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© 1995 Oxford University Press 4552-4558

Footnote

Expression of the gene for the POU domain transcription factor Tst-1/Oct6 is regulated by an estrogen-dependent enhancer

Expression of the gene for the POU domain transcription factor Tst-1/Oct6 is regulated by an estrogen-dependent enhancer Karin Renner , Elisabeth Sock , John R. Bermingham,Jr 1 and Michael Wegner*

Zentrum für Molekulare Neurobiologie, Universität Hamburg, Martinistrasse 52, D-20246 Hamburg , Germany and 1 Department of Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla , CA 92093-0648, USA

Received July 12, 1996; Revised and Accepted September 27, 1996 DDBJ/EMBLGenBank accession no. X99229

ABSTRACT

Expression of the POU domain protein Tst-1/Oct6 during development of glia and neurons is subject to a tight multifactorial control. Here we show that 17 [beta] -estradiol increases the level of endogenous Tst-1/Oct6 in glial cells. This effect was mediated at the level of gene expression by an enhancer present in the 5 ' flanking region of the mouse gene for Tst-1/Oct6, ~ 5 kb upstream of the transcriptional start site. The enhancer contained as the functional element a sequence motif that closely resembled a classical estrogen response element. It consisted of an imperfect palindrome with a spacing of 3 bp, and was bound in vitro by activated estrogen receptor. Furthermore, this element was able to confer estrogen responsiveness when introduced into a heterologous promoter. In the Tst-1/Oct6 gene enhancer, a TPA response element was found in close proximity to the estrogen receptor binding site. As a consequence, TPA and estrogen activated transcription of the Tst-1/Oct6 gene in a synergistic manner.

INTRODUCTION

POU domain proteins, in particular members of the class III and IV subfamilies, are expressed in very distinct spatio-temporal patterns in the developing and adult nervous system ( 1 - 4 ). It is believed that a combinatorial code of these proteins is involved in specifying the fate and identity of neuronal and glial cell populations. One of these POU domain proteins is the class III protein Tst-1/Oct6 which is also known as SCIP ( 5 - 9 ). In line with its supposed role during differentiation of glial and neuronal cells ( 10 , 11 ), targeted disruption of the Tst-1/Oct6 gene in mice led to severe disturbances of the normal myelination program in Schwann cells and to a fatal breathing defect associated with aberrant differentiation and migration of specific neurons ( 12 , 13 ).

If Tst-1/Oct6 is a key regulator of neural differentiation, its expression should be under tight control. Indeed, it was shown that axonal contact stimulates expression of the Tst-1/Oct6 gene in Schwann cells by an increase in the intracellular level of cyclic-AMP ( 14 ). Induction of Tst-1/Oct6 gene expression through elevation of cyclic-AMP levels could also be obtained by application of forskolin to Schwann cell cultures ( 8 , 11 ).

Other factors which are important in gliogenesis or neurogenesis, and therefore could represent potential regulators of Tst-1/Oct6 gene expression, are steroid hormones and retinoids. It has been known for a long time that dietary changes in the levels of 17[beta]-estradiol, triiodothyronine and vitamin A each have profound influences on the myelination process ( 15 - 17 ). Furthermore, steroid hormones and retinoids participate in the timing of oligodendrocyte development ( 18 - 20 ). While retinoic acid has been shown to modulate the expression of Tst-1/Oct6 in mouse P19 cells ( 7 ), no such analyses have been performed for steroid hormones. Here, we provide the first evidence for a regulation of Tst-1/Oct6 by 17[beta]-estradiol and describe the underlying mechanism.

