The glucocorticoid receptor acts as an antirepressor in receptor-dependent in vitro transcription

Eur. J. Biochem. 215, 505-511 (1993) 0 FEBS 1993

The glucocorticoid receptor acts as an antirepressor in receptor-dependent in vitro transcription Per ERIKSSON and Orjan WRANGE Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden

(Received February 24, 1993) - EJB 93 0294/1

Glucocorticoid-receptor-dependent and glucocorticoid-response-element-dependent in vitro transcription was established using a crude nuclear extract and purified glucocorticoid receptor from rat liver. The capacity of glucocorticoid receptor to stimulate in vitro transcription was only detectable when basal transcription, i.e. transcription in the absence of glucocorticoid receptor, had been repressed. Transcriptional repression was achieved either by adding purified histone H1, or by lowering the amount of DNA template relative to the amount of crude nuclear extract. Glucocorti- coid receptor caused a 1.1 2 0.7-fold stimulation of transcription from the mouse-mammary-tumor- virus promoter when basal transcription was not repressed, and a 7.0 ? 1.5-fold stimulation when basal transcription had been repressed by addition of histone H1. Similar results were obtained when using a minimal promoter consisting of two glucocorticoid-response elements and a TATA box. Our data suggest that glucocorticoid receptor stimulates in vitro transcription by an antirepression mechanism.

The glucocorticoid receptor (GR) belongs to a family of ligand-activated transcription factors. The hormone/receptor complex recognizes and binds to specific DNA sequences that act as glucocorticoid-response elements (GRE) in vivo if positioned in the vicinity of a promoter. Positively as well as negatively regulated glucocorticoid-responsive promoters have been described (Evans, 1988; Beato, 1989).

Several studies have suggested a role for chromatin in maintaining a glucocorticoid-inducible promoter in a repressed state, and a possible function of GR is to unwind the chromatin, i.e. to act as an antirepressor. A strictly phased nucleosome(s), organizing the GRE, is altered or dissociated by hormone-induced GR activation (Richard-Foy and Hager, 1987; Carr and Richard-Foy, 1990; Reik et al., 1991) as observed by an increased sensitivity to DNase I (Zaret and Yamamoto, 1984). Furthermore, the nuclear-factor-1 (NF-1) site, in the mouse-mammary-tumor-virus (MMTV) promoter, is only occupied after GR induction, as if opening of chromatin was required to allow NF-1 binding (Cordingley et al., 1987). Removal of 90% of the nucleosomes from a MMTV promoter, by co-injecting competing non-specific DNA into Xenopus oocytes, resulted in a strong stimulation of MMTV transcription in a GR-independent fashion (Perlmann and Wrange, 1991). These results suggest that the function of GR in positive gene regulation is to unwind chromatin structure. The chromatin would thus act as a repressor and GR would act as a hormone-inducible antirepressor. This model for GR- activated transcription does not exclude the possibility that

GR has additional functions, such as attracting component(s) of the transcription machinery. This would mean that GR activates transcription concomitantly by antirepression and activation as recently suggested by another study (Archer et al., 1992).

A strong direct-activation capacity of GR, e.g. by protein/ protein interaction with component(s) of the transcription machinery, might be revealed by in vitro transcription experiments. However, the presence of inhibitors in crude extracts usually used in these experiments makes it difficult to evaluate if an activating activity or an antirepressor activity is responsible for the observed transcriptional stimulation (Freedman et al., 1989). Croston et al. (1991) have described a strategy to approach this problem. They identified histone H1 as a repressor for in vitro transcription and showed that its inhibitory effect is counteracted by certain sequence-specific transcription factors that will act as antirepressors. The antirepression of histone H1 was also observed when the DNA template was reconstituted into nucleosomes (Laybourn and Kadonaga, 1991).

We have used this strategy (Croston et al., 1991) to address to what extent GR mediates antirepression and/or direct activation in in vitro transcription experiments. For this pur- pose we used purified histone H1, nuclear extracts depleted of histone H1 and purified GR, all prepared from rat liver. We show that GR has an antirepression activity both for the natural MMTV promoter and a simple synthetic promoter harbouring two GRE sequences in addition to a TATA box.

Correspondence fo 0. Wrange, Department of Cell and Molecu- lar Biology, Medical Nobel Institute, Karolinska Institutet, BOX 60400. S-104 01 Stockholm. Sweden MATERIALS AND METHODs

Fax: +46 8 31 35 29. Abbreviafions. GR, glucocorticoid receptor; GRE, glucocor-

ticoid-response element; MMTV, mouse mammary tumor virus ; NF-1, nuclear-factor one; AdML, adenovirus 2 major late.

Construction and preparation of DNA fragments The plasmids containing the adenovirus-2-major-late pro-

moter (AdML promoter in pBalEl, a gift from T. Edlund)

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and the MMTV promoter (pLSwt, 5.4 kb, a gift from E. Buetti and H. Diggelman) as well as the DNA probes used for S 1-nuclease mapping have been described previously (Buetti and Kuhnel, 1986; Perlmann and Wrange, 1991).

