(Received for publication, July 27, 1995; and in revised form, September 18, 1995)
From the
Glucocorticoids influence numerous cell functions by regulating gene activity. The glucocorticoid receptor (GR) is a ligand-activated transcription factor and, like any other transcription factor, does not modulate gene activity just by binding to DNA. Interaction with other proteins is probably required to enhance the establishment of a functional transcription initiation complex. To identify such proteins, we analyzed the in vitro interaction of the glucocorticoid receptor bound to a double glucocorticoid response element with nuclear proteins and describe here three interacting proteins with different molecular weights. One of them, which we named GRIP 170 (GR-interacting protein), was purified and microsequenced, and it turned out to be an unknown protein. When tested in a cell-free transcription assay, the fraction highly enriched for GRIP 170 does not influence basal promoter activity but does enhance GR induction.
Glucocorticoids are active in inducing gluconeogenesis in liver,
promote the development of various organs, cause apoptosis of lymphoid
cells, and are necessary for the growth of many cell types in
vitro. The glucocorticoid receptor (GR) ()belongs to
the superfamily of steroid hormone receptors and was the first
transcription factor to be isolated and studied in
detail(1, 2) . Most glucocorticoid-regulated genes
contain a short palindromic DNA sequence known as glucocorticoid
response element (GRE). Depending on the type of GRE and on the
promoter context, binding of GR to these elements can result in either
gene induction or repression.
For regulating gene expression, the
glucocorticoid receptor, like any other transcription factor, has to
interact either directly with the transcription/initiation complex or
indirectly via adapters or intermediary
factors(3, 4) . Nuclear receptors have been
demonstrated to interact with both types of factors. For some nuclear
receptors, but not for GR, interaction with TFB has been
shown (5, 6, 7) . The estrogen receptor was
demonstrated to interact with TAF
30, a component of the
TF
D complex (8) and with transcription
intermediary factor 1, a putative mediator of the ligand-dependent
activation function(9) . Other, unknown factors have been
demonstrated to interact with nuclear receptors in
vitro(10, 11) , or in vivo(12) or to enhance receptor-DNA binding in
vitro(13) . In addition, receptor interaction with
proteins involved in alterations of chromatin structure or function,
Swi3 and Spt6, has been shown(14, 15) .
We have been interested in analyzing mechanisms for a synergistically increased steroid response mediated by multiple binding sites for different or identical transcription factors. Cooperative DNA binding has been shown for the glucocorticoid and the progesterone receptor in the case of duplicated binding sites(16, 17, 18, 19) . GR can also synergize with various transcription factors(20) . Synergistic activation with the ubiquitous transcription factor OTF-1/Oct-1 is found in the context of the MMTV promoter, and this effect seems to be mediated by cooperative DNA binding(21) .
Replacement of the DNA binding domain of GR with the GAL4-DNA binding domain abolished cooperative binding to duplicated upstream activating sequence elements in contrast to functional synergy, which could still be observed(19) . Functional synergy may involve two transcription factors simultaneously (and cooperatively) touching a target protein. Therefore, we designed a strategy for the search of GR-interacting factors, which upon isolation should enhance GR mediated in vitro transcription. Our strategy is based on the use of DNA-bound GRs to a double GRE to avoid interactions irrelevant to transcriptional control and to possibly increase the binding affinity to the target protein. Using this procedure we identified three GR-interacting proteins (GRIPs).
Electroblotting was carried out for 2.75 h in
a semidry blotting system with a constant current of 1 mA/cm gel using STGM buffer (0.06% SDS, 12.5 mM Tris, 50
mM glycine, 5% methanol) as electrode buffer.
The Western blot analysis was carried out as follows. After separation of nuclear proteins by SDS-PAGE using a 7% polyacrylamide gel, the proteins were electroblotted to a PVDF membrane (Immobilon P, Millipore). After blocking, membranes were incubated overnight either with a 1:100 dilution of a polyclonal serum raised against hGR, or with a 1:200 dilution of an affinity-purified polyclonal antibody raised against Hbrm. Purified goat anti rabbit antibody was used as secondary antibody (Aurion, Germany). The immune complexes were visualized by the Immuno Gold detection kit according to the manufacturer (Aurion, Germany).
