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The mammalian transcriptional RBP (CBF1) targets TFIID and TFIIA to prevent activated

Ivan Olave, Danny Reinberg,1 and Lynne D. Vales2 Department of Biochemistry and 1Howard Hughes Medical Institute, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 USA

RBP is a cellular that functions as a transcriptional repressor in mammalian cells. RBP has elicited great interest lately because of its established roles in regulating expression, in and mouse development, and as a component of the Notch signal transduction pathway. This report focuses on the mechanism by which RBP represses transcription and thereby regulates expression of a relatively simple, but natural, . The results show that, irrespective of the close proximity between RBP and other transcription factors bound to the promoter, RBP does not occlude binding by these other transcription factors. Instead, RBP interacts with two transcriptional coactivators: dTAFII110, a subunit of TFIID, and TFIIA to repress transcription. The domain of dTAFII110 targeted by RBP is the same domain that interacts with TFIIA, but is disparate from the domain that interacts with Sp1. Repression can be thwarted when stable transcription preinitiation complexes are formed before RBP addition, suggesting that RBP interaction with TFIIA and TFIID perturbs optimal interactions between these coactivators. Consistent with this, interaction between RBP and TFIIA precludes interaction with dTAFII110. This is the first report of a repressor specifically targeting these two coactivators to subvert activated transcription. [Key Words: RBP; transcriptional repression; TFIIA/TFIID targeting] Received November 17, 1997; revised version accepted April 1, 1998.

The role of the cellular RBP protein in gene regulation Barr encoded EBNA2 (Grossman et al. 1994; Henkel et al. has been established fairly recently. Earlier genetic stud- 1994; Laux et al. 1994; Waltzer et al. 1994; Zimber-Strobl ies demonstrated a pivotal role for the Drosophila homo- et al. 1994); EBNA3A,B,C (Robertson et al. 1996); and the log of RBP in development (Nash 1965, 1970; Furukawa mammalian Notch (Fortini and Artavanis-Tsa- et al. 1992; Schweisguth and Posakony 1992; Tun et al. konas 1994; Hsieh et al. 1996)] and KyoT2 (Taniguchi et 1994). Initial studies on mammalian RBP contributed al. 1998). biochemical and genetic characterizations, although The identification of RBP as a transcriptional repressor RBP was thought to be a recombinase at that time in mammalian cells was established during studies of (Hamaguchi et al. 1989, 1992; Matsunami et al. 1989; virus . The important role of RBP in regu- Kawaichi et al. 1992). Since then, RBP has been recog- lating gene expression in the mature cell is highlighted nized to be a that represses mamma- by its sequestration during virus infection. Both adeno- lian gene expression (Dou et al. 1994; Kannabiran et al. virus and Epstein-Barr virus evolved to sequester RBP for 1997; Plaisance et al. 1997; Oswald et al. 1998), but ac- viral advantage. The adenoviral gene encoding the capsid tivates Drosophila gene expression (Brou et al. 1994). protein polypeptide IX (pIX) is expressed only after viral RBP has recently been implicated in the Notch signal DNA replication in infected cells. We found that the pIX transduction pathway in Drosophila, which may bridge promoter contained a repressive element that was spe- the role of RBP in gene expression and development (For- cifically bound by cellular RBP. RBP was shown to re- tini and Artavanis-Tsakonas 1994; Bailey and Posakony press pIX expression before viral DNA replication oc- 1995; Lecourtois and Schweisguth 1995; Hsieh et al. curs; this repression was dependent on the presence of 1996; Eastman et al. 1997). That RBP has a pivotal regu- the RBP-specific DNA-binding site within the pIX pro- latory role in gene expression is highlighted by its inter- moter. Furthermore, purified RBP protein was shown to action with viral and cellular proteins that modulate its repress pIX transcription in a reconstituted transcription activity [Drosophila Hairless (Brou et al. 1994); Epstein– assay performed in vitro (Dou et al. 1994). RBP was shown to be usurped during Epstein-Barr vi- 2Corresponding author. rus infection by the potent transcriptional E-MAIL [email protected]; FAX (732) 235-4783. EBNA2. EBNA2 lacks DNA-binding activity, but a com-