MATERIALS AND METHODS

Plasmids

Approximately 10 kb of 5' flanking region from the mouse gene for Tst-1/Oct6 were isolated from a mouse genomic library derived from J1 ES cells ( 12 ). A Bgl II site was introduced at the translational start site of Tst-1/Oct6 [defined as +49 according to ( 21 )]. Using Sal I and Bgl II restriction endonucleases, this fragment was cloned into the luciferase plasmid pGL2basic (Promega), yielding the reporter plasmid p10Kluc. Successively shortened versions of p10Kluc were generated by using the following restriction sites: Nhe I (p6.5Kluc, containing sequences from approximately -6.5 K to +49), Avr II (p3Kluc, containing sequences from approximately -3 K to +49), Hin dIII (p2Kluc, containing sequences from approximately -2 K to +49), Bam HI (p0.6Kluc, containing sequences from -538 to +49), Not I (p0.3Kluc, containing sequences from -286 to +49), Sac II (pminluc, containing sequences from -93 to +49) and Ngo A IV (p0.1Kluc, containing sequences from -27 to +49). As shown in Figure 3 , several fragments from the region between the Nhe I and Avr II sites (sequences from approximately -6.5 to -3 K) were also inserted immediately in front of the minimal promoter of the Tst-1/Oct6 gene present in pminluc. The resulting derivatives of pminluc were designated as pNAminluc, pXAminluc, pNXminluc, pDSminluc, pDXminluc, pBSminluc and pXSminluc. Plasmid pBXminluc contained juxtaposed to the Tst-1/Oct6 minimal promoter a 397 bp Bst EII- Xba I fragment (DDBJ/EMBL/GenBank accession no. X99229) which is naturally situated ~5 kb further upstream. A mutant version of this 397 bp fragment was derived by introducing an Spe I site into the first half-site of an identified estrogen response element, and cloned in an identical manner, yielding pBXminluc mut. The estrogen response element was also inserted in single copy immediately in front of the rat prolactin minimal promoter present in p36luc ( 22 ) using oligonucleotide M1 5'-AATCACCATCAG AGGTCATCCTGCCCA GTTTCTAAA, where bold letters represent the identified estrogen response element. The resulting plasmid M1p36luc, as well as its mutant versions M2p36luc-M5p36luc, are shown in Figure 4 . Plasmids pRSV-ER and pCMV/Tst-1 have been described before ( 22 , 23 ).

Cell culture, transfections and luciferase assays

U87-MG glioblastoma, 33B oligodendroglioma and COS-7 cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). One day before transfection, U87-MG cells were plated at a density of 5 * 10 5 per 60 mm plate. U87-MG cells were transfected by the calcium phosphate technique ( 24 ) with 2 [mu]g of luciferase reporter plasmid and 0.5 [mu]g of expression plasmid for the estrogen receptor. The total amount of plasmid was kept constant. At 3 h post-transfection, cells were treated for 1 min with 30% (v/v) glycerol in phosphate buffered saline (PBS) and placed in fresh medium. Estrogen and phorbol-12-myristate-13-acetate (TPA) were added to some plates after an additional period of 20 h to a final concentration of 10 -6 M and 0.1 [mu]g/ml, respectively. During transfections cells were kept in DMEM suplemented either with 10% charcoal-stripped FCS or 0.5% untreated FCS with comparable results. Cells were harvested 48 h after transfection, and extracts were assayed for luciferase activity ( 22 ). For protein extracts COS-7 cells were transfected in DMEM/10% FCS at a density of 2 * 10 6 per 100 mm plate using DEAE-dextran ( 25 ).

Preparation of nuclear extracts and recombinant proteins

Nuclear extracts were prepared from 33B oligodendroglioma cells, and transiently transfected COS-7 cells as described ( 26 ). Shortly, cells from two 100 mm plates were washed twice with PBS, scraped from the plates in hypotonic buffer, swollen on ice and lysed by the addition of 1% Nonidet P-40 and vortexing. Nuclei were pelleted and extracted for 15 min at 4oC under constant rotation in 200 [mu]l ice cold 10 mM HEPES pH 7.9, 400 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM DTT, 1% Nonidet P-40, 2 [mu]g/ml pepstatin, 2 [mu]g/ml leupeptine and 1 [mu]g/ml aprotinin. Jun-D was expressed as a glutathione S -transferase fusion protein in the bacterial strain DH5[alpha], and purified as described ( 22 ).

Electrophoretic mobility shift assay

In general, 0.5 ng of 32 P-labeled probe (oligonucleotides M1-M6, for sequence see Figs 4 and 6 A) was incubated for 20 min at room temperature with 2.5 [mu]g of nuclear extract from COS-7 cells, 50 fmol of recombinant human estrogen receptor or 50 fmol of purified Jun-D in a 20 [mu]l reaction mixture containing 10 mM HEPES (pH 7.9), 5% glycerol, 25 mM NaCl, 2 mM DTT, 0.1 mM EDTA and 1 [mu]g of poly(dI-dC) as unspecific competitor. Reactions were loaded onto native 4% polyacrylamide gels and electrophoresed in 0.5 * TBE (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.3) at 180 V for 1.5 h.

