Molecular and Cellular Endocrinology, 69 (1990) 167-178 Elsevier Scientific Publishers Ireland, Ltd.
MOLCEL
167
02235
Transcription factors different from the estrogen receptor stimulate in vitro transcription from promoters containing estrogen response elements M. Kaling, T. Weimar-Ehl,
M. Kleinhans
and G.U. Ryffel
*
Kernforschungszentrum Karlsruhe, Institut ftir Genetik und ftir Toxikolagie von Spaltstoffen, Karlsruhe, F. R.G. (Received
Key words: Rat hepatoma
13 November
FTO-ZB cell; Transcription,
1989; accepted
in vitro; Estrogen
4 December
response
1989)
element;
Vitellogenin
Bl promoter;
USF
Summary The estrogen response element (ERE) directly linked to a TATA box induces CAT activity in a hormone-dependent manner in Fe 33 cells, the rat hepatoma cell line FTO-2B, stably transfected with the human estrogen receptor (ER). The same promoter construct mediates the stimulation of in vitro transcription. This stimulation is dependent on the presence of the ERE. Induction of transcription in a variety of nuclear extracts derived from mammalian cells is of the same magnitude irrespective of the presence of ER. Similarly, transcription in vitro mediated by Bl vitellogenin 5’ flanking sequences in different nuclear extracts is not due to the interaction of the ER with the ERE. Competition analyses with a variety of oligonucleotides reveal that proteins different from the ER, which recognize ERE-like DNA elements, functionally interact with the ERE in vitro. These experiments suggest that ubiquitous proteins related or even identical to the transcription factor USF (MLTF) activate in vitro transcription in an ERE-dependent manner.
Introduction Estrogen-mediated gene expression is brought about by ligand-induced binding of estrogen receptor (ER) to a specific DNA sequence - the estrogen response element, ERE in the 5’ flanking region of target genes (for reviews see Yamamoto, 1985; Evans, 1988; Ryffel et al., 1988; Beato, 1989). We have identified in the vitellogenin A2 gene
Address for correspondence: M. Kaling, Kemforschungszentrum Karlsruhe, Institut fur Genetik und ftir Toxikologie von Spaltstoffen, Postfach 3640, D-7500 Karlsruhe 1, F.R.G. * Present address: Institut ftr Zellbiologie (Tumorforschung), Universitltsklinikum Essen, Hufelandstr. 55, D-4300 Essen, F.R.G. 0303-7207/90/$03.50
0 1990 Elsevier Scientific
Publishers
Ireland,
of Xenopus the ERE as being the 13 bp palindromic sequence GGTCA NNN TGACC (Klein-Hitpass et al., 1986). This sequence is sufficient to confer estrogen inducibility to a heterologous promoter (Klock et al., 1987; Martinez et al., 1987; Klein-Hitpass et al., 1988b) and binds the estrogen receptor as a homodimer in a liganddependent manner (Kumar and Chambon, 1988). Single-point mutations introduced into the ERE destroy its function and decrease its affinity for the receptor (Klein-Hitpass et al., 1988b, 1989). Estrogen response can also be achieved through synergism of two closely adjacent mutated EREs, which are not functional on their own but together constitute an estrogen response unit, ERU (Martinez et al., 1987; Klein-Hitpass et al., 1988a). Cato et al. (1988) showed that an ERE and a Ltd.
progesterone response element form a response unit that functions synergistically, when the two different steroid hormones estrogen and progesterone are present. Furthermore, synergism of hormone response elements with other regulatory elements, known to increase basal activity in a hormone-independent manner, has been reported (SchUle et al., 1988; Strlihle et al., 1988). All these findings establish that the activity of a given hormone response element can be modulated by other regulatory sequences. Such effects have been explained by postulating an interaction of the regulatory proteins involved. Interaction between steroid hormone receptors and other tram-acting factors might represent one of the many possible mechanisms which lead to activation of transcription of a given gene. These molecular mechanisms are still poorly understood, although some of these factors, particularly the steroid hormone receptors, are well characterized (for reviews see Evans, 1988; Green and Chambon. 1988). A prerequisite for a direct experimental approach to study the events leading to gene regulation by steroid hormones seemed to us the development of an in vitro transcription system, which initiates transcription in an ER- and/or ERE-dependent manner. In vitro transcription systems have been successfully employed to characterize tram-acting factors and their cognate cis elements (Dynan et al., 1985; Jones et al., 1985; Fletcher et al., 1987) to identify tissue-specific transcription factors (Gorski et al., 1986; Bodner and Karin. 1987; Scheidereit et al., 1987: Schorpp et al., 1988). or to characterize the specific role of transcription factors in transcription initiation (Hai et al., 1988). In the present study we report on the development of an in vitro transcription system, the activity of which depends on the presence of an ERE. Quite unexpectedly we observed that ubiquitous rruns-acting factors and not the ER are responsible for this activity in vitro. Materials and methods Construction of plasmids Plasmids pl-T and pl-TG replacing the HindIII-BglII