2GRE-OVEC, 6.9 kb, was constructed from pMTV ( - 200 to - 148)* which contained two tandemly arranged GRE sequences from the -190 to -167 position of the MMTV promoter (Eriksson and Wrange, 1990). A Sstl- BglII fragment, containing the two GRE sequences, was sub- cloned into a pGEM-1 vector. Thc Ssrl-Sull fragment of this construct was then transferred to an OVEC-1 vector (Westin et al., 1987). OVEC-REF, with a 28-bp deletion near the initiation site, was described previously (Westin et al., 1987). RNA analysis was performed by S1-nuclease mapping using a 5'-end-labelled Sull -BamHl double-stranded fragment of OVEC-1 as a probe.

GR preparation GR - ['H]triamcinolone-acetonide complex was purified

from rat liver as described previously (Eriksson and Wrange, 1990), with the exception that for the last Mono Q (Phar- macia LKB Biotechnology Inc.) purification a salt gradient containing 20 mM Tris/HCl, pH 7.8, 0.0-1.0 M KCl, 2% (by vol.) glycerol was used. GR was eluted at approximately 0.18 M KCI. This modification was made since glycerol above 10% (by vol.) inhibited the in vim transcription activity of our nuclear extracts (data not shown). This change in purification procedure did not alter or impair the DNA-binding ability of GR as tested by DNase-1 footprinting and mobility-shift assay (Li, Q. and Wrange, O., unpublished results). GR purity in different preparations varied 70-95% (Fig. 1). GR was quantified assuming that one molecule of tritiated hormone is bound to one GR subunit (Wrange et al., 1989). Since our GR preparation has approximately 50% DNA-binding activity and has a 2000-fold DNA-binding selectivity (Perlmann et al., 1990) this means that approximately 3 pmol GR must be added to saturate the GRE of 400 ng MMTV template which contains 3-4 GR-homodimer-binding sites.

Histone H1 and nuclear extract The nuclear extract was prepared from rat liver essen-

tially as described by Gorski et al. (1986). The temperature was kept at 0°C throughout the entire procedure. One liver, approximately 12.5 g from an adrenalectomized rat (to mini- mize intranuclear GR), was homogenized in 75 ml 2.2 M sucrose, 10 mM Hepes, pH 7.6, 15 mM KCl, 10% (by vol.) glycerol, 0.15 mM spermine, 0.5 mM spermidine, 2 mM EDTA, 0.5 mM dithiothreitol, 0.7 pg/ml pepstatin, 0.7 pg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride and 16.7 pg/ml aprotinin (Trasylol). The homogenate was layered on top of three 10-ml cushions consisting of the same buffer and centrifuged at 22000 rpm for 45 min in a Sorvall AH- 627 rotor. The nuclear pellet was resuspended and diluted to A2,,, 10 in lOmM Hepes, pH7.6, 1OOmM KCl, 3 mM MgC12, 0.1 mM EDTA, 10% (by vol.) glycerol, 0.1 mM phenylmethylsulfonyl fluoride and 1.67 pg/ml aprotinin. 1/10 volume 4 M (NH,),SO, was added and after 30 min on ice the nuclear extract was clarified by a 60-min centrifugation at 35000 rpm in a Sorvall A-641 rotor. The clarified superna- tant, i.e. the nuclear extract, was adjusted to 2.27 M by the addition of solid (NH,),SO,. The precipitate was allowed to form during a 30-min incubation on ice and was collected by

a 25-min centrifugation at 35000 rpm in a Sorvall A-641 rotor. The protein pellet was suspended in 25 mM Hepes, pH 7.6,40 mM KC1, 0.1 mM EDTA, 1 mM dithiothreitol and 10% (by vol.) glycerol and dialyzed twice for 2 h against 500ml of the same buffer. Any remaining precipitate was removed by a 5-min centrifugation at 15OOOXg in Eppen- dorf tubes. The protein concentration in these extracts was 4-8 mg/ml as estimated by the formula pg/ml = 183 A230 -75.8 A,,, (Kalb and Bernlohr, 1977).

Histone H1 was purified by a modified procedure of the previously described protocol (Croston et al., 1991). The su- pernatant, remaining after the 2.27-M (NH,),SO, precipitation of the nuclear extract, was dialyzed for 18 h against 25 mM Hepes, pH 7.6, 0.1 mM EDTA, 1 mM dithiothreitol and 10% (by vol.) glycerol. The solution was then subjected to chromatography on a Mono S (Pharmacia LKB Biotech- nology Inc.) column and eluted with a 0.1 - 1 .O-M KCl gradient. Histone H1, which was eluted at approximately 0.5-M KCl, was identified by SDSPAGE (Eriksson and Wrange, 1990) with purified calf thymus histones as markers (Sigma). The pooled fractions of histone H1, had a protein concentration of 0.7 pg/pl which corresponds to approximately 22 pmol Hl/p1 (Kalb and Bernlohr, 1977).