Western blot analysis

Aliquots of 20 [mu]l of nuclear extract (~2.5 mg/ml) were size fractionated on an SDS-10% polyacrylamide gel and transferred to nitrocellulose membranes. Nitrocellulose filters were blocked for 1 h at room temperature with 5% non-fat milk in PBS + 0.1% Tween-20 (PBST). After rinsing the membranes with PBST, they were incubated for 1 h at room temperature with 1:3000 dilutions of rabbit antisera against Tst-1/Oct6 ( 27 ) or c-Fos (Santa Cruz Biotechnology) in PBST. Following three washes with PBST, membranes were incubated for 20 min at room temperature with horseradish-peroxidase coupled protein A in PBST at a 1:3000 dilution. After extensive washing, the antigen was detected with the enhanced chemiluminescence detection system (Amersham) as specified by the manufacturer.

RESULTS AND DISCUSSION

The oligodendroglioma cell line 33B expresses significant amounts of endogenous Tst-1/Oct6 as previously shown ( 25 ). Here we tested whether treatment of 33B cells with 17[beta]-estradiol or the estrogen-antagonist tamoxifen would influence the endogenous levels of Tst-1/Oct6. Addition of 17[beta]-estradiol for 12 h to 33B cell cultures led to a significant increase in the amount of Tst-1/Oct6 present in nuclear extracts (Fig. 1 ). Exposure to tamoxifen, on the other hand, caused a dramatic reduction in Tst-1/Oct6 levels. At the same time, levels of c-Fos protein remained unaffected in these cells by either estrogen or tamoxifen treatment. Thus it seems that the endogenous amount of Tst-1/Oct6 is regulated by estrogen in 33B cells.


Figure 1 . Estrogen-dependent expression of Tst-1/Oct6 in 33B oligodendroglioma cells. Nuclear extracts from 33B cells which were either kept in the presence of DMEM supplemented with 0.5% FCS (-, lane 1), or were additionally stimulated for 12 h with 1 [mu]M 17[beta]-estradiol (E 2 , lane 2), or 1 [mu]M tamoxifen (T, lane 3) were separated on SDS-10% polyacrylamide gels. Tst-1/Oct6 (upper panel) and c-Fos (lower panel) were detected using polyclonal rabbit antisera as primary, and horseradish-peroxidase coupled protein A (both diluted 1:3000) as secondary antibody. An extract from CV1 cells transiently transfected with pCMV/Tst-1 served as a control (lane 4). Size of markers are indicated on the right of the gel in kilodaltons.

To investigate the possibility of a transcriptional regulation by estrogen, we analyzed whether the 5' flanking region of the Tst-1/Oct6 gene when fused to a luciferase reporter and transfected into glial cells would respond to 17[beta]-estradiol. U87-MG glioblastoma cells were used for transfections. Because these cells lack endogenous estrogen receptor as evidenced in Western blot analyses (data not shown), this protein had to be supplied by co-transfection. The original construct contained ~10 kb of 5' flanking region from the mouse Tst-1/Oct6 gene. When transfected into U87-MG glioblastoma cells, the activity of this reporter construct could be stimulated ~10-fold in the presence of both estrogen and its receptor (p10Kluc in Fig. 2 ). No stimulation was observed, however, when only the hormone or the receptor were present.


Figure 2 . Estrogen-dependent stimulation of the transcriptional activity of the Tst-1/Oct6 5' flanking region. Luciferase reporter plasmids carrying successively shorter parts of the 5' flanking region of the mouse Tst-1/Oct6 gene were transfected into U87-MG glioblastoma cells with (+ER) or without (-ER) the expression plasmid for estrogen receptor pRSV/ER. Estrogen (E 2 ) was added to the media 24 h before harvesting where indicated. The following reporter plasmids were used: p10Kluc, p6.5Kluc, p3Kluc, p2Kluc, p0.6Kluc, p0.3Kluc, pminluc and p0.1Kluc. Luciferase activities in extracts from transfected cells were determined in four independent experiments, each performed in duplicate. Data are presented as fold inductions which were calculated for each reporter plasmid by comparing luciferase activities with values from cells which were transfected with reporter plasmid and empty RSV expression plasmid and were kept in the absence of added 17[beta]-estradiol.