were constructed by fragment of synO-T
and synO-TG, respectively, carrying the Xenopus HP1 sequence (Schorpp et al., 1988) with the polylinker of pBlCAT3 ( HindIII-BglII fragment). All other constructs are derivatives of pl-T and pl-TG. Synthetic double-stranded oligonucleotides encompassing the EREwt, the EREmt3 or the USF sequences and carrying Hind111 and BumHI restriction ends replaced the polylinker in PI-T and pl-TG, thus generating plasmid containing the respective DNA element. Dimers of EREwt and EREmt3 were constructed by ligating the BumHI restriction fragments of pA2((331/-319)ztkCATS’ and pA2(C~330/-319)ztk-CAT8+ (KleinHitpass et al., 1988a) into BgIII linearized and dephosphorylized pl-T and pl-TG. Thus the polylinker is not removed from these constructs. Plasmids carrying Bl gene 5’ flanking sequences were constructed in the following way: a 6.4 kb EcoRI subclone of the vitellogenin Bl gene genomic cloneXXlv201 (Wahli et al., 1982) encompassing 5’ flanking sequences and the cap site of the gene was digested with BamHI and PvuII. Two of the resulting fragments, about 1.8 kb were isolated on low melting agarose and ligated with a Hind111 linker (8mer). Subsequent digestion with Hind111 and BglII yielded a 520 bp fragment which was isolated and cloned into HindIII-BglII digested pl-TG to generate pBl(-562/-37)-TG. Alternatively the same fragment was ligated into synO-TATA Bl (Ryffel et al., 1989) thereby replacing the HP1 sequence. This resulted in plasmid pB1((562/-24)-G. For construction of pB1 (-299/ -37)-TG and pBl(-299/ -24)-G the plasmid pBl(-562/ -37)-TG was digested with Hind111 and XhoI. The DNA fragment resulting from restriction with both enzymes was isolated, digested with DdeI and blunt ended by filling in with Klenow polymerase. This gave a 300 bp fragment to which a Hind111 linker was ligated. Digestion with Hind111 and BgIII resulted in a fragment encompassing the desired 5’ flanking sequences of the vitellogenin Bl gene. It was isolated and cloned into Hind111 - BglII digested pi-TG or synO-TATA Bl. The control plasmid consisting of HP1 in front of a shortened -G box (HPlcaslOO) was constructed by digesting synO-TG with X/z01 and filling in the overhangs with Klenow polymerase. After digestion with Hind111 the vector band was
169
isolated and ligated with a 180 bp fragment encompassing HP1 and a shortened -G box. This fragment was obtained by digesting synO-TG with PuuII. The correct PuuII fragment was isolated, cleaved with FokI, and the overhanging ends were blunt ended with Klenow polymerase. Subsequent digestion with Hind111 resulted in the 180 bp fragment, which was isolated for cloning. The structure of all clones was verified by DNA sequencing according to Sanger et al. (1977). Cell culture and transfections FTO-2B and Fe 33 cells were grown in a 1 : 1 DMEM/Ham’s F12 medium mixture containing 10% fetal calf serum (FCS) and penicillin/streptomycin (100 U/ml). Cells were transfected using the DEAE-dextran procedure described earlier (Druege et al., 1986). After transfection, cells were either treated with 1 x lo-’ M diethylstilbestrol or were left untreated and cultured in medium depleted by charcoal treatment of any components which act as estrogens. Nuclear extract preparations Rat liver nuclear extract was prepared essentially as described by Gorski et al. (1986). Nuclear extracts from cells grown in cell culture were
+ 260167
33118; Fe33
+ 331167 +HEO
prepared as described by Ryffel et al. (1989). 18 and 1 h prior to harvesting, cells were treated with 1 X lo-’ M 17b-estradiol. The concentration of estrogen receptor in every extract was determined using the Abbott ER enzyme immunoassay (EREIA), according to the specifications of the supplier. In vitro transcription In vitro transcription reactions were performed essentially as described previously (Dobbeling et al., 1988; Ryffel et al., 1989).
Introduction of estrogen responsiveness into FTO-2B rat hepatoma cells Estrogen-induced activation of the vitellogenin genes is restricted to hepatocytes (Wahli and Ryffel, 1985). Therefore we decided to establish an estrogen-responsive hepatoma cell line as an experimental system to set up an in vitro transcription assay. We have chosen the rat hepatoma cell line FTO-2B in which we have previously observed an estrogen response upon transient transfection with the human estrogen receptor expression vector HE0 (Druege et al., 1986). Via stable
+ 331167
26016:
331,67+ +HEO
FTO-2B
Fig. 1. FTO-2B cells become estrogen responsive upon stable transfection with a human estrogen receptor expression vector. Transient transfection of vitellogenin-tk-CAT fusion genes into Fe 33 (left panel) and FTO-2B cells (right panel). Cells were transfected with either 5 pg salmon sperm DNA and 5 pg pA2(-331/-87)tk-CAT8+, or 5 pg pA2(-260/-87)tk-CAT8+ (Klein-Hitpass et al., 1986), or with 5 pg pKCR2-ER (HEO) (Green et al., 1986) and 5 pg pA2(-331/-87)tk-CAT8+. Cells were kept in medium containing 10% charcoal-treated FCS without (-) or with (+) 1 X10-’ M diethylstilbestrol (DES). After 4 days, cell extracts (200 ng protein) were assayed for CAT activity.
