for support and helpful discussion of this work, and Alan Wolffe (NICHD, NIH) for critical review of this paper.


    FOOTNOTES
 
Address requests for reprints to: Dr. S. Stoney Simons, Jr., Building 8, Room B2A-07, NIDDK/LMCB, NIH, Bethesda, Maryland 20892.

Received for publication August 8, 1999. Revision received February 18, 2000. Revision received April 5, 2000. Accepted for publication April 7, 2000.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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