In vitro transcription A typical in vitro transcription assay consisted of a

40-pl reaction containing 25 mM Hepes, pH 7.5, 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 5-10% (by vol.) glycerol, 6 mM MgCL, 10 pl nuclear extract, 400 ng su- percoiled-DNA template, 30 U ribonuclease inhibitor (Pro- mega), 0.6 mM ATP, CTP, GTP and UTP. GR was routinely incubated with the DNA template for 30 min at 25OC. After this incubation, the nuclear extract was added either in the presence or absence of histone H1. After 15 min on ice and subsequent addition of nucleotides, the reactions were incubated at 26°C for 45 min and terminated by adding 200 mM NaCl, 20 mM EDTA and 1 % SDS. A control incubation was performed by adding 9 pmol histone H1 to the template fol- lowed by a 15-min incubation at 0°C. 2-4pmol GR was then added and the sample was incubated at 25 "C for 15 min. Nucleotides and nuclear extract were added as above. After phenol extraction and ethanol precipitation the DNA was removed by DNase-1 treatment (RQ1 DNase 1, Promega). RNA analysis, using nuclease-S1 mapping, was performed as described by Perlmann and Wrange (1991). RNA synthe- sis was inhibited by 0.5 pg/ml a-amanitine, showing that the transcription reaction is mediated by RNA polymerase I1 (data not shown).

Quantification of bands on gels was performed using an Image Quant v3.0 Fast-Scan system (Molecular Dynamics; Johnston et al., 1990). Background activity was subtracted for each quantification. In experiments using the GR-responsive MMTV promoter the AdML promoter was used as a positive control. This constitutively active promoter was not stimulated by GR. However, its ability to be inhibited by GR prevented its use as an internal control when quantifying the GR-induced stimulation of the MMTV promoter. In experiments using the 2GRE-OVEC promoter, OVEC-REF was used as internal standard for quantification of GR stimulation.

RESULTS Experimental approach

We have used a nuclear extract from rat liver as a source for thc basal transcription factors in our in vitro transcription

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Nuclear extract

M GR H1 ' + H l -HI.

9 7 4 -I - 66.2 0

4 5 0 _1_x

31.0 - 21.5 0

14.4 - Fig. 1. SDS/PAGE of purified glucocorticoid receptor, purified histone H1 and nuclear extracts used in the in vih.0 transcription assays after staining with Coomassie brilliant blue. M, markers. Numbers refer to the molecular mass (kDa) of the standard proteins: phosphorylase b ; bovine serum albumine; ovalbumin; carbonic an- hydrase; soybean trypsin inhibitor; lysozyme (highest molecular mass first). GR, purified glucocorticoid receptor (94 m a ) . H1, purified histone H1. Nuclear extract, two lanes showing rat liver nuclear extract with (+Hl) or without (-H1) the same relative amount of H1 as the 18-pmol H1 extract used in the in vitro transcription experiments. The arrows show the migration of the double band consti- tuting histone H1.

experiments. Preparation of this extract involves an ammo- nium-sulfate precipitation which separates the transcription factors from histone H1 (Croston et al., 1991). Further purification of histone H1 resulted in a fraction of high purity as shown by SDSPAGE (Fig. 1). This figure also shows that a mixture of purified histone H1 and nuclear extract, with the same relative amounts used in the in vitro transcription experiments, i.e. 18 pmol H1, contained detectable amounts of H1 while this was not the case in the control nuclear extract without added H1.

We established the amount of histone H1 required to re- press basal transcription of the GRE-containing promoters to a level where transcription was barely detectable. This oc- cured at 9 - 18 pmol H1 in a standard transcription reaction containing 400ng template (data not shown). In vivo, the amount of histone H1 corresponds to approximately one H1 molecule/35 -45 bp chromatin-linker DNA (Croston et al., 1991). For the 400ng DNA template used in our experiments, it would correspond to 13-17 pmol DNA-binding sites for histone H1. This suggests that in our in vitro transcription assays, approximately 1 H1 moleculehinding site was required to suppress basal transcription. This is in agreement with previous results by Croston et al. (1991).

GR acts as an antirepressor counteracting histone-Hl-mediated repression of in vitro transcription from the MMTV promoter

Fig. 2 shows an in vitro transcription experiment, in the presence or absence of purified histone H1, using the MMTV promoter, a wild-type GR-activated promoter. The MMTV- regulatory region (Fig. 2B) contains two glucocorticoid-responsive domains located within positions -190 to -167 and -134 to -76, that altogether bind 6-8 GR subunits at full occupancy (Wrange et al., 1989; Perlmann et al., 1990).