I 70
transfection of HE0 using nro selection we obtained a number of estrogen-responsive clones and chose the one showing the highest level of induction - Fe 33 - for further characterization. Fe 33 expresses high CAT activity in hormonetreated medium upon transient transfection with the estrogen-inducible indicator plasmid pA2(-331/ -87)tk-CAT8+, containing an ERE (Klein-Hitpass et al., 1986) (Fig. 1, left panel). In hormone-free medium, the same plasmid mediates only basal level transcription. The non-inducible plasmid pA2(-260/-87)tk-CAT8’. lacking an ERE, gave only low CAT activity under both conditions. Cotransfection of pA2(-331/ -87)tk-CAT8+ and the ER-cDNA expression plasmid HE0 does not result in an increased CAT activity compared to transfection with the indicator plasmid alone {Fig. 1). This indicates that the concentration of ER in Fe 33 is not a limiting factor in estrogen induction. As expected, in the stem cell line FTO2B cotransfection of the ER is absolutely essential for a hormone effect (Fig. 1, right panel).
thymidine kinase promoter (Klein-Hitpass et al., 1988bf. The tk promoter fragment used (- 105/ +51) is known to contain DNA sequence elements recognized by several transcription factors (Jones et al., 1987). It has been shown that the glucocorticoid receptor functions synergistically with a number of different l’rans-acting factors (Schule et al., 1988; Strahle et al.. 1988). To ex-
a
PI-T
ERE and TA TA box are sufficient to mediate estrogen inducihility in oioo In transient transfection studies we have shown that the 13 bp ERE as found in the Xenopus vitellogenin A2 gene confers inducibility to the
Fig. 2. The ERE confers estrogen inducibility to a TATA box. (u) Schematic drawing (not to scale) of pl-T. the basal vector used for construction of plasmids for transient transfections. Nucleotide sequences of oligonucleotides inserted into Hind111 and &/II sites of PI-T are shown on the bottom. The 13 bp palindromic ERE as found in the Xenopus vitellogenin A2 gene and its homologues are boxed. Mutations relative to the wild-type sequence are shown in small tetters. The gap into the USF sequence was introduced to obtain optimal alignment with the other seqtences. (b) Transient transfections into Fe 33 cells were performed by the DEAE-dextran procedure (Druegc et al., 1986). Plasmids are PI-T derivatives carrying the oligonucleotide sequences indicated on the left (EREwtd and EREmt3d: dimers of EREwt and EREmt3, respectively) instead of the polyhnker. Extracts of cells either treated with I x 10 -’ M DES in 70% ethanol ( +. hatched bars) or with 70% ethanol alone ( -. white bars) were prepared 40 h after transfection and assayed for CAT activity (200 ng protein each). Induction factors (IF), indicated on the right. are corrected for mock transfection and are expressed relative to the value obtained with the vector PI-T (pl) alone.
Hind;ll
b
%
IF
L&F
+
11
EREmt3 d
’
EREwtd
l
EREmt3
+
EREwi
+
37
I I
41 09
21 I
Pi
+
10 I
/ 2 reiaf,ve
3 CAT
4 act,v,,y
5
3 6
171
chide such contributions in our system, we placed ERE sequences without any other known regulatory elements immediately upstream of a TATA box in the vector pl-T. This plasmid contains the CAT gene as indicator (Fig. 2~). Results of transient transfections of these constructs into Fe 33 cells are shown in Fig. 2b. The ERE wild-type sequence (EREwt) linked to the TATA box confers a slight but reproducible estrogen-dependent increase in CAT activity (2fold), whereas the mutated element (EREmt3) did not mediate any effect upon estrogen treatment. This agrees with our earlier results using the tk promoter (Klein-Hitpass et al., 1988b). An EREwt dimer directly linked to the TATA box mediates a 4-fold induction in the presence of hormone (Fig. 2b). This only additive effect is in contrast to the synergistic interaction of EREs in the tk promoter context reported previously (Klein-Hitpass et al., 1988a). A 4-fold increase in CAT activity is also observed when testing a dimer of EREmt3 in transient transfection (Fig. 26). This is similar to the value found previously using the tk promoter. As a control we used the vector pl-T without any regulatory element except the TATA box. The activity of this construct was not induced by estrogen. To further prove that the estrogen response depends on the presence of the ERE we inserted into pl-T an oligonucleotide corresponding to the USF recognition sequence. This sequence, found in the adeno major late promoter (Carthew et al., 1985), bears a high degree of similarity to the ERE (see Fig. 2~). However, it is not active in inducing CAT activity in a hormone-dependent manner (Fig. 26). Taken together, we conclude that a single ERE is sufficient to confer estrogen inducibility to a TATA box. In contrast to results obtained when using the tk promoter induction is only moderate and we did not observe synergism in hormone response using EREwt dimers. The ERE is a positive regulatory element in an in vitro transcription system Having established the minimal requirements for hormone response in vivo, we used these, namely the estrogen-responsive cell line Fe 33 as a source of nuclear extract and the minimal promo-