Figure 3 . Mapping the estrogen response element in the 5' flanking region of the Tst-1/Oct6 gene. (A) Various parts of a 3.5 kb segment which is naturally positioned 6.5-3 kb upstream of the Tst-1/Oct6 coding region were cloned in front of the minimal promoter of the Tst-1/Oct6 gene present in pminluc. The resulting luciferase plasmids (pNAminluc, pXAminluc, pNXminluc, pDSminluc, pDXminluc, pBSminluc, pXSminluc) were transfected into U87-MG glioblastoma cells with (+ER) or without (-ER) the expression plasmid for estrogen receptor pRSV/ER. (B) Similarly, a 397 bp fragment naturally situated ~5 kb in front of the Tst-1/Oct6 coding region was cloned in wild-type (pBXminluc) or mutant version (pBXminluc mut) into pminluc, and transfected into U87-MG cells. Estrogen (E 2 ) was added to the media 24 h before harvesting where indicated. Luciferase activities in extracts from transfected cells were determined in four independent experiments, each performed in duplicate. Data are presented as fold inductions which were calculated for each reporter plasmid by comparing luciferase activities with values from cells which were transfected with reporter plasmid and empty RSV expression plasmid and were kept in the absence of 17[beta]-estradiol. The following enzymes were used in the generation of the various fragments: N, Nhe I; D, Nde I; B, Bst EII; X, Xba I; S, Sac I; A, Avr II.


Figure 4 . Mutagenesis of the estrogen response element from the 5' flanking region of the Tst-1/Oct6 gene. The ERE from the Tst-1/Oct6 gene was cloned in front of the rat prolactin minimal promoter of the luciferase reporter plasmid p36luc in its authentic sequence (M1) or after various base substitutions (M2-M5). The resulting luciferase plasmids were transfected into U87-MG glioblastoma cells with (+ER) or without (-ER) the expression plasmid for estrogen receptor pRSV/ER. Where indicated, 17[beta]-estradiol (E 2 ) was added to the media 24 h before harvesting. Luciferase activities in extracts from transfected cells were determined in three independent experiments, each performed in duplicate. Data are presented as fold inductions which were calculated for each reporter plasmid by comparing luciferase activities with values from cells which were transfected with reporter plasmid and empty RSV expression plasmid and were kept in the absence of added 17[beta]-estradiol.


To map the region responsible for mediating this estrogen- dependent transcriptional activation, we deleted various parts from the distal end of the Tst-1/Oct6 5' flanking region, thus generating a series of reporter plasmids carrying successively shortened fragments. With the exception of the shortest (p0.1Kluc), all fragments conferred similar basal levels of luciferase expression (data not shown). This indicates that a region of 142 bp immediately preceding the Tst-1/Oct6 coding region is fully sufficient to mediate basal transcription. We will refer to this region as the minimal Tst-1/Oct6 gene promoter.

Deletion of ~3.5 kb from the most distal part of the 5' flanking region led to no significant alteration in the estrogen response (p6.5Kluc in Fig. 2 ). However, when the 5' flanking region was further shortened by additional 3.5 kb, estrogen responsiveness was lost almost completely dropping from a 13-fold induction for p6.5Kluc to a mere 2.5-fold stimulation for p3Kluc. None of the reporter plasmids carrying even shorter 5' flanking regions was inducible by estrogen, with luciferase activities being indistinguishable in the presence and absence of 17[beta]-estradiol and its receptor. Therefore, an estrogen responsive element must be located between 6.5 and 3 kb upstream of the translational start site.