Template DNAs were preincubated
with GR, GRIP 170, and in each case with an equal amount of the GRIP
170 buffer for 10 min on ice in a volume of 12.5 µl. Then HeLa cell
nuclear extract (40 µg), nucleotides were added to make a
final volume of 25 µl, and transcription was carried out for 45 min
at 30 °C. Transcripts were treated as described(29) ,
separated by 6.5% denaturing polyacrylamide gel electrophoresis, and
visualized by autoradiography. Quantitation was made with a
PhosphorImager (Molecular Dynamics), and relative transcription was
determined with respect to the transcription of the control template.
Figure 1:
Direct interaction of
GRP-dGRE complex with several nuclear proteins. 100
µg of nuclear extract of dexamethasone-induced HeLa-cells (N, even-numbered lanes) and 100 µg of bacterial
extract (B, odd-numbered lanes) were separated by
SDS-PAGE and blotted on an Immobilon membrane.
P-labeled
dGRE was cross-linked to a protein fraction in the presence of
poly(dI-dC) for 5 min at 280 nm. Cross-linked protein fractions were as
follows: 10 µg of GR fraction (lanes 1-4); 10 µg of
nuclear extract (N, lanes 5 and 6); 10
µg of bacterial extract (B, lanes 7 and 8), and no protein (lanes 9 and 10).
Competition of the cross-link reaction with 25-fold excess of unlabeled
GRE-DNA reduces intensities or completely inhibits labeling of the
170-, 120-, and 95-kDa bands (lane 4). The bands were
visualized by autoradiography and labeled according to their apparent
molecular masses (170, 120, 115, and 95
kDa).
Although from our competition tests in the cross-linking reaction we
knew that HeLa-GR was bound to the double GRE, we wanted to confirm
this result with a glucocorticoid receptor from another source. We used
human GR expressed with a baculovirus vector in insect cells. After
purification by DNA affinity column (28) we used this protein
to generate a GRP-GRE probe. As seen in Fig. 2A the baculovirus-expressed GR identifies nearly the
same bands as the HeLa-GR including the 170-kDa band.
Figure 2:
Specificity of the
GRP-dGRE probe. 100 µg of nuclear extract of
dexamethasone-induced HeLa cells were separated by SDS-PAGE and blotted
on Immobilon membranes. A,
P-labeled dGRE was
cross-linked either to GR fraction (GRfr; HeLa nuclear extract
fraction enriched for GR) or to baculovirus-expressed GR (GRbv) in the presence of poly(dI-dC). The membrane was
incubated with the indicated cross-linked probes. The bands were
visualized by autoradiography. B, Western blot analysis of the
nuclear extract. The membranes were incubated with an antibody against
Hbrm (
Hbrm) or against hGR (
GR), and the
antibody complexes were visualized by Immuno Gold detection. C, nuclear extract (N) and 40 µg of a GRIP
170-enriched Q-Sepharose fraction (Q 15) were
SDS-PAGE-separated and blotted. The blot was incubated with
GRbv
P-dGRE in the absence of competitor (lanes 1 and 2) or in the presence of 25-fold excess unlabeled
GRbv
dGRE (lanes 3 and 4) or with
GRbr
P-sGRE (lanes 5 and 6).
Toward
the isolation and characterization of GRIP 170 we fractionated HeLa
nuclear extract (Fig. 3) by Q-Sepharose chromatography and
assayed for the presence of GRIP 170 with the HeLa
GRP-dGRE probe. In contrast to the unfractionated
nuclear extract, pooled QS fractions, eluted with 390-540 mM salt, contained only three out of the four labeled bands, GRIP
170, the 120-kDa protein, and the 95-kDa protein (Fig. 4A).
This pool from 2 g of nuclear extract was further purified on a Mono S
column, allowing the isolation of GRIP 170 containing fractions eluting
between 350 and 450 mM salt. This cation exchange
chromatography led to the separation of GRIP 170 from the two other
interacting factors, since they show longer retention times (not
shown). Further purification was achieved with heparin-Sepharose where
GRIP 170 could not be eluted in a linear salt gradient between 0 and
500 mM salt. The following one-step elution at 1.5 M salt revealed the GRIP 170 activity. GRIP 170 was recovered in two
fractions, which were diluted to a final salt concentration of 150
mM NaCl and concentrated on Mono Q column. The anion exchange
chromatography leads to three positive fractions eluting between 400
and 475 mM salt, respectively. Chromatography by these four
columns resulted in about 1000-fold purification of GRIP 170. The final
purification was achieved by SDS-PAGE and subsequent blotting on a PVDF
membrane. Fig. 4B shows the pattern of protein bands
after Coomassie staining (lane 1). In parallel an aliquot was
blotted after SDS-PAGE and incubated with baculovirus-expressed GR
after UV-cross-linking to a
P-dGRE oligonucleotide. Since
the 170-kDa band was labeled (Fig. 4B, lane
2), we excised and analyzed the corresponding band on the PVDF
membrane by N-terminal microsequencing. Additionally, a preparative
SDS-PAGE was run, and after Coomassie staining the 170-kDa band was
excised and digested by endoproteinase Lys-C. The resulting peptides
were purified by reverse phase HPLC and sequenced in addition to the
full-length protein. Three different sequences could be read and
compared with the Swiss Prot computer data base. No homology to known
protein sequences could be detected.