ter conferring the hormone response, to set up an in vitro transcription system. To monitor the extent of in vitro transcription in a rapid and efficient way, we inserted into the single XhoI site of the various pl-T derivatives the 400 bp G-free cassette described earlier (Sawadogo and Roeder, 1985; Schorpp et al., 1988). In the absence of GTP, transcripts from these templates are generated exclusively downstream of the TATA box along the G-free cassette and can therefore readily be analyzed. A shortened -G box of 200 bp under the control of the adeno major late promoter was included into each reaction as internal standard for quantification (Kugler et al., 1988; see Schorpp et al., 1988). As the preparation of nuclear extract involves dialysis, the concentration of estrogen is reduced. We therefore, added to all assays 17P-estradiol to a final concentration of lo-’ M. All promoter constructs are able to induce transcription in vitro when tested in a nuclear extract derived from Fe 33 cells (Fig. 3). Compared to the plasmid pl-TG, in which the ERE is replaced by a polylinker sequence, the EREwt mediates a moderately increased transcriptional activation, which upon dimerization (EREwtd) is further increased. We conclude that the ERE activates transcription in vitro. The possible interpretation that the polylinker acts as a negative element can be excluded, since this polylinker is present in the active dimer constructs. Furthermore, no negative effect of the same polylinker has been observed previously (Schorpp et al., 1988; Ryffel et al., 1989). Surprisingly, EREmt3 combined with a TATA box is also active in directing transcription in vitro, although the same promoter construct is not estrogen response in vivo (see Fig. 2b). The activity of the mutant sequence is even higher than the one of the wild-type sequence. Again dimerization results in an increase in signal strength. An example of an experiment without added estradiol is included in Fig. 3. The extent of transcription under these non-hormone conditions is essentially the same as seen in the presence of hormone. Taken together the above data show that the ERE is a positive regulatory element in in vitro transcription and that the activity is independent of the presence of estrogen.
172
+
estrogen
- estrogen
Fig. 3. In vitro transcription from wild-type and mutant ERE templates. In vitro transcriptions were carried out in Fe 33 nuclear extract (for ER concentration see bottom of Fig. 4). Products of transcriptions were separated on a sequencing gel. Templates added to the reactions are listed on top of each lane by indicating the regulatory element replacing the polylinker in pl-TG (lane pl. see Fig. 2a for abbreviations and sequences). 17/3-Estradiol was added to the reactions shown in the left five lanes to a final concentration of 1 x IO-’ M. whereas the reactions of the right five lanes were left untreated. Signals were obtained by transcription from the experimental template (e. 400 ng per reaction) and from the internal control (c, adeno major late promoter driving a shortened -G box, 200 ng per reaction). HaeIII-digested pBR322 was used as a size marker (lane M).
Transcriptional activity in vitro is independent of the presence of estrogen receptor To determine whether the increase in transcriptional activity in Fe 33 nuclear extract is brought about by interaction of the estrogen receptor with its target DNA sequence, the ERE, we tested the various templates in a number of nuclear extracts with different amounts of ER. The ER concentration in each extract was determined using the Abbott ER enzyme immunoassay. Concentrations of receptor per ml of in vitro transcription assay
vary by a factor of up to about 500 (see bottom of Fig. 4). Extracts from hormone-responsive cell lines, known to express either the endogenous (MCF-7) or a stably transfected (Fe 33: this paper: Le 42: Druege et al., 1986) ER contain high receptor levels, whereas in extracts of non-responsive cell lines (FTO, Ltk ) the ER concentration is close to or below the limit of detection. In HeLa cell nuclear extract we did not measure the ER concentration, but its absence has been reported previously (Green et al., 1986). Fig. 4 illustrates that all the extracts are functional in initiating transcription from EREwt and EREmt3 monomer and dimer promoter constructs. Comparison of the signal strengths obtained in the various nuclear extracts with the four templates demonstrates: (i) Transcriptional activation is dependent of the presence of estrogen receptor. Extracts containing high levels of ER (Fe 33, Le 42, MCF-7) and nuclear extracts preparations from the ER-deficient cell lines and from male rat liver are equally in transcribing the different templates. (ii) In all extracts tested, the weakest regulatory element is the EREwt monomer mediating only a 2- to 3-fold induction of transcription. A monomer of a mutated ERE (EREmt3) on the other hand is a strong regulatory sequence which is responsible for an up to 12-fold increase in signal strength compared to pl-TG. (iii) Dimerizaton of the wild-type sequence (EREwtd) results in a dramatic increase in transcription rate compared to the monomer sequence. Two EREs act synergistically in stimulating transcription in vitro from a minimal promoter. The synergism is evident in all extracts tested. With a dimer EREmt3, the increase in transcriptional activity is at most additive. These data indicate that transcriptional activity in vitro mediated by ERE and TATA box is due to the interaction of transcription factors, which are different from the estrogen receptor and which are present ubiquitously in all extracts tested. Oligonucleotides with sequence requirements different from the ER compete for ERE-mediated in vitro transcription The in vitro transcription experiments suggested to us that not the estrogen receptor but
173 0 m -
30.-: z .?