This conclusion is also supported by the fact that an Nhe I- Avr II fragment spanning this 3.5 kb region was able to confer estrogen responsiveness to the minimal Tst-1/Oct6 gene promoter which by itself could not be activated by 17[beta]-estradiol (compare pminluc with pNAminluc in Fig. 3 A). To determine the location of the estrogen response element in greater detail, we divided the 3.5 kb region into a distal Nhe I- Xba I and a proximal Xba I- Avr II subfragment. Only the distal fragment transferred estrogen responsiveness to the minimal Tst-1/Oct6 gene promoter, not however, the proximal (compare pNXminluc with pXAminluc in Fig. 3 A). In addition to these fragments, several other parts of the 3.5 kb region were analyzed in luciferase assays. All fragments which were still able to respond to 17[beta]-estradiol when inserted into a luciferase reporter in front of the minimal Tst-1/Oct6 gene promoter overlapped in a short 397 bp region demarcated by Bst EII and Xba I sites (Fig. 3 A).

This 397 bp fragment elicited an estrogen-response, when placed in front of the minimal Tst-1/Oct6 gene promoter (Fig. 3 B). The observed 6.5-fold stimulation was comparable with the one observed for most responsive fragments and approximately half as high as the 11-fold stimulation obtained with the whole 3.5 kb fragment (compare pNAminluc with pBXminluc in Fig. 3 ). Sequence analysis of the Bst EII- Xba I fragment revealed the presence of a motif (AGGTCANNNTG C CC A ) which differed only in two positions from the perfect palindromic estrogen response element AGGTCANNNTGACCT of the Xenopus vitellogenin A2 promoter ( 28 ). To investigate whether this motif is involved in mediating the estrogen responsiveness of the 397 bp enhancer, we changed the first half-site from AGGTCA to ACTAGT. This mutation led to a concomitant loss of the ability to confer estrogen responsiveness to the Tst-1/Oct6 gene promoter (Fig. 3 B).

In another set of experiments, we transferred the motif in its naturally occurring sequence, orientation and context to the rat prolactin minimal promoter, placing it directly in front of the TATA box of the luciferase reporter p36luc. As shown in Figure 4 , the presence of a single copy of the motif from the 5' flanking region of the Tst-1/Oct6 gene led to a robust 11-fold estrogen- dependent stimulation of luciferase gene expression which could not be detected in its absence. Therefore the identified motif is not only necessary, but also sufficient to mediate the estrogen response, and thus constitutes a bona fide estrogen response element (ERE).

Next we attempted a detailed analysis of the ERE from the 5' flanking region of the Tst-1/Oct6 gene by exchanging two bases in either the first half of the motif, the second half, or the spacer element in the middle. Changing the first half-site from AGGTCA to ACATCA led to a complete loss of the estrogen response (M2p36luc). Similarly, changing the second half site from TGCCCA to TGCTGA caused a severe reduction in the estrogen- dependent induction of luciferase activity (M5p36luc). However, when the second half site was mutated in such a way that a perfect palindrome similar to the ERE from the vitellogenin promoter was generated, estrogen response increased from an 11- to a 20-fold induction (compare M1p36luc with M4p36luc). As expected, mutation of two bases in the spacer region between both half-sites did not alter the responsiveness of this element toward estrogen (M3p36luc).

Given the similarity between the ERE from the 5' flanking region of the Tst-1/Oct6 gene and the prototypic ERE from the vitellogenin gene it seemed reasonable to assume that the estrogen effect could be mediated by direct binding of the estrogen receptor to this sequence motif. To analyze this possibility we prepared nuclear extracts from 17[beta]-estradiol treated COS cells which were either mock-transfected or transiently transfected with an expression vector for the estrogen receptor. These nuclear extracts were used in gel shift analyses with the ERE from the Tst-1/Oct6 gene in its natural context serving as a probe. Proteins from mock-transfected COS cell nuclear extracts were able to interact with the probe despite the absence of estrogen receptor in these extracts (Fig. 5 A). As expected, the resulting complex proved to be refractory to the presence of a monoclonal antibody directed against the estrogen receptor (Fig. 5 A) and was found to be non-specific in competition studies (data not shown).