If no histone H1 was added to the nuclear extract, the addition of GR, in amounts required to saturate the GRE sequences of the MMTV template, had no effect on MMTV

A DESIGN OF THE IN VlTRO TRANSCRPTION REACTION. EXTRACT

DNA + / - H I STOP 4 & z 0 30 45 90 rnm

t NUCLEOTDES

6 DNA CONSTRUCT

P.ITV -200 -180 -160 -140 -120 -100 -80 -60 -40 -20 *I +20

C 9 pmol Hl I8 pmol H1 0 2.7 5.4 0 2.7 5.4 0 2.7 5.4 pmol GR

C AdML

I i

-h- wn cMMTV

1 2 3 4 5 6 7 8 9

D RELATIVE TRANSCRIPTION ACTIVITY

MML

MMTV

0 2 . 7 5 . 4 . ( 0 2 .75 .41 . ,O 2 . 7 5 . 4 1 prnolGR

9 prrm HI 18 pmol H1

E INDUCTION OF MMTV PROMOTER (-FOLD INCREASE)

9 pmol H1 - 18 pmol H i

0 2 7 5 4 0 2 7 5 4 0 2 7 5 4 p m d G R

Fig. 2. GR acts as an antirepressor by counteracting histone-Hl- mediated repression of in vitro transcription from the MMTV promoter. (A) The design of the in vitro transcription experiment. (B) The control region of the MMTV promoter with response elements for GR. NF-1 and Oct-1 are indicated. (C) S1-nuclease protec- tion analysis of transcripts after in vitro transcription of 400 ng MMTV and 20 ng AdML DNA. (D) Phosphoimager quantification of the results shown in (C). Black bars refer to the activity of AdML promoter and open bars to the activity of the MMTV promoter. (E) GR induction of the MMTV promoter. For each extract (containing 0, 9 or 18 pmol histone Hl), the activity in the presence of GR was divided by the activity in the absence of GR. For all bands, the background density was first subtracted.

transcription (1.1 k0.7-fold increase, mean 2 SD, n = 3; Fig. 2C, lanes 1-3). This was in contrast to the distinct GR- dependent 7.0 k 1.5-fold stimulation (mean 2 SD, n = 3) of MMTV transcription observed when titrating basal transcription by adding 9-18 pmol purified histone H1 to the nuclear extract (Fig. 2C, lanes 4-9). The AdML promoter, which does not contain any GRE, showed no GR-dependent stimu-

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lation in the presence or absence of histone H1. Instead, the AdML-promoter transcription was reduced by GR. The quantification of transcriptional activity by phosphoimager analysis is shown in Fig. 2D. This figure demonstrates that the basal activity of the MMTV promoter was repressed in the presence of histone H1 and that GR-stimulated transcription did not reach the basal transcriptional activity obtained in the absence of added histone H1. The data show that histone-H1 -mediated repression is counteracted by purified GR in the GRE-containing MMTV promoter but not in the AdML promoter. This suggests that GR acts as a GRE-dependent antirepressor.

Usually GR was incubated with the template before addition of histone H1 and nuclear extract. The concomitant addition of GR and histone H1 to the template resulted in a similar GR-dependent antirepression (data not shown). Incuba- tion of histone H1 and template before the addition of GR resulted in a lower and more variable GR-induced antirepression effect on the MMTV promoter. In these experiments, the addition of 9pmol H1 repressed transcription to 31 t 12% (mean t SD, n = 5) compared to the control in the absence of H1. When GR was added after H1 the transcriptional activity was increased to 72 t 18% (mean 2 SD, n = 5). We conclude that the repression obtained by adding histone H1 is at least partially reversed by GR.

GR-dependent antirepression is also observed in a simple synthetic promoter containing two GRE sequences and a TATA box

The MMTV promoter contains several other factor-binding DNA elements in addition to the two GRE sequences (Fig. 2B); both a NF-1 and two octa-factor-responsive elements are located between the TATA box and the GRE sequences (Nowock et al., 1985; Briiggemeier et al., 1991). To investigate whether the results obtained using the MMTV promoter are valid with a simpler promoter, we constructed the 2GRE-OVEC promoter that contains two GRE sequences upstream of a P-globin TATA box (Fig. 3B). The two GRE sequences were derived from the -200 to -148 DNA segment of the MMTV promoter containing a GR-induced DNase-I footprint at -190 to -167; it does not contain any other factor-binding sites, according to analysis by linker- scanning mutagenesis of this segment (Buetti and Kuhnel, 1986). Two such segments, when inserted as direct repeats, placed the two GR-binding segments between residues - 147 to -65, relative to the transcription-start site, with a spacing of 58 bp as measured from the centre of each GRE.