-
5
-
EREwt EREmt3 EREwtd EREmt3d
2 .o .E 20; : z 0 .? E m L
1 o-
Fe 33
FTO
Le42
180-450
MOLCEL
167
02235
Transcription factors different from the estrogen receptor stimulate in vitro transcription from promoters containing estrogen response elements M. Kaling, T. Weimar-Ehl,
M. Kleinhans
and G.U. Ryffel
*
Kernforschungszentrum Karlsruhe, Institut ftir Genetik und ftir Toxikolagie von Spaltstoffen, Karlsruhe, F. R.G. (Received
Key words: Rat hepatoma
13 November
FTO-ZB cell; Transcription,
1989; accepted
in vitro; Estrogen
4 December
response
1989)
element;
Vitellogenin
Bl promoter;
USF
Summary The estrogen response element (ERE) directly linked to a TATA box induces CAT activity in a hormone-dependent manner in Fe 33 cells, the rat hepatoma cell line FTO-2B, stably transfected with the human estrogen receptor (ER). The same promoter construct mediates the stimulation of in vitro transcription. This stimulation is dependent on the presence of the ERE. Induction of transcription in a variety of nuclear extracts derived from mammalian cells is of the same magnitude irrespective of the presence of ER. Similarly, transcription in vitro mediated by Bl vitellogenin 5’ flanking sequences in different nuclear extracts is not due to the interaction of the ER with the ERE. Competition analyses with a variety of oligonucleotides reveal that proteins different from the ER, which recognize ERE-like DNA elements, functionally interact with the ERE in vitro. These experiments suggest that ubiquitous proteins related or even identical to the transcription factor USF (MLTF) activate in vitro transcription in an ERE-dependent manner.
Introduction Estrogen-mediated gene expression is brought about by ligand-induced binding of estrogen receptor (ER) to a specific DNA sequence - the estrogen response element, ERE in the 5’ flanking region of target genes (for reviews see Yamamoto, 1985; Evans, 1988; Ryffel et al., 1988; Beato, 1989). We have identified in the vitellogenin A2 gene
Address for correspondence: M. Kaling, Kemforschungszentrum Karlsruhe, Institut fur Genetik und ftir Toxikologie von Spaltstoffen, Postfach 3640, D-7500 Karlsruhe 1, F.R.G. * Present address: Institut ftr Zellbiologie (Tumorforschung), Universitltsklinikum Essen, Hufelandstr. 55, D-4300 Essen, F.R.G. 0303-7207/90/$03.50
0 1990 Elsevier Scientific
Publishers
Ireland,
of Xenopus the ERE as being the 13 bp palindromic sequence GGTCA NNN TGACC (Klein-Hitpass et al., 1986). This sequence is sufficient to confer estrogen inducibility to a heterologous promoter (Klock et al., 1987; Martinez et al., 1987; Klein-Hitpass et al., 1988b) and binds the estrogen receptor as a homodimer in a liganddependent manner (Kumar and Chambon, 1988). Single-point mutations introduced into the ERE destroy its function and decrease its affinity for the receptor (Klein-Hitpass et al., 1988b, 1989). Estrogen response can also be achieved through synergism of two closely adjacent mutated EREs, which are not functional on their own but together constitute an estrogen response unit, ERU (Martinez et al., 1987; Klein-Hitpass et al., 1988a). Cato et al. (1988) showed that an ERE and a Ltd.
progesterone response element form a response unit that functions synergistically, when the two different steroid hormones estrogen and progesterone are present. Furthermore, synergism of hormone response elements with other regulatory elements, known to increase basal activity in a hormone-independent manner, has been reported (SchUle et al., 1988; Strlihle et al., 1988). All these findings establish that the activity of a given hormone response element can be modulated by other regulatory sequences. Such effects have been explained by postulating an interaction of the regulatory proteins involved. Interaction between steroid hormone receptors and other tram-acting factors might represent one of the many possible mechanisms which lead to activation of transcription of a given gene. These molecular mechanisms are still poorly understood, although some of these factors, particularly the steroid hormone receptors, are well characterized (for reviews see Evans, 1988; Green and Chambon. 1988). A prerequisite for a direct experimental approach to study the events leading to gene regulation by steroid hormones seemed to us the development of an in vitro transcription system, which initiates transcription in an ER- and/or ERE-dependent manner. In vitro transcription systems have been successfully employed to characterize tram-acting factors and their cognate cis elements (Dynan et al., 1985; Jones et al., 1985; Fletcher et al., 1987) to identify tissue-specific transcription factors (Gorski et al., 1986; Bodner and Karin. 1987; Scheidereit et al., 1987: Schorpp et al., 1988). or to characterize the specific role of transcription factors in transcription initiation (Hai et al., 1988). In the present study we report on the development of an in vitro transcription system, the activity of which depends on the presence of an ERE. Quite unexpectedly we observed that ubiquitous rruns-acting factors and not the ER are responsible for this activity in vitro. Materials and methods Construction of plasmids Plasmids pl-T and pl-TG replacing the HindIII-BglII