Figure 3: Enrichment of GRIP 170. 2 g of nuclear extract was separated by Q-Sepharose, and the fractions containing GRIP 170 (380-530 mM) were further purified by cation exchange chromatography (Mono-S). The resulting positive fractions (350-450 mM) were applied to heparin-Sepharose column. A linear gradient between 0 and 500 mM did not elute GRIP 170, but the activity was present in a 1.5 M salt fraction. Final concentration of GRIP 170 was performed by Mono Q separation, resulting in a 1.3-mg pool containing highly enriched GRIP 170.
Figure 4:
Purification of GRIP 170. A, 100
µg of nuclear extract of dexamethasone-induced HeLa cells (N), 100 µg of bacterial extract (B), and 50
µg of a Q-Sepharose pool containing partially purified GRIP 170
were separated by SDS-PAGE and blotted on an Immobilon membrane. The
membrane was probed with a cross-linked GRP-dGRE
complex as described under ``Experimental Procedures.'' GRIP
170, GRIP 120, and GRIP 95 are pointed out. B, 35 µg of a
Mono Q fraction containing highly enriched GRIP 170 were separated by
SDS-PAGE and were blotted on an Immobilon membrane. The membrane was
stained with Coomassie Blue (lane 1) or probed with a
cross-linked GRbv
P-dGRE complex (lane 2) as
described under ``Experimental Procedures.'' C, EMSA
with
P-sGRE was carried out in the presence of bovine
serum albumin (lane 1), with 0.6 µg of the GRIP 170
fraction (lanes 2 and 4) or with 0.35 µg of
baculovirus-expressed GR (lanes 3 and 4). The
retarded complex (GR) and the free probe (f.p.) are
indicated.
Another step toward the functional analysis of a protein interacting with a transcription factor would be the use of an in vitro transcription system. Therefore, we prepared nuclear extracts from HeLa cells (23) that efficiently mediate induction by purified glucocorticoid receptor(29) . Human GR was expressed in insect cells with a baculovirus expression system and purified(28) . DNA templates used contain the full-length MMTV promoter up to position -240 bp of the transcriptional start site. This promoter region encompasses the natural GRE sequences (31) and was fused to a G-less cassette of 380 bp in length. As an internal control the reaction contained a second template with a truncated MMTV promoter, lacking the GRE sequences but containing the nuclear factor I and octamer transcription factor I binding sites. This promoter up to -80 bp was fused to a G-less cassette of 280 bp in length and provides a standard for basal gene transcription. In vitro transcription in the presence of radioactive nucleotides yielded a specific transcript for both templates (Fig. 5A). The upper band in each pair of bands represents read-through transcripts, which are almost not affected by the presence of GR or GRIP 170 fraction, whereas the lower band represents the correctly initiated transcripts for both DNA templates and is enhanced in the presence of GR with the full-length MMTV template. PhosphorImager quantitation was used to determine the relative transcription activity. Addition of the GRIP 170 fraction to the basal transcription reaction results, if in anything, in a marginal increase in transcription on both promoters (Fig. 5, A, lanes 2 and 4, and B). Similarly, no effect was seen with an adenovirus major late promoter as template (data not shown). Addition of baculovirus-expressed GR generates a transcriptional induction as expected(29) . The combined addition of GR and GRIP 170 fraction shows a synergistic increase in transcription (Fig. 5, A, lane 6, and B). This induction very likely was mediated by GRIP 170 because all of the reactions contained the same amount of GRIP 170 buffer, since a different fraction not containing GRIP 170 did not change transcription efficiency, and finally GRIP 170 ``shoulder fractions'' are less effective as the peak fraction (Table 1).