As shown in Fig. 3C and D, a histone-H1-mediated effect for 2GRE-OVEC transcription was obtained similar to that observed with the MMTV promoter. Quantifications (Fig. 3 E and F) illustrate that GR-dependent transcriptional stimulation was increased from 1.8 t 0.5-fold (mean t SD, n = 11) in the absence of histone H1 to 6.2 t 1 .4-fold (mean t SD, n = 11) in the presence of 9-18 pmol histone H1. In these experiments, the transcriptional activity of the 2GRE-OVEC promoter was normalized to the activity of the reference plasmid, which contains no GRE and a 28-bp deletion around the transcription-initiation site (Fig. 3B). The reference plasmid, as well as an OVEC-1 promoter lacking GRE (data not shown), showed no response to GR, thus indicating that a specific interaction between GR and the GRE was required for transcriptional induction. We conclude that GR also acted as an antirepressor for the 2GRE-OVEC promoter since a significant increase in GR-dependent stimulation of tran-

A DESIGN OF THE IN VITRO TRANSCRIPTION REACTION:

r G R EXTRACT DNA +/- H1 STOP

6 i o 65 si, min t

NUCLEOTDES

TATA DNA CONSTRUCTS

& Y 2 G R E - WE C

Y

T*TA S W O I F + REF .. .. ... . - L O -120 -1w -80 -60 -40 -20 +I +20 +40

C 9 pmol H1 1

0 2.1 4.2 0 2.1 4.2 pmol GR

D RELATIVE TRANSCRIPTION ACTIVITY

150000 0 XREOVEC

9 pmol H I

100000

50000

0 2 1 4 2 0 2 1 4 2 pmolGR

E F REUTIVE TRANSCRIPTION ACTIVIM OF 2GREQVEC INDUCTION OF XREQVEC

NORMALIZED TO REF NORMALIZE0 TO REF I-FOLD INCREASE1

9 pmol H1

100000 n

Fig. 3. GR-dependent antirepression of a synthetic promoter which contains two GRE sequences and a TATA box. (A) Design of the in vitro transcription experiment. (B) Control regions of the 2GRE-OVEC and REF constructs. (C) Quantified S1 mapping of transcripts after in vitro transcription of 400 ng 2GRE-OVEC and 80 ng REF plasmids. (D) Phosphoimager quantification of the result shown in C. Black bars refer to the transcription activity of OVEC- REF and open bars to the activity of 2GRE-OVEC. For all bands, the background density was first subtracted. (E) Transcription activity of 2GRE-OVEC from D after being normalized to OVEC-REF. For each extract used (0 or 9 pmol Hl), the density of the 2GRE- OVEC band was normalized to variations in the reference band. (F) Transcription activity (-fold GR induction) of the 2GRE-OVEC promoter. For each extract (including either 0 or 9pmol Hl), the activity of the 2GRE-OVEC promoter in the presence of GR, as determined in (E) was divided by the activity of the 2GRE-OVEC promoter in the absence of GR.

scription, in relative terms, is achieved by first reducing basal transcription. The GR-dependent transcription in the presence of histone H1 did not reach the level of basal transcription in the absence of H1 (Fig. 3D); this corroborates the suggestion that GR acts as an antirepressor. Binding of 0.25-0.5 pmol TFIID (Promega) to the DNA template had no influence on the GR-dependent transcriptional activity, regardless of the presence or absence of histone H1 (data not shown).

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c RELATIVE TRANSCRIPTION ACTIVITY OF 20 ng AdML

A DESIGN OF THE IN VlTRO TRANSCRIPTION REACTION:

A D N A t/- GR EXTRACT STOP

1 1 0 60 105 rnin

t NUCLEOTIDES

D RELATIVE TRANSCRIPTION ACTIVITY OF 50~1600 rig MMTV

30000 1 B 50 100 200 400 800 1600 ng MMTV

' - +" - +" - + " - + " - + " - +'GR

CAdML

E INDUCTION OF MMTV (. FOLD INCREASE]

2 n + MMTV

Fig.4. GR-dependent in vitro transcription of the MMTV promoter after reducing the amount of template DNA relative to the amount of protein extract. (A) Design of the in vitm transcription experiment. The MMTV promoter is shown in Fig. 2B. (B) Quantified S1 mapping of in iitro transcription of 50-1600 ng MMTV and 20 ng AdML in the presence or absence of 4.4 pmol GR and a constant amount of nuclear extract (10 PI ) . (C) Phosphoimager quantification of AdML activity indicated in (B) is shown. (D) Phosphoimager quantification of MMTV activi{y indicated in (B) is shown. (E) Activity (-fold GR induction) of the MMTV promoter. For each template concentration, the activity in the presence of GR was divided by the activity in the absence of GR.

Reduction of the amount of template DNA leads to reduced basal transcription and enhanced GR-dependent stimulation

In the i r i 1irr.o transcription experiments using 2GRE- OVEC (Fig. 3). a 1.8+0.5-fold (mean 2 SD, n = 11) GR- dependent transcriptional stimulation was observed also in the absence of added histone H1. This low but reproducible induction could be a result of GR-dependent antirepression, e.g. mediated by residual amounts of histone H1 or other components in the nuclear extract acting as inhibitors or re- pressors of the in vitro transcription proce this signal represents a direct GR-dependen than an antirepression, possibly by contacting basal transcription factor( s ) thus stimulating the formation of a preinitiation complex.

We investigated this further by titrating the amounts of GRE-containing DNA template in the absence of added histone H1 (Fig. 4). 20 ng AdML plasmid was incubated with 50- 1600 ng MMTV DNA while keeping the concentration of protein extract constant. In two independent experiments, the highest GR-dependent transcriptional stimulation (5.2 -C 3.S-fold, mean ? SD) of the MMTV promoter was obtained at an MMTV-DNA template level of 100 ng (Fig. 4E). This amount of template resulted in suboptimal basal transcription of both the AdML (Fig. 4C) and the MMTV promoter (Fig. 4D). At MMTV template levels of 200 and 400 ng, which produced optimal b 1 transcription, the relative GR-induced enhancement was significantly lower ( 1.9 -f 0.3-fold, 1 .8 -C 0.8-fold, respectively, mean -C SD, Fig. 4E). It appears GR is counteracting the inhibitory effect observed for transcription at the lower amounts of template (50- 100 ng) and that increased amount of template (up to approximately 400 ng) titrates out inhibitory factors thus resulting in an increased basal transcription. The amount of positive GR-mediating factors are not limiting for

100-400 ng template since the level of GR-induced transcription is not altered within this range. These experiments are consistent with GR acting as an antirepressor. This GR effect was similar to the results obtained in the experiments shown in Fig. 2 and Fig. 3 but with the difference that the basal transcription activity was reduced by decreasing the amount of template instead of by adding purified histone HI. In both cases the GR stimulation was only observed for the GRE-containing promoter (compare Fig. 4 C and D).

At higher amounts of DNA template (800-1600 ng), the transcriptional activity of both the MMTV and the AdML promoter was reduced and the GR-dependent stimulation was lost, probably because of titrating out positive factors essential for transcription.

DISCUSSION

Our results show that antirepression is the cause of GR- dependent transcriptional activation in our in vitro system. This conclusion is based on histone H1 being a non-specific repressor for in vitro transcription as demonstrated by Cros- ton et al. (1991).

A GR-mediated stimulation of transcription was obtained also in the absence of added histone H1. This GR effect was increased, in relative terms, by decreasing the amount of template in relation to the amount of protein extract thus resulting in decreased basal transcription (Fig. 4). In this case, the inhibitors counteracted by GR are inherent components of the nuclear extract. The inhibition of in vitro transcription due to a low level of template is well established (Manley et al., 1980). We conclude that this GR-dependent and GRE- dependent transcriptional stimulation is to a major extent also caused by antirepression.

A weak but histone-H1 independent GR effect was observed using the 2GRE-OVEC promoter (1.8 -C 0.5-fold,

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mean ? SD, n = 1 I , Fig. 3) as well as for some experiments using the MMTV promoter (1.8-1.9-fold, Fig. 4). Our experiments do not resolve whether this weak GR stimulation is caused by antirepression or by a direct activation, for ex- ample by a protein-protein interaction with component(s) of the transcriptional-initiation complex. Addressing this issue requires further purification of the in vitro transcription system.

The mechanistic basis for the observed GR-mediated inhibitory effect for the AdML promoter is unknown but inhibition due to non-specific DNA-binding of GR or squelching of an AdML-specific factor are possible explanations. Squelching, however, does not explain the observed GRE- dependent GR stimulation since addition of a wide range of GR concentrations did not lead to a reproducible reduction of transcription from the MMTV, 2GRE-OVEC or the OVEC-REF promoter.

We were unable to address the ligand dependence of the GR-induced transcription since our receptor is purified as a complex with the hormone in an activated DNA-binding form. It has been established that the hormone ligand cannot be reversibly dissociated from activated GR without further GR reactivation. Such an in vitro reactivation may be achieved using a crude reticulocyte lysate but only with trace amounts of GR and is thus unsuitable for our in vitro transcription experiments (Hutchinson et al., 1992). However, we have demonstrated that dissociation of the hormone from our preparations of purified GR results in concomitant loss of the DNA-binding activity (Wrange et al., 1989). The requirement for a GRE to obtain receptor-induced in vitro transcription thus implies that the GR effect observed in our experiments is indeed hormone dependent.

How are histone H1 and GR interacting when generating the positive GR-dependent and GRE-dependent effect? DNase-I footprinting experiments, using the MMTV promoter and purified histone Hl , as well as a band-shift assay, using a single GRE of 29 bp, demonstrated that histone H1 binds to DNA without sequence specificity (Eriksson, P. and Wrange, O., unpublished results). These experiments also demonstrated that bound GR will stay bound to specific sequences of the GRE after addition of histone H1. This result, and the fact that GR could partially reverse the histone-H1 induced repression of the MMTV promoter, argues against the idea that histone-H1 repression reflects a non-specific and insoluble H1 -DNA aggregation. We favour the hypothe- sis that histone H1 is inhibiting transcription by its general and non-specific binding to DNA and that this DNA binding is perturbed locally by the binding of GR. Whether this effect requires only GR or if other factors are necessary, i. e. co- antirepressors, remains to be investigated. A recent report by Yoshinaga et al. (1992), showed that a yeast SWI3-specific antibody inhibits the stimulation of in vitro transcription in Drosophila embryo extracts exerted by an Escherichia-coli- expressed GR derivative. They also showed that GR and SWI factors form a complex, offering an explanation for the GRE dependence of the transcription. They hypothesized that SWI factors act, in concert with the GR derivative, by re- moving various proteins from the template, i.e. acting as a coantirepressor. A link of SWI proteins to chromatin is of- fered by the localisation of supressor mutants in yeast swi- strains to chromatin components, e.g. a HMG-1-like protein and histone H3 (Peterson and Herskowitz, 1992). Croston et al. (1992) have demonstrated that GAL4-VP16 antirepression, which is detectable in Drosophila embryo extract, requires a co-antirepressor activity.

What functional domain(s) of GR are required for the observed antirepression? This is difficult to address, since the N-terminal domain has drastic effects on DNA-binding affinity and specificity (Wrange and Gustafsson, 1978 ; Eriks- son and Wrange, 1990). Furthermore, the isolated DNA- binding domain has an affinity for DNA which is 1-2 orders of magnitude lower (HLd et al., 1990) than intact GR (Perlmann et al., 1990).

A report by Tsai et a1.(1990) described a GR-stimulated in virro transcription system utilising GR expressed in a ba- culovirus system. This GR preparation did not require hormone ligand to bind DNA. They used a nuclear extract from HeLa cells prepared according to Dignam et al. (1983), a procedure which does not involve separation of H1 from the transcription factors. Based on promoter-commitment experiments, they suggested that the GR-induced transcription was mediated by GR contacting the basal transcription machinery, i.e. by direct activation. Our conclusion that GR acts mainly as an antirepressor is in apparent conflict with their result. These authors did not evaluate the presence of repres- sors, e.g. histone H1, in their in virro transcription experiments.

Our results, suggesting that GR acts mainly as an antirepressor in vitro, are in accordance with in vivo results con- cerning the interplay of GR and chromatin. It remains to be shown to what extent our results reflect the in vivo events involving chromatin. Laybourn and Kadonaga (1991) demonstrated that transcriptional properties of H1 -DNA com- plexes were similar to those of H1-containing in vitro reconstituted nucleosomes. Furthermore, a report by Bresnick et al. (1 992) has demonstrated a glucocorticoid-hormone-dependent reduction of histone H1 in the MMTV promoter in vivo and the concomitant appearance of an NF-1 footprint. The removal of histone H1 may be an important event in creating a promoter ready for the subsequent binding of other transcription factors.

We are indebted to Ulla Bjork for excellent technical assistance. Dr James Kadonaga is gratefully acknowledged for helpful advice and for kindly communicating unpublished results essential for this study. We are indebted to Gunnar Westin for providing the plasmids OVEC-1 and OVEC-REF. Drs Urban Lendahl, Christer Hoog, Qiao Li. Lars Wieslander, Bjorn Vennstrom and Patrik Blomquist are acknowledged for constructive criticism of this manuscript. This work was supported by grants from the Swedish Cancer Society (No 2222-B92-08XAC), the foundations of Fredrik Lundberg, Ingabritt and Arne Lundberg and M. Bergvall.

REFERENCES Archer, T. K., Lefebrvre, P., Wolford, R. G. & Hager, G. L. (1992)

Transcription factor loading on the MMTV promoter: a bimodal mechanism for promoter activation, Science 255, 1573- 1576.

Beato, M. (1989) Gene regulation by steroid hormones, Cell 56, 335 - 344.

Bresnick, E. H., Bustin, M., Marsaud, V., Richard-Foy, H. & Hager, G. L. (1 992) The transcriptionally-active MMTV promoter is depleted of histone H1, Nucleic Acids Rex 20, 273-278.

Briiggemeier, U., Kalff, M., Franke, S., Scheidereit, C. & Beato, M. ( 1 991) Ubiquitous transcription factor O F - 1 mediates induction of the MMTV promoter through synergistic interaction with hormone receptors, Cell 64, 565-572.

Buetti, E. & Kuhnel, B. (1986) Distinct sequence elements involved in the glucocorticoid regulation of the mouse mammary tumor virus promoter identified by linker scanning mutagenesis, J. Mof. Biol. 190, 379-389.

511

Carr, K. D. & Richard-Foy, H. (1990) Glucocorticoids locally disrupt an array of positioned nucleosomes on the rat tyrosine aminotransferase promoter in hepatoma cells, Proc. Narl Acad. Sci. USA 87, 9300-9304.

Cordingley, M. G., Riegel. A. T. & Hager, G. L. (1987) Steroid- dependent interaction of transcription factors with the inducible promoter of mouse mammary tumor virus in vivo, Cell 48, 261 - 270.

Croston, G. E.. Kerrigan, L. A., Lira, L. M., Marshak, D. R. & Kadonaga, J. T. (1991) Sequence-specific antirepression of histone HI-mediated inhibition of basal RNA polymerase I1 transcription, Science 251, 643-649.

Croston, G . E., Laybourn, P. J., Paranjape, S. M. & Kadonaga, J. T. (1 992) Mechanism of transcriptional antirepression by GALA- VP16, Genes & Del: 6 , 2270-2281.

Dignam. J. D.. Lebovitz, R. M. & Roeder, R. G. (1983) Accurate transcription initiation by RNA polymerase I1 in a soluble extract from isolated mammalian nuclei, Nucleic Acids Res. 11, 1475- 1489.

Eriksson, P. & Wrange. 0. (1990) Protein-protein contacts in the GR homodimer influence its DNA binding properties. J. Bid. Chem. 265, 3535 -3542.

Evans, R. M. (1988) The steroid and thyroid hormone receptor su- perfamily. Science 240, 889 - 895.

Freedman, L. P., Yoshinaga, S. K., Vanderbilt, J. N. & Yamamoto, K. R. (1989) In vitro transcription enhancement by purified de- rivatives of the GR, Science 245. 298-301.

Gorski. K., Carneiro, M. & Schibler, U. (1986) Tissue-specific in vitro transcription from the mouse albumin promoter, Cell 47, 767-776.

Hutchinson, K. A., Czar, M. J., Scherrer, L. C. & Pratt, W. B. (1992) Monovalent cation selectivity for ATP-dependent association of the glucocorticoid receptor with hsp70 and hsp90, J . Biol. Chem. 267, 14047-14053.

Hard, T.. Dahlman. K.. Carlstedt-Duke, J., Gustafsson, J . - k & Rigler, R. (1990) Cooperativity and specificity in the interactions between DNA and the GR DNA binding domain, Biochemistry 29, 5358-5364.

Johnston, R. F.. Pickett, S. C. & Barker, D. L. (1990) Autoradio- graphy using storage phosphor technology, Electrophoresis 11.

Kalb, V. F. & Bernlohr. R. W. (1977) A new spectrophometric assay for protein in cell extracts, A n d . Biochem. 82, 362-371.

Laybourn, P. J. & Kadonaga, J. T. (1991) Role of nucleosomal cores and histone H1 in regulation of transcription by RNA polymerase 11, Science 254, 238-245.

355 - 360.

Manley, J. L., Fire, A,. Cano, A,, Sharp, P. A. & Gefter, M. L. (1980) DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract, Proc. Narl Acad. Sci. USA 77, 385553859,

Nowock, J., Borgmeyer, U., Piischel, A. W., Rupp, R. A. W. & Sippel, A. E. (1985) The TGGCA protein binds to the MMTV- LTR, the adenovirus origin of replication, and the BK virus enhancer, Nucleic Acids Res. 13, 2045-2061.

Perlmann, T., Eriksson, P. & Wrange, 0. (1990) Quantitative analysis of the GR-DNA interaction at the mouse mammary tumor virus glucocorticoid response element, J. Biol. Chem. 265, 17222- 17229.

Perlmann, T. & Wrange, 0. (1991) Inhibition of chromatin assembly in Xenopus oocytes correlates with derepression of the mouse mammary tumor virus promoter, Mol. Cell. Biol. 11, 5259- 5265.

Peterson, C. L. & Herskowitz, I. (1992) Characterization of the yeast SWII. SW12 and SW13 genes, which encode a global activator of transcription, Cell 68, 573-583.

Reik, A., Schiitz, G. & Stewart, A. F. (1991) Glucocorticoids are required for establishment and maintenace of an alteration in chromatin structure : induction leads to a reversible disruption of nucleosomes over an enhancer, EMBO J. 10, 2569-2576.

Richard-Foy, H. & Hager, G. L. (1987) Sequence-specific position- ing of nucleosomes over the steroid-inducible MMTV promoter,

Tsai, S. Y., Srinivasan, G., Allan, G. F., Thompson, E. B., O’Malley, B. W. & Tsai, M.-J. (1990) Recombinant human GR induces transcription of hormone response genes in vitro, J. Biol. Chem. 265, 17055 - 17061.

Westin, G., Gerster, T., Miiller, M. M., Schaffner, G. & Schaffner, W. (1987) OVEC, a versatile system to study transcription in mammalian cells and cell-free extracts, Nucleic Acids Res. 15, 6787-6798.

Wrange, 0. & Gustafsson, J.-A. (1978) Separation of the hormone- and DNA-binding sites of the hepatic GR by means of proteo- lysis, J. B i d . Chem. 253, 856-865.

Wrange, O., Eriksson, P. & Perlmann, T. (1989) The purified activated GR is a homodimer, J. Biol. Chem. 264, 5253-5259.

Yoshinaga, S. K., Peterson, C. I. , Herskowitz, I. & Yamamoto, K. R. (1992) Roles of SWI1, SW12 and SW13 proteins for transcriptional enhancement by steroid receptors, Science 258, 1598- 1604.

Zaret, K. S. & Yamamoto, K. R. (1984) Reversible and persistent changes in chromatin structure accompany activation of a glucocorticoid-dependent enhancer element, Cell 38, 29-38.

EMBO J. 6 , 2321 -2328.

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The glucocorticoid receptor acts as an antirepressor in receptor-dependent in vitro transcription