were constructed by fragment of synO-T
and synO-TG, respectively, carrying the Xenopus HP1 sequence (Schorpp et al., 1988) with the polylinker of pBlCAT3 ( HindIII-BglII fragment). All other constructs are derivatives of pl-T and pl-TG. Synthetic double-stranded oligonucleotides encompassing the EREwt, the EREmt3 or the USF sequences and carrying Hind111 and BumHI restriction ends replaced the polylinker in PI-T and pl-TG, thus generating plasmid containing the respective DNA element. Dimers of EREwt and EREmt3 were constructed by ligating the BumHI restriction fragments of pA2((331/-319)ztkCATS’ and pA2(C~330/-319)ztk-CAT8+ (KleinHitpass et al., 1988a) into BgIII linearized and dephosphorylized pl-T and pl-TG. Thus the polylinker is not removed from these constructs. Plasmids carrying Bl gene 5’ flanking sequences were constructed in the following way: a 6.4 kb EcoRI subclone of the vitellogenin Bl gene genomic cloneXXlv201 (Wahli et al., 1982) encompassing 5’ flanking sequences and the cap site of the gene was digested with BamHI and PvuII. Two of the resulting fragments, about 1.8 kb were isolated on low melting agarose and ligated with a Hind111 linker (8mer). Subsequent digestion with Hind111 and BglII yielded a 520 bp fragment which was isolated and cloned into HindIII-BglII digested pl-TG to generate pBl(-562/-37)-TG. Alternatively the same fragment was ligated into synO-TATA Bl (Ryffel et al., 1989) thereby replacing the HP1 sequence. This resulted in plasmid pB1((562/-24)-G. For construction of pB1 (-299/ -37)-TG and pBl(-299/ -24)-G the plasmid pBl(-562/ -37)-TG was digested with Hind111 and XhoI. The DNA fragment resulting from restriction with both enzymes was isolated, digested with DdeI and blunt ended by filling in with Klenow polymerase. This gave a 300 bp fragment to which a Hind111 linker was ligated. Digestion with Hind111 and BgIII resulted in a fragment encompassing the desired 5’ flanking sequences of the vitellogenin Bl gene. It was isolated and cloned into Hind111 - BglII digested pi-TG or synO-TATA Bl. The control plasmid consisting of HP1 in front of a shortened -G box (HPlcaslOO) was constructed by digesting synO-TG with X/z01 and filling in the overhangs with Klenow polymerase. After digestion with Hind111 the vector band was
169
isolated and ligated with a 180 bp fragment encompassing HP1 and a shortened -G box. This fragment was obtained by digesting synO-TG with PuuII. The correct PuuII fragment was isolated, cleaved with FokI, and the overhanging ends were blunt ended with Klenow polymerase. Subsequent digestion with Hind111 resulted in the 180 bp fragment, which was isolated for cloning. The structure of all clones was verified by DNA sequencing according to Sanger et al. (1977). Cell culture and transfections FTO-2B and Fe 33 cells were grown in a 1 : 1 DMEM/Ham’s F12 medium mixture containing 10% fetal calf serum (FCS) and penicillin/streptomycin (100 U/ml). Cells were transfected using the DEAE-dextran procedure described earlier (Druege et al., 1986). After transfection, cells were either treated with 1 x lo-’ M diethylstilbestrol or were left untreated and cultured in medium depleted by charcoal treatment of any components which act as estrogens. Nuclear extract preparations Rat liver nuclear extract was prepared essentially as described by Gorski et al. (1986). Nuclear extracts from cells grown in cell culture were
+ 260167
33118; Fe33
+ 331167 +HEO
prepared as described by Ryffel et al. (1989). 18 and 1 h prior to harvesting, cells were treated with 1 X lo-’ M 17b-estradiol. The concentration of estrogen receptor in every extract was determined using the Abbott ER enzyme immunoassay (EREIA), according to the specifications of the supplier. In vitro transcription In vitro transcription reactions were performed essentially as described previously (Dobbeling et al., 1988; Ryffel et al., 1989).
Introduction of estrogen responsiveness into FTO-2B rat hepatoma cells Estrogen-induced activation of the vitellogenin genes is restricted to hepatocytes (Wahli and Ryffel, 1985). Therefore we decided to establish an estrogen-responsive hepatoma cell line as an experimental system to set up an in vitro transcription assay. We have chosen the rat hepatoma cell line FTO-2B in which we have previously observed an estrogen response upon transient transfection with the human estrogen receptor expression vector HE0 (Druege et al., 1986). Via stable
+ 331167
26016:
331,67+ +HEO
FTO-2B
Fig. 1. FTO-2B cells become estrogen responsive upon stable transfection with a human estrogen receptor expression vector. Transient transfection of vitellogenin-tk-CAT fusion genes into Fe 33 (left panel) and FTO-2B cells (right panel). Cells were transfected with either 5 pg salmon sperm DNA and 5 pg pA2(-331/-87)tk-CAT8+, or 5 pg pA2(-260/-87)tk-CAT8+ (Klein-Hitpass et al., 1986), or with 5 pg pKCR2-ER (HEO) (Green et al., 1986) and 5 pg pA2(-331/-87)tk-CAT8+. Cells were kept in medium containing 10% charcoal-treated FCS without (-) or with (+) 1 X10-’ M diethylstilbestrol (DES). After 4 days, cell extracts (200 ng protein) were assayed for CAT activity.