Figure 5 . Electrophoretic mobility shift assays. (A) The radiolabeled ERE from the Tst-1/Oct6 gene (M1) was incubated with 2.5 [mu]g of nuclear extracts from COS cells which were either mock-transfected (NE, lanes 2 and 3) or transfected with pRSV/ER (NE/ER, lanes 4 and 5), and kept in the presence of 17[beta]-estradiol. For some binding reactions nuclear extracts were preincubated with a mouse monoclonal antibody (clone TE-111, Dianova) raised against estrogen receptor ([alpha]ER, lanes 3 and 5). Lane 1, probe alone. ( B ) Radiolabeled M1 was incubated with 2.5 [mu]g of nuclear extraxts from COS cells which expressed ligand-bound estrogen receptor in the presence of 10-fold (uneven-numbered lanes) or 100-fold (even-numbered lanes) molar excess of various competitor DNAs. Lanes 1 and 2, M1 competitor; lanes 3 and 4, M2 competitor; lanes 5 and 6, M3 competitor; lanes 7 and 8, M4 competitor; lanes 9 and 10, M5 competitor; lane 11, without competitor. Sequences of M1-M5 are shown in Figure 4. ( C ) The ERE from the Tst-1/Oct6 gene was incubated in wild-type (M1) or mutant (M2-M5) version in the presence (+) or absence (-) of nuclear extracts from COS cells which expressed ligand-bound estrogen receptor (NE/ER). ( D ) Radiolabeled M6 containing both the ERE and an adjacent TRE was incubated with 50 fmol purified Jun-D (JUN-D, lane 2), 50 fmol recombinant human estrogen receptor (ER, lane 3) or both (lanes 4-7). Antibodies (Ab) directed against estrogen receptor (ER, lane 5), Jun-D (JD, lane 6) or an unrelated antigen (NS, lane 7) were added to the reactions as indicated above the lanes. Lane 1, probe alone. Throughout the figure, complexes specific for estrogen receptor and Jun-D are marked by `ER' and `JUN' respectively, while the ternary complex of both proteins binding to DNA is labelled as `TC'. Only the part of the gel with the estrogen receptor complex is shown in (B) and (C).


Figure 6 . Synergistic stimulation of Tst-1/Oct6 gene expression by 17[beta]-estradiol and TPA. ( A ) Localization of TPA and estrogen response elements (TRE and ERE) in the 397 bp enhancer. Only the first 60 bases are shown, which also constitute the sequence of the M6 oligonucleotide. ( B ) Luciferase plasmids pNAminluc, pBXminluc and pBXminluc mut were transfected into U87-MG glioblastoma cells with or without the expression plasmid for estrogen receptor pRSV/ER (ER). 17[beta]-estradiol (E 2 ) or TPA were added to the media 24 h before harvesting as indicated. Luciferase activities in extracts from transfected cells were determined in two independent experiments, each performed in duplicate. Data are presented as fold inductions which were calculated for each reporter plasmid by comparing luciferase activites with values from cells which were transfected with reporter plasmid and empty RSV expression plasmid and were kept in the absence of added 17[beta]-estradiol and TPA.

Transfection of COS cells with an expression plasmid for the estrogen receptor led to the appearance of a second complex which had a significantly lower mobility than the complex from mock-transfected COS cells. This complex was specifically shifted to an even lower mobility in the presence of antibodies directed against the estrogen receptor. This finding not only shows the presence of estrogen receptor in the complex, but also proves that the ERE from the 5' flanking region of the Tst-1/Oct6 gene can indeed be recognized by estrogen receptor. In a series of competition experiments we compared the relative affinity of the estrogen receptor with the various mutant versions of the ERE used in previous transfection studies (Fig. 5 B). Competition efficiencies correlated well with the ability of the respective site to confer estrogen responsiveness to a heterologous minimal promoter. M3 which contained base substitutions in the variable spacer region proved to be almost as good a competitor as the wild-type M1 sequence, while M4 which contained a fully palindromic ERE was an even better one. Mutations in the second half-site (M5), and especially in the first half-site (M2), on the other hand, were highly effective in obliterating competitor function. Similar results were also obtained in experiments in which the various versions of the ERE from the Tst-1/Oct6 gene were used as probe instead of competitor (Fig. 5 C). Whereas M3 and M4 bound the estrogen receptor at levels slightly below and above M1, respectively, no complex containing the estrogen receptor could be observed with M2 or M5 as a probe. Thus, binding of estrogen receptor correlated well with the estrogen inducibility mediated by the respective site.