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Olave et al. plex formed between RBP and EBNA2 facilitates EBNA2 ous report showing that RBP does not occlude Sp1 or tethering to the DNA via the RBP-specific DNA-binding TBP binding, RBP does not occlude binding of Sp1, activity (Grossman et al. 1994; Henkel et al. 1994; Laux TFIIA, and TFIID. All four proteins bind the pIX pro- et al. 1994; Waltzer et al. 1994; Zimber-Strobl et al. moter concomitantly. Instead of repressing by occlusion, 1994). Transcriptional activation by EBNA2 is expedited our results show that RBP mediates repression by direct not only by RBP-mediated access to specific promoters, interaction with two coactivators: TFIIA and the but also by EBNA2 masking of the RBP repression do- TAFII110 subunit of TFIID. Moreover, the component of main (Hsieh and Hayward 1995). TFIID targeted by RBP is the same TAF that interacts Transcriptional repression in eukaryotes has been ap- with TFIIA as well as with the activation domains of preciated relatively recently (for review, see Herschbach Sp1, the only activator required for optimal pIX expres- and Johnson 1993; Johnson 1995). Several sion. However, our results also show that the domain of have been identified and their critical role in regulating TAFII110 targeted by RBP is the same as that which in- gene expression has been established. Similar to the teracts with TFIIA, but distinct from that which inter- precedents set by studies of prokaryotic repressors, dif- acts with Sp1. Furthermore, interaction between RBP ferent eukaryotic repressors that target distinct compo- and TFIIA precludes interaction of TAFII110. Therefore, nents and stages of the transcription process have been RBP interaction with TFIID and TFIIA alters optimal documented recently. Some repressors target activated interaction between these two coactivators, not to dis- transcription by competing with activators for overlap- lodge them from the promoter, but instead to subvert ping DNA-binding sites (e.g., Oct-1 and C/EBP; Wu et al. activated transcription. 1997). Other repressors interact with activators to mask activation domains (e.g., MDM2 and ; Oliner et al. 1993). Activator–repressor interactions can also serve to Results tether the repressor to specific promoters. Then the re- pressor can effectively compete with other activators for Repression depends on the position of the RBP site basal transcription factors (e.g., MDM2 and TFIIE–TBP; in vivo Thut et al. 1997) or compete with a basal transcription factor for interaction with other activators (e.g., pRb and Figure 1a shows the simple adenoviral pIX promoter con- TBP; Weintraub et al. 1995). Some repressors complex taining a single Sp1 site, a single RBP site, and a consen- with basal transcription factors to exclude interaction sus TATA box. Our previous studies showed that this with other factors (Dr1 and TBP; Inostroza et al. 1992), region is sufficient for pIX regulation, that RBP-mediated whereas others can access promoters directly and target repression required the RBP-binding site, and that RBP candidate basal transcription factors to silence gene ex- binding did not dislodge Sp1 or TBP from the pIX pro- pression (e.g., Kruppel and TFIIB–TFIIE␤; Sauer et al. moter (Dou et al. 1994). We initiated our studies to iden- 1995). RBP is a transcriptional repressor with specific tify the molecular basis of RBP repression by testing DNA-binding activity, and RBP binding was known to whether the position of the RBP site immediately up- be required for repression, yet the molecular basis of stream of the TATA box was fortuitous or important in RBP-mediated repression was not known. repression. Our initial studies of the adenoviral pIX gene sug- The single RBP site was repositioned at several dis- gested that RBP-mediated repression does not involve tinct sites within the promoter region of the pIX gene. In competition between RBP and other transcription fac- each case, the wild-type RBP site was substituted with tors for promoter access (Dou et al. 1994). The pIX pro- linker sequence and the optimal distance between the moter is relatively simple, containing a single site for the Sp1 site and the TATA consensus was maintained (Fig. cellular transcription factor Sp1 and a consensus TATA 1a). In two cases, the RBP site was repositioned upstream box (Babiss and Vales 1991). The RBP-specific DNA- of the Sp1 site. RBP/20nt/Sp1 contains an RBP site 20 binding site lies between these two positions immedi- nucleotides upstream of the Sp1 site, whereas RBP/Sp1 ately upstream of the TATA box. However, RBP binding contains an RBP site immediately upstream of the Sp1 did not dislodge either Sp1 or TBP from their respective site, similar to its normal position immediately up- sites. With this as a basis, we pursued the molecular stream of the TATA box. In two other cases, the RBP site mechanism by which RBP successfully silences pIX tran- was maintained between Sp1 activator and TATA and scription. placed either immediately downstream of Sp1 (Sp1/RBP) Using a transcription assay reconstituted with recom- or centered between the two sites (RBP center). The re- binant factors and highly purified native factors, we es- positioned RBP site restores RBP-specific DNA-binding tablished conditions for Sp1-activated pIX transcription activity in each case (data not shown). The Sub9 control and RBP-mediated repression. Repression was dependent contains only the linker substitution in lieu of the RBP on the presence of the RBP-binding site and was also site. dependent on the position of the RBP site within the pIX We first compared the levels of pIX expression from promoter. However, RBP-mediated repression could be each of these constructs in transfected F9 cells (Fig. 1b). thwarted when a transcription preinitiation complex in- As shown previously with virus infections, the presence termediate composed of Sp1, TFIIA, and TFIID was of the RBP site is repressive to pIX gene expression; wild- formed on the pIX promoter. Consistent with our previ- type pIX expression is barely detectable as opposed to the

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Transcription repression by RBP through TFIIA and TFIID

However, the possibility existed that RBP binding may occlude the TFIID complex, thereby resulting in repres- sion in the wild-type case and relief from repression in the RBP/Sp1 case in which RBP is distanced from the TATA box. We first tested RBP co-binding with TFIID and Sp1 at the wild-type pIX promoter using DNase I footprinting analyses (Fig. 2a). These results show that RBP and Sp1 co-bind the pIX promoter (cf. lanes 1–4), RBP and TFIID co-bind the pIX promoter (cf. lanes 1, 2, 5, and 6), and Sp1, TFIID, and RBP co-bind the pIX pro- moter (cf. lanes 1, 2, 7, and 8). Therefore, repression is not mediated by RBP occlusion of TFIID in the wild-type case. We similarly tested each of the constructs containing a repositioned RBP site. In the case of Sp1/RBP and RBP center, which exhibited repression in the presence of RBP in vivo (Fig. 1b), RBP binding at the repositioned site led to diminished Sp1 binding (data not shown). There- fore, the presence of RBP destabilized Sp1 binding in the Sp1/RBP and RBP center cases; this, no doubt, contrib- uted to pIX repression from Sp1/RBP and RBP center in a manner disparate from the wild-type case in which all three factors co-bind. On the other hand, RBP and Sp1 Figure 1. The position of the RBP-binding site determines RBP-mediated repression in vivo. (a) Schematic representation did co-bind the RBP/Sp1 promoter similar to the wild- of the different pIX promoter constructs used in this study. The type case (Fig. 2b). Therefore, relief from RBP repression wild-type (WT) pIX promoter, the Sub9 promoter containing a in the RBP/Sp1 case was not attributable to Sp1 occlu- linker substitution of the RBP-binding site, and the pIX pro- sion of RBP binding, for example. This result strongly moter containing repositioned RBP-binding sites are shown (see suggested that pIX repression was dependent on the po- text for description). (Arrows) Start sites of pIX transcription sition of the single RBP site within the pIX promoter. (+1); (boxes) position of the TATA box, Sp1, and RBP sites as indicated. In all cases, the distance between the Sp1 site and the TATA box was conserved so that Sp1-activated transcription Repression depends on the position of the RBP site would be expected to be the same in all cases. (b) Results from in vitro an RNase T2 protection assay with an antisense probe that spans the start site of pIX transcription and equivalent aliquots Because the comparative analysis shown in the previous of RNA isolated from F9 cells transfected with the pIX con- section was performed in vivo, the possibility existed structs indicated at the top of the lanes. In all cases, cells were that chromatin structure may affect pIX gene expression cotransfected with an expression vector for RBP and internal from the constructs containing repositioned RBP sites, control for transfection efficiency (SV/dl17). The levels of pIX particularly RBP/20nt/Sp1 and RBP/Sp1, which were re- mRNA and internal control are scored within the same assay lieved from repression. To address this possibility and to (see Materials and Methods). (Arrow) Protected fragment ex- establish conditions to examine RBP repression at