I 70
transfection of HE0 using nro selection we obtained a number of estrogen-responsive clones and chose the one showing the highest level of induction - Fe 33 - for further characterization. Fe 33 expresses high CAT activity in hormonetreated medium upon transient transfection with the estrogen-inducible indicator plasmid pA2(-331/ -87)tk-CAT8+, containing an ERE (Klein-Hitpass et al., 1986) (Fig. 1, left panel). In hormone-free medium, the same plasmid mediates only basal level transcription. The non-inducible plasmid pA2(-260/-87)tk-CAT8’. lacking an ERE, gave only low CAT activity under both conditions. Cotransfection of pA2(-331/ -87)tk-CAT8+ and the ER-cDNA expression plasmid HE0 does not result in an increased CAT activity compared to transfection with the indicator plasmid alone {Fig. 1). This indicates that the concentration of ER in Fe 33 is not a limiting factor in estrogen induction. As expected, in the stem cell line FTO2B cotransfection of the ER is absolutely essential for a hormone effect (Fig. 1, right panel).
thymidine kinase promoter (Klein-Hitpass et al., 1988bf. The tk promoter fragment used (- 105/ +51) is known to contain DNA sequence elements recognized by several transcription factors (Jones et al., 1987). It has been shown that the glucocorticoid receptor functions synergistically with a number of different l’rans-acting factors (Schule et al., 1988; Strahle et al.. 1988). To ex-
a
PI-T
ERE and TA TA box are sufficient to mediate estrogen inducihility in oioo In transient transfection studies we have shown that the 13 bp ERE as found in the Xenopus vitellogenin A2 gene confers inducibility to the
Fig. 2. The ERE confers estrogen inducibility to a TATA box. (u) Schematic drawing (not to scale) of pl-T. the basal vector used for construction of plasmids for transient transfections. Nucleotide sequences of oligonucleotides inserted into Hind111 and &/II sites of PI-T are shown on the bottom. The 13 bp palindromic ERE as found in the Xenopus vitellogenin A2 gene and its homologues are boxed. Mutations relative to the wild-type sequence are shown in small tetters. The gap into the USF sequence was introduced to obtain optimal alignment with the other seqtences. (b) Transient transfections into Fe 33 cells were performed by the DEAE-dextran procedure (Druegc et al., 1986). Plasmids are PI-T derivatives carrying the oligonucleotide sequences indicated on the left (EREwtd and EREmt3d: dimers of EREwt and EREmt3, respectively) instead of the polyhnker. Extracts of cells either treated with I x 10 -’ M DES in 70% ethanol ( +. hatched bars) or with 70% ethanol alone ( -. white bars) were prepared 40 h after transfection and assayed for CAT activity (200 ng protein each). Induction factors (IF), indicated on the right. are corrected for mock transfection and are expressed relative to the value obtained with the vector PI-T (pl) alone.
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chide such contributions in our system, we placed ERE sequences without any other known regulatory elements immediately upstream of a TATA box in the vector pl-T. This plasmid contains the CAT gene as indicator (Fig. 2~). Results of transient transfections of these constructs into Fe 33 cells are shown in Fig. 2b. The ERE wild-type sequence (EREwt) linked to the TATA box confers a slight but reproducible estrogen-dependent increase in CAT activity (2fold), whereas the mutated element (EREmt3) did not mediate any effect upon estrogen treatment. This agrees with our earlier results using the tk promoter (Klein-Hitpass et al., 1988b). An EREwt dimer directly linked to the TATA box mediates a 4-fold induction in the presence of hormone (Fig. 2b). This only additive effect is in contrast to the synergistic interaction of EREs in the tk promoter context reported previously (Klein-Hitpass et al., 1988a). A 4-fold increase in CAT activity is also observed when testing a dimer of EREmt3 in transient transfection (Fig. 26). This is similar to the value found previously using the tk promoter. As a control we used the vector pl-T without any regulatory element except the TATA box. The activity of this construct was not induced by estrogen. To further prove that the estrogen response depends on the presence of the ERE we inserted into pl-T an oligonucleotide corresponding to the USF recognition sequence. This sequence, found in the adeno major late promoter (Carthew et al., 1985), bears a high degree of similarity to the ERE (see Fig. 2~). However, it is not active in inducing CAT activity in a hormone-dependent manner (Fig. 26). Taken together, we conclude that a single ERE is sufficient to confer estrogen inducibility to a TATA box. In contrast to results obtained when using the tk promoter induction is only moderate and we did not observe synergism in hormone response using EREwt dimers. The ERE is a positive regulatory element in an in vitro transcription system Having established the minimal requirements for hormone response in vivo, we used these, namely the estrogen-responsive cell line Fe 33 as a source of nuclear extract and the minimal promo-