When looking at the sequence of the 397 bp enhancer, we noticed the presence of a perfect TPA responsive element (TRE) in the immediate vicinity of the identified ERE (Fig. 6 A). Using oligonucleotide M6 which comprised both ERE and TRE, we addressed the question whether TRE-binding AP-1 proteins and estrogen receptor could bind simultaneously to DNA. Because nuclear extracts yielded a highly complex pattern in gel shift experiments with M6 (data not shown), we used recombinant human estrogen receptor and the purified AP-1 component Jun-D. Estrogen receptor and Jun-D each generated specific complexes when incubated with the probe (Fig. 5 D). Simultaneous incubation of M6 with both proteins yielded a new complex in addition to the ones observed with either protein alone. This ternary complex was recognized by antibodies specific for estrogen receptor as well as by anti-Jun-D antiserum, not however, by an unrelated antibody. From the stoichiometry of complexes it can be concluded that binding of Jun-D and estrogen receptor is not cooperative. However, this finding does not exclude the existence of cooperative binding between estrogen receptor and one of the many other cellular AP-1 complexes of different composition.

Because functional synergism between estrogen and TPA has been observed in a number of other systems ( 29 - 31 ), transient transfections were performed to elucidate a potential crosstalk between these two agents in the regulation of the Tst-1/Oct6 gene promoter (Fig. 6 B). Luciferase reporter plasmids which contained both the ERE and the TRE were not only stimulated by the presence of ligand-bound estrogen receptor, but also by the presence of TPA (pNAminluc and pBXminluc). Remarkably, the presence of both TPA and the activated estrogen receptor led to a more than additive stimulation of luciferase expression showing the existence of synergism between ERE and adjacent TRE. As previously observed ( 32 ) the strength of the observed synergism increased with the distance between response elements on the one side and TATA-box containing minimal promoter on the other.

When only the TRE but not the ERE is present in the luciferase reporter (pBXminluc mut), TPA-dependent activation remained unchanged. Concomitant with a loss in estrogen responsiveness, however, synergistic activation by estrogen and TPA was no longer observed in this construct. These data clearly show that synergism is strictly dependent on the presence of the ERE.

Thus, the observed synergism between estrogen and TPA is mechanistically different from other cases described in the literature. For the ovalbumin gene promoter synergistic activation by TPA and estrogen is mediated by a single site which is different from both a classical TRE or ERE, and is bound by a complex of both AP-1 and estrogen receptor ( 29 ). In the human c-fos gene, synergism is mediated by a composite element which contains overlapping degenerate recognition sites for AP-1 and estrogen receptor ( 31 ). In our case, synergism rather seems to stem from both AP-1 and estrogen receptor binding to adjacent sites. This, of course, does not exclude the possibility that both factors also interact with each other directly.

Synergism with other signal transducers such as TPA seems to be typical for estrogen function in glial cells. For instance, estrogen-dependent stimulation of Schwann cell proliferation requires a simultaneous increase in intracellular cAMP levels ( 33 ). In addition to its influence on proliferation, estrogen also has diverse effects on the expression of myelin-specific genes ( 34 ). In agreement with these functions for estrogen in glial cells, estrogen receptor has been shown to exist in both Schwann cells and oligodendrocytes ( 33 - 35 ).

Here we have presented evidence for a regulation of the Tst-1/Oct6 gene by estrogen. Steroid-dependent regulation of gene expression via hormone-responsive distant enhancers has also been observed for a number of other POU domain proteins such as Pit-1 and Oct-3/4 ( 36 , 37 ), and provides a way of communication between steroid receptors and POU domain proteins different from the joint regulation of common target genes ( 38 - 41 ). We suggest that the observed estrogen-dependent transcriptional activation of Tst-1/Oct6, which is itself an important factor in the development of myelinating glia ( 8 , 10 , 12 ), is a key component in estrogen regulation of in vivo myelination ( 16 ).

ACKNOWLEDGEMENTS

We thank Dr D. B. Evans and Ciba-Geigy AG, Basel, Switzerland for the generous gift of recombinant human estrogen receptor. In addition to basic support from the BMBF, this work was supported by a grant from the Deutsche Forschungsgemeinschaft to M.W. (We1326/5-1).

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