the pected for pIX mRNA. The larger protected fragment derives molecular level, each of these constructs was similarly from the internal control. The smaller protected fragment de- compared for Sp1-activated pIX levels and susceptibility rives from transcription that initiates within vector sequences upstream of the pIX promoter and utilizes the acceptor splice to RBP repression in transcription reactions performed in site downstream of the pIX start site; this protected fragment vitro. First, optimal levels of pIX transcription were re- serves as another internal control for transfection efficiency (see constituted with purified recombinant transcription fac- Materials and Methods). tors (TFIIB, TFIIE, and TFIIF), highly purified TFIIH and RNA II isolated from HeLa cells, purified epitope-tagged TFIID, and the purified recombinant co- clearly detectable levels of pIX expression from the Sub9 activators TFIIA and PC4, in the absence and presence of construct that lacks the RBP-binding site (cf. lanes 1 and Sp1 activator; each reaction included template with the 2). The two constructs that maintained RBP binding be- wild-type pIX promoter and control template with the tween Sp1 and TATA retained susceptibility to repres- major late promoter, which does not contain Sp1 or RBP sion (lanes 5, 6). On the other hand, the two constructs sites (see Materials and Methods). Figure 3a shows the containing a single RBP site upstream of Sp1 gave rise to levels of basal and Sp1-activated pIX transcription ob- similar pIX expression as that observed with Sub9 (cf. tained under these conditions. Sp1 activation of pIX tran- lanes 2–4). This result suggested that repression was re- scription is clearly detectable and dependent on the co- lieved when the RBP DNA target site was upstream of activators TFIIA and PC4. the activator. Figure 3b shows the purification scheme followed for Our previous results showed that RBP, Sp1, and TBP the isolation of recombinant RBP. Figure 3c shows the can co-bind the wild-type pIX promoter (Dou et al. 1994). preparations of recombinant (lane 4) and native RBP

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Olave et al.

Figure 2. RBP, Sp1, and TFIID co-bind the pIX promoter. (a) DNase I footprinting analysis on the coding strand of the wild- type pIX promoter as a function of rRBP, Sp1, and epitope-tagged TFIID proteins ei- ther alone or in combination as indicated on the top of each lane. (b) Similar analysis for rRBP and Sp1 on the coding strands of either wild-type or RBP/Sp1 pIX promoter constructs. Brackets enclose the protected region of the pIX promoter associated with binding by each protein. (G and G+A) Max- am–Gilbert sequencing reactions.

(lane 6) utilized in the transcription assays. The addition fected by RBP addition in vivo and in vitro and RBP/Sp1 of increasing amounts of purified human (data not is also relieved from repression in vivo and in vitro even shown) or recombinant RBP to the transcription reac- though RBP and Sp1 co-bind. Most importantly, the pat- tions resulted in pIX repression (Fig. 3d). On the other tern of repression observed in vitro as a function of the hand, although the levels of Sp1 activation of pIX tran- position of the RBP-specific DNA-binding site mimics scription from