ter conferring the hormone response, to set up an in vitro transcription system. To monitor the extent of in vitro transcription in a rapid and efficient way, we inserted into the single XhoI site of the various pl-T derivatives the 400 bp G-free cassette described earlier (Sawadogo and Roeder, 1985; Schorpp et al., 1988). In the absence of GTP, transcripts from these templates are generated exclusively downstream of the TATA box along the G-free cassette and can therefore readily be analyzed. A shortened -G box of 200 bp under the control of the adeno major late promoter was included into each reaction as internal standard for quantification (Kugler et al., 1988; see Schorpp et al., 1988). As the preparation of nuclear extract involves dialysis, the concentration of estrogen is reduced. We therefore, added to all assays 17P-estradiol to a final concentration of lo-’ M. All promoter constructs are able to induce transcription in vitro when tested in a nuclear extract derived from Fe 33 cells (Fig. 3). Compared to the plasmid pl-TG, in which the ERE is replaced by a polylinker sequence, the EREwt mediates a moderately increased transcriptional activation, which upon dimerization (EREwtd) is further increased. We conclude that the ERE activates transcription in vitro. The possible interpretation that the polylinker acts as a negative element can be excluded, since this polylinker is present in the active dimer constructs. Furthermore, no negative effect of the same polylinker has been observed previously (Schorpp et al., 1988; Ryffel et al., 1989). Surprisingly, EREmt3 combined with a TATA box is also active in directing transcription in vitro, although the same promoter construct is not estrogen response in vivo (see Fig. 2b). The activity of the mutant sequence is even higher than the one of the wild-type sequence. Again dimerization results in an increase in signal strength. An example of an experiment without added estradiol is included in Fig. 3. The extent of transcription under these non-hormone conditions is essentially the same as seen in the presence of hormone. Taken together the above data show that the ERE is a positive regulatory element in in vitro transcription and that the activity is independent of the presence of estrogen.
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Fig. 3. In vitro transcription from wild-type and mutant ERE templates. In vitro transcriptions were carried out in Fe 33 nuclear extract (for ER concentration see bottom of Fig. 4). Products of transcriptions were separated on a sequencing gel. Templates added to the reactions are listed on top of each lane by indicating the regulatory element replacing the polylinker in pl-TG (lane pl. see Fig. 2a for abbreviations and sequences). 17/3-Estradiol was added to the reactions shown in the left five lanes to a final concentration of 1 x IO-’ M. whereas the reactions of the right five lanes were left untreated. Signals were obtained by transcription from the experimental template (e. 400 ng per reaction) and from the internal control (c, adeno major late promoter driving a shortened -G box, 200 ng per reaction). HaeIII-digested pBR322 was used as a size marker (lane M).
Transcriptional activity in vitro is independent of the presence of estrogen receptor To determine whether the increase in transcriptional activity in Fe 33 nuclear extract is brought about by interaction of the estrogen receptor with its target DNA sequence, the ERE, we tested the various templates in a number of nuclear extracts with different amounts of ER. The ER concentration in each extract was determined using the Abbott ER enzyme immunoassay. Concentrations of receptor per ml of in vitro transcription assay
vary by a factor of up to about 500 (see bottom of Fig. 4). Extracts from hormone-responsive cell lines, known to express either the endogenous (MCF-7) or a stably transfected (Fe 33: this paper: Le 42: Druege et al., 1986) ER contain high receptor levels, whereas in extracts of non-responsive cell lines (FTO, Ltk ) the ER concentration is close to or below the limit of detection. In HeLa cell nuclear extract we did not measure the ER concentration, but its absence has been reported previously (Green et al., 1986). Fig. 4 illustrates that all the extracts are functional in initiating transcription from EREwt and EREmt3 monomer and dimer promoter constructs. Comparison of the signal strengths obtained in the various nuclear extracts with the four templates demonstrates: (i) Transcriptional activation is dependent of the presence of estrogen receptor. Extracts containing high levels of ER (Fe 33, Le 42, MCF-7) and nuclear extracts preparations from the ER-deficient cell lines and from male rat liver are equally in transcribing the different templates. (ii) In all extracts tested, the weakest regulatory element is the EREwt monomer mediating only a 2- to 3-fold induction of transcription. A monomer of a mutated ERE (EREmt3) on the other hand is a strong regulatory sequence which is responsible for an up to 12-fold increase in signal strength compared to pl-TG. (iii) Dimerizaton of the wild-type sequence (EREwtd) results in a dramatic increase in transcription rate compared to the monomer sequence. Two EREs act synergistically in stimulating transcription in vitro from a minimal promoter. The synergism is evident in all extracts tested. With a dimer EREmt3, the increase in transcriptional activity is at most additive. These data indicate that transcriptional activity in vitro mediated by ERE and TATA box is due to the interaction of transcription factors, which are different from the estrogen receptor and which are present ubiquitously in all extracts tested. Oligonucleotides with sequence requirements different from the ER compete for ERE-mediated in vitro transcription The in vitro transcription experiments suggested to us that not the estrogen receptor but
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