Figure 5: Influence of GR and GRIP 170 on in vitro transcription of the MMTV promoter. A, where indicated, 0.35 µg of GR preparation and/or 0.6 µg of the purified GRIP 170 material were used as described under ``Experimental Procedures.'' Cell-free transcription reactions were performed with the GR-responsive plasmid pMMTV 240/380 and the control plasmid pMMTV 80/280. Correctly initiated transcripts are indicated (-240/380 and -80/280, respectively). Lanes 1 and 2 show a longer exposure of lanes 3 and 4 to visualize the unchanged basal transcription by GRIP 170 on both templates. B, data of six independent experiments, as shown in A, were normalized to receptor-mediated activity; mean values and standard errors are shown.
Here we identified three proteins (170, 120, and 95 kDa) that
interact with a specifically designed glucocorticoid receptor probe.
The probe consists of a P-labeled DNA harboring a double
GRE with UV-cross-linked GR molecules. This probe serves several
requirements. First, the GR can be easily and efficiently labeled.
Second, the double GRE arrangement provides several DNA bound GR
molecules, which increase the binding affinity to the target protein.
This assumption is based on the finding that transactivation is
stronger with two synergizing GREs as compared with individual GRE
sequences and is confirmed by the failure of the GR-
P-sGRE
probe to detect GRIP 170 on a blot. The GR
P-dGRE
probe also avoids detection of proteins interacting with the inactive,
DNA-unbound GR such as hsp 90, p59, and
others(1, 2, 32) . In addition, blotted HeLa
GR was not detected, which may be explained by an incorrect folding
after denaturation and renaturation or by the occupied dimerization
domain of the dimerized DNA-bound GR molecules.
Receptor interaction
and transcriptional activation has been shown for factors comprising
the TATA box-binding protein and its associated factors and
TFB complexes(5, 8) . There is no direct
proof that the GRIP 170 factor, binding to the
GR
P-dGRE probe, is identical to the factor within
the GRIP 170 fraction causing the increase in GR-mediated in vitro transcription. Nevertheless, the co-purification of these two
functions via four different columns and the reduced activity of GRIP
170 ``shoulder fractions'' argue for the transcriptional
activity of GRIP 170. From the fact that the transcription extract is
prepared from HeLa nuclei one would predict that GRIP 170 should be
present and that the addition of GRIP 170 should not change the
transcriptional activity. One explanation might be that GRIP 170 is
limiting in the presence of added GR and, therefore, that additional
amounts of GRIP 170 optimize the GR-mediated transcription. Comparison
of blotted protein from nuclear extracts prepared from
glucocorticoid-treated or from untreated cells did not reveal any
hormone-dependent change in GRIP binding intensities (not shown).
Our sequence analysis rules out the possibility that GRIP 170 might
belong to the TAF or TF group as far as these are known.
Similarly, we would rule out that GRIP 170 belongs to the group of
factors associated with the inactive GR, such as hsp 90, hsp 59, or hsp
70(33, 34, 35, 36) . Receptor
interaction and/or potentiation of receptor function has been shown for
proteins involved in alterations of chromatin structure or
function(14, 15, 30, 37) . Again,
the GRIP 170 sequence is different from these proteins (Swi, Snf, or
Spt6).
Other factors have been shown to increase the receptor binding affinity to DNA by in vitro binding tests. Glucocorticoid and androgen receptors require RAF 130 for optimal DNA binding(13) ; for the estrogen receptor hsp 70 and 48- and 45-kDa proteins increase the DNA association rate(12) ; and in the case of GR low molecular weight factors have been seen with a similar function(38) . Such an activity was not seen with GR and GRIP 170.
Receptor-interacting proteins with molecular weight comparable with that of GRIP 170 have been identified in conjunction with the estrogen receptor and named ERAP 160 or RIP 160(10, 11) . ERAP 160 and RIP 160 may be identical and bind to the estrogen receptor only in the presence of estrogen. For GR such a ligand-dependent binding would not be detectable, since GR will be activated through the purification procedure(39, 40) . Thus, based on the molecular weight, GRIP 170 could be similar or identical to ERAP 160/RIP 160. For the GRIP 170 fraction we found a GR-dependent transcriptional increase in the in vitro transcription assay, an activity not identified for previously described receptor-associated or interacting factors (10, 11, 12, 13) . Although in vitro binding to GR depends on the presence of two GREs, it remains to be shown, whether GRIP 170 is required to mediate synergy or to mediate the intrinsic induction from a single GRE in vivo.