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The EMBO Journal vol.10 no.9 pp.2513-2521, 1991
Interference and synergism of glucocorticoid receptor and octamer factors
Stefan Wieland, Udo Dobbeling1 and Sandro Rusconi Institut fOr Molekularbiologie II der Universitat Zurich. ETH/HPM Honggerberg, 8093 Zurich, Switzerland 'Present address: Institut fOir Pharmakologie und Biochemie, Universitat Zarich Irchel, Winterthurerstrasse 190, 8057 Zurich, Switzerland Communicated by W.Schaffner
We have analysed the interplay of glucocorticoid receptor (GR) and the lymphocyte-specific factor Oct-2A with transient co-transfection assays. Our data confirm our previously described observation that GR and the apparently unrelated factors belonging to the Octamer family can synergize when permitted to bind in cis. However, when GR binding sites are not present in the reporter genes, we observe that the action of the cloned factor Oct-2A expressed in HeLa cells is strongly inhibited in the presence of active GR molecules. We can demonstrate that this GR-mediated inhibition of Oct-2A action is neither due to competitive binding to DNA target sites nor to a reduction of DNA binding competent Oct-2A in the transfected cells. We observe that the phenomenon is not reciprocal, since co-expression of Oct-2A does not inhibit GR-dependent transcription activation. Furthermore, we provide evidence that the observed GR- Oct-2A interference may be dependent on the type of cell line hosting the co-transfected molecules. We consider it likely that the GR-mediated inhibition is due to the exhaustion of some rate-limiting co-activators. Key words: enhancer and promoter/glucocorticoid receptor/ octamer factors/transcriptional co-activator/transient expression
Introduction Transcriptional promoters and enhancers are composed of clusters of DNA motifs situated proximally or at a distance from the initiation region (for a review see Muller et al., 1988a; Johnson and McKnight, 1989; Mitchell and Tjian, 1989). Transcriptional control is thought to be mediated by the specific binding of nuclear transcription factors to their cognate DNA elements, whereby the simultaneous binding of transcription factors results in a synergistic stimulation of transcription (Schule et al., 1988; Schatt et al., 1990). The molecular cloning and functional characterization of several transcription factors has yielded a general picture of a trans-activator, which would consist of a DNA binding domain, an optional multimerization domain, and most importantly, one or more activation domains (reviewed by Mitchell and Tjian, 1989). Other factors seem to function without directly binding to the DNA molecule, but rather (C Oxford University Press
by interacting with another DNA binding factor. They have been shown to contain protein -protein interaction domains and activation domains (reviewed by Ptashne and Gann, 1990). It has become apparent that there are several classes of activation domains (Ptashne and Gann, 1990; Tasset et al., 1990). An important family is represented by the acidic domains which have the potential of folding into amphipathic helices (Ma and Ptashne, 1987; Ptashne, 1988). Other classes include glutamine- or proline-rich domains (Courey and Tjian, 1988; Mermod et al., 1989). It is not clear by which mechanism the activation domains exert their transcription activating function but the most common speculation is that they do so by contacting components of the basic transcription apparatus like the TATA-box binding factor TFIID (Stringer et al., 1990) or some subunit of the RNA polymerase II holoenzyme (Kelleher et al., 1990). This contact would result in the stabilization of initiation complexes and the subsequent increase of initiation rate. A number of experiments have suggested that this interaction between transcription factors and basic transcription apparatus components does not need to be direct (Tasset et al., 1990; reviewed by Ptashne and Gann, 1990). In fact, an elevated concentration (overexpression in vivo or enrichment in vitro) of transcription factors results in selective transcriptional interference, both in vitro (Berger et al., 1990; Kelleher et al., 1990) and in vivo (Tasset et al., 1990). This phenomenon was initially named 'squelching' (Gill and Ptashne, 1988) or 'transcriptional interference' (Meyer et al., 1989) and has led to the conclusion that some sort of rate-limiting co-activator(s) (or 'adapter') may be required for the efficient interaction between activating domains and the basic transcription apparatus. Titration of common co-activators is not the only way by which interference among transcription factors can occur. For example, it has been reported that transcriptional interference may occur either via direct interaction between transcription factors (Jonat et al., 1990; Lucibello et al., 1990; Schule et al., 1990; Yang et al., 1990) or their competition for overlapping DNA target sites (Oro et al., 1988; Drouin et al., 1989). Rat glucocorticoid receptor (GR) and the lymphocytespecific Octamer factor 2A (Oct-2A) used in our study, also belong to the category of nuclear transcription factors. GR is ubiquitously distributed (reviewed by Yamamoto, 1985), whereas Oct-2A is found mainly in lymphoid tissues (Muller et al., 1990 and references therein). Both factors comprise several activating domains (see for GR: Giguere et al., 1986; Hollenberg and Evans, 1988; Tora et al., 1989; Tasset et al., 1990 and for Oct2A: Gerster et al., 1990; Muller et al., 1990). Several isoforms of the 60 kDa Oct-2 are found in few cell types, notably in lymphoid cells (Wirth et al., 1991), with Oct-2A being the predominant form in human B-cells (W.Schaffner, personal communication). In addition, there is a ubiquitously distributed octamer factor, the 90 kDa Oct-I (Sturm et al., 1988) which displays the same DNA
2513
S.Wieland, U.Dobbeling and S.Rusconi
binding specificity as Oct-2A and is encoded by a separate gene. In this study we demonstrate that the presence of functional GR strongly reduces the transcription stimulation by Oct-2A in HeLa cells, but apparently not in lymphoid cells. Different activating domains of GR seem to participate to various degrees in this competition. Although DNA binding per se does not seem to play a role, we find that an intact Zn finger region (DNA binding domain) must be included to provide efficient competition. We can furthermore demonstrate that the GR-mediated inhibition: (i) is not due to interference with Oct-2A production in HeLa cells or to impairment of Oct-2A competence for DNA binding; (ii) is not due to competitive binding for the Octamer-specific promoter element; (iii) is not reciprocal and (iv) does not apply to the ubiquitous factor Oct-i, thus suggesting that different members of the Octamer transcription factor family function via distinct pathways.
Results Reporter genes and trans-activators We have used different reporter genes which allow the quantitative detection of rabbit ,B-globin transcripts in transiently transfected cells (Westin et al., 1987). The parental vector is the promoter-deprived, (-globin derivative OVEC (Westin et al., 1987), in which the region between -425 and -37 is substituted by a short linker sequence (Figure 1A, top) to allow the insertion of different DNA segments (Figure lA). For our experiments, the target sites for Oct-2A (Dl) and GR (P1) were cloned at this promoter position. Additional features include elements at an enhancer position (SV40 as a constitutive enhancer and P4 as a GRdependent enhancer, see constructs D1-3-SV and D1-f--P4 in Figure lA). Neither of these reporter genes is strongly transcribed in HeLa cells, since the specific trans-activators are not active or not present in the required amount. Therefore, we provided exogenous trans-activators GR or Oct-2A by co-transfecting the expression vectors illustrated in Figure 1B in different combinations and amounts. We included in all transfections a reference gene which yields a distinguishable signal (OVEC-REF2, modified from Westin et al., 1987; see Materials and methods for further details). Interference of GR with Oct-2A action in non-lymphoid cells As described by Muller et al. (1988b), efficient transcription from the Octamer-dependent reporter gene Dl-( depends on the presence of co-transfected Oct-2A trans-activating plasmid (Figure 2A, lanes 1 versus 2). The basal level (lane 1) is most likely due to the endogenous Oct-l trans-activator (Kemler et al., 1991). The fundamental observation is that this relatively strong stimulation is inhibited when active GR ('competitor') is included during the transient expression (lanes 3 and 5). Note that the amount of GR expression plasmid added is five times less than the amount of Oct-2A expression plasmid (see Figure 2 legend, and Materials and methods). GR-mediated inhibition of Oct-2A function is dependent on the presence of a hormone agonist such as dexamethasone (lanes 3 and 5) and is less efficient in the presence of the antagonist RU486 (lanes 4 and 6). Intact receptor does not seem to be required, since the expression of a GR fragment lacking the amino-terminal 'potentiator' (see Figure lB for details) still results in the efficient 2514
inhibition of Oct-2A action (lane 5). In fact, the constitutively active GR fragments GR3-556 (described later in Figure 3A) and GR407-556 (see Discussion and Figure 5A) are capable of inhibition too. Transcription from the reference gene (signal marked REF in Figure 2) is unaffected by the presence of either trans-activator. We can demonstrate that the GR-mediated inhibition is not due to a decreased level of Oct-2A competent for DNA binding (Figure 2B). The amount of Oct-2A was determined by analysing the nuclear extract from the same cells which yielded the RNA assayed in Figure 2A. Gel-retardation signals indicate that all the samples transfected with Oct-2A contain the same amount of Oct-2A competent for DNA binding (including extracts from cells which co-expressed active GR, compare lanes 2-7). Another possibility could have been that GR competed for binding to the Oct-2A target site. A mutant GR which does not bind to DNA but can still interfere with Oct-2A action would ultimately have answered this question. Unfortunately, so far we have not been able to define such a mutant GR (S.Wieland, E.Zandi and R.Lanz, unpublished observations). Therefore, we used again a gel-retardation analysis in which we tested the effect of GR on the binding of Oct-2A to its target (Figure 2C). The experiments were performed with nuclear extracts (NE) containing Oct-2A either alone or together with the bacterially made (BM) GR fragment 438-556. The small GR fragment was chosen because it is a very strong and specific GRE binder (Freedman et al., 1988; our unpublished observations) and it yields a shifted band which is easily distinguishable from those of the Octamer proteins (Oct-I and Oct-2A). Two distinct labelled probes ('octamer' and 'GRE') were used for this assay. The data demonstrate that even with a large excess of GR (lanes 3 and 7) there is no appreciable cross-reaction of GR with the unrelated octamer sequence motif. Furthermore, a large excess of GR fragment does not impair the DNA binding functions of Oct-2A (lane 3). We conclude therefore that the inhibition of Oct-2A action by GR is not due to interference with Oct-2A expression or its DNA binding properties, but more likely to competition for another type of rate-limiting function. Characterization of the GR-mediated inhibition of Octamer factor action In the course of our experiments we did not observe a substantial difference in the squelching characteristics of wild type GR and the constitutively active deletion mutant GR3-556. Therefore, we subsequently present data obtained with the hormone-independent GR3-556 which allows for an easy control of experiments (e.g. use of point mutants and avoiding overlapping reactions from hormone activatable resident GR). Since GR-mediated inhibition of Oct-2A transactivation appears to be due to competition for additional rate-limiting factors (see Discussion), we wanted to determine whether this trans-inhibition is reciprocal. Figure 3A confirms that GR is very efficient in interfering with Oct-2A action (lanes 3, 4 and 5). This experiment shows that co-transfection of a relatively small amount of GR-encoding plasmid results in a significant reduction of the Oct-2Adependent transcriptional activation (see lane 3). Lanes 9 and 10 show that the converse is not true. In fact, it appears that upon co-expression of Oct-2A the stimulation by GR is even better. It has been demonstrated that the reporter genes stimulated by Oct-2A can also be activated by the ubiquitous factor
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Fig. 1. Structure of the reporter gene constructs and the corresponding trans-activator/competitor expression vectors. (A) Reporter gene constructs. At the top we show the parental plasmid OVEC (Westin et al., 1987) comprising a modified rabbit 0-globin gene still including its proper transcription initiator region and TATA-box, while having the promoter region (-435 to -37) replaced by a synthetic linker. In Dl-,B, Dl-,B-SV and D1-,B-P4 an octamer sequence (ATTTGCAT, boxed) (Muller et al., 1990) has been introduced as a promoter element immediately upstream of the ,B-globin TATA-box. In P1-,B one palindromic GRE (boxed with arrows) (P1, Severne et al., 1988) is inserted at this position. Some constructs contain a remote (- 1.8 kb) enhancer in addition to the octamer site at the promoter position: D1-O-SV contains a SV40 enhancer (SV, Banerji et al., 1981) and DI-0-P4 a cluster of four GRE palindromes (P4, Schatt et al., 1990; Wieland et al., 1990a,b). Symbols: wavy lines, plasmid sequences; solid line, rabbit ,B-globin non-coding gene sequences; hatched box, coding sequences; bent arrow, transcription initiation site. (B) Expression vectors for trans-activators and competitors. At the top, the eukaryotic expression vector pSTC (Severne et al., 1988; Rusconi et al., 1990; Schatt et al., 1990) comprising the cytomegalovirus (CMV, black rectangle) promoter/enhancer linked to thymidine kinase (TK) leader sequences including the initiator codon AUG and two additional amino acids is shown. Wild type rat GR (GR3-795, amino acids 3-795) and the deletion mutants GR3-556, GR407-795 and GR407-556 (amino acids 3-556, 407-795 and 407-556, respectively) were cloned in-frame with respect to the TK initiation codon. In the construct encoding GR3-795, the three major operationally defined domains of GR are indicated (Beato, 1989): P, potentiator domain (white box); D, DNA binding domain (hatched box); H hormone binding domain (filled box). Constructs at the bottom represent the cDNA inserts encoding the B-cell-specific trans-activator Oct-2A (Muller et al., 1988b) and the ubiquitous Octamer factor Oct-I (Sturm et al., 1988) as re-cloned by Kemler et al. (1991). Vertically hatched boxes within the shadowed rectangle indicate the homeo- and POU-specific domains of the Oct factors. Other symbols: thin line, TK leader sequences; thick broken line, rabbit 0-globin splice and polyadenylation signals; SVori, 120 bp fragment spanning the origin of DNA replication of SV40 (Rusconi et al., 1990); other symbols as in (A). For further details about single components of the vectors see Materials and methods.
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Fig. 2. Interference of GR with Oct-2A-mediated transcription in non-lymphoid cells. (A) SI nuclease mapping of RNA from transiently transfected HeLa cells. Lane 3 shows the interference of wild type GR with Oct-2A-mediated transcription (compare with lane 2). 10 itg of the reporter gene Dl-,B was co-transfected along with 5 Ag of the expression vector encoding the trans-activator Oct-2A (lanes 2-7). In lanes 3-7, 1 yg of the indicated trans 'competitor' GR expressing plasmids were added on top of Oct-2A. After two days incubation in the presence of the synthetic GR agonist dexamethasone (DEX, 5 x 10-7 M) or the antagonist RU486 (5 x 10-8 M), cytoplasmic RNA was extracted and analysed. When the level obtained in lane 2 is defined as 100%, the relative transcription levels in lane 1-7 are: 3%, 100%, 9%, 32%, 24%, 73%, 95%, respectively. For details of transfection conditions and quantification see Materials and methods. Symbols: RT, read-through transcription; CT, correctly initiated transcription; REF, transcription from the reference gene. (B) Gel-retardation assays for Oct-2A present in nuclear extracts (Muller et al., 1989) from the same transfection series as presented in (A). The amount of Oct-2A is unaffected by co-expression of GR. Symbols: free DNA, radiolabelled unbound DNA fragment; Oct-i, fragment complexed with endogenous Oct-I protein; Oct-2A, complex of transiently expressed Oct-2A. (C) Gelretardation assays with bacterially made GR and Oct-2A from nuclear extracts. Lane 4 shows that GR and Oct-2A do not interfere with each other for binding to their corresponding recognition sequences. In the top row '+' and '+ +' indicate the amount (20 ng and 200 ng, respectively) of bacterially made (BM) GR fragment of 438-556 present in the bandshift rection. In the second row '+' indicates the addition of 5 yd of nuclear extract containing Oct-2A (NE) to the bandshift reaction. Symbols: Oct-i, shifted complex of endogenous Oct-i; Oct-2A, shifted complex of transiently expressed Oct-2A; GR, shifted complex, of bacterially made GR (amino acids 438-556); free 'octamer' and free 'GRE', radiolabelled DNA fragments containing an 'octamer' (58 bp fragment) or a 'GRE palindrome' (36 bp fragment), respectively. For details of the DNA fragments used and bandshift conditions see Materials and methods.
Oct-I in HeLa cells (Kemler et al., 1991). This finding and the fact that the two proteins are closely related lead us to wonder whether Oct-I can also be down-regulated by GR. Figure 3B shows a similar transfection series to that presented in Figure 3A but involving co-transfection of Oct-I as the activator of the octamer promoter. Lanes 3 and 4 show that Oct-I action, while being much weaker than Oct-2A, cannot be squelched by GR under conditions which lead to significant reduction of Oct-2A action. Conversely, GRmediated transcription is not squelched by high amounts of Oct-I (lanes 6, 7 and 8). There is even a more substantial increase of transcription on a GR-dependent promoter than with Oct-2A (compare Figure 3A lanes 7-10 with Figure 3B lanes 6-8). Finally, we found that the presence of a GRE cluster at a remote position in the reporter gene can apparently compensate for GR-mediated inhibition (Figure 3C). Addition of an element such as the SV40 enhancer stimulates
2516
transcription, but does not prevent inhibition by GR (Figure 3C, left panel). We know that the reduction of transcription for DI-,B-SV is not due to inhibition of the SV40 enhancer, since our reference gene (which itself contains the SV40 enhancer element at a remote position, see Materials and methods) is not significantly inhibited. The presence of a GR-specific enhancer results in a mitigation of the transinhibition (lanes 8 and 9). This implies that while GR competes for a rate-limiting co-factor which is necessary for the stimulation of an Octamer-dependent promoter (see examples above), the same co-factor(s) can be resupplemented through 'trapping' GR molecules with specific high affinity binding sites at a remote position (2 kb from the promoter). The high affinity GR binding sites need to be placed on the same plasmid bearing the test promoter, since we observed, as expected, that co-transfection of a separate plasmid bearing multiple high affinity GREs (i.e. in trans to the Oct-dependent promoter) does not relieve the
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Fig. 3. Features of the GR-Octamer factor interplay. (A) Interference of GR with Oct-2A is not reciprocal: ectopically expressed Oct-2A in HeLa cells cannot squelch GR-mediated transcription. RNase protection mapping of cytoplasmic RNA isolated from transiently transfected HeLa cells. Cotransfections included 10 Atg of the indicated reporter gene (D1-3 or P1-,B) together with the plasmids pSTC:Oct-2A (5 jig) and pSTC:GR3-556 (1 Ag) expressing the trans-activator Oct-2A and the constitutively active GR mutant (amino acids 3-556), respectively. The quantity of co-transfected competitor plasmid is given as well as the type of competitor. Radiographic signals were quantified as described in Materials and methods. If the results of transfections represented in lanes 2 (D1-j3/Oct-2A) and 7 (P1-,B/GR3-556) are defined as 100%, the following transcription levels are observed: Oct-2A mediated transcription (lanes 1-5): 7%, 100%, 30%, 20%, 18%; and for GR-dependent transcription (lanes 6-10): 11%, 100%, 75%, 110%, 180%. Symbols: CT, correctly initiated transcripts; REF, transcripts derived from the reference gene; para, a pSTC vector encoding a non-functional GR deletion mutant (Rusconi et al., 1990). (B) GR and ubiquitous Oct-I do not interfere with each other in HeLa cells. The same transfection series as in (A) was performed involving Oct-I instead of Oct-2A. For the reporter gene D1-,B, transcription in the presence of pSTC:Oct-l (lane 2) is considered 100%. Accordingly, the signal of lane 6 is considered 100% for GR-mediated transcription. From this the following transcription levels are observed: Oct-i-mediated transcription (lanes 1-4): 40%, 100%, 109%, 200%; and for transcription activation by GR (lanes 5-8): 22%, 100%, 150%, 900%. Symbols are as in (A). (C) Compensation of titrated co-activators by specific enhancers. RNA from transiently transfected HeLa cells was analysed by RNase protection. 10 jig of the reporter genes D1-O-SV or D1-,B-P4 have been co-transfected along with 5 ytg pSTC:Oct-2A and the indicated amounts of competitor GR (GR3-556) or mock GR (para). The 100% level is defined for both D1-,-SV and D1-0-P4 as transcription in the presence of Oct-2A (lanes 2 and 7, respectively). This gives the following relative transcription levels in percentages for lanes 1-4: 22%, 100%, 50%, 90%; and for lanes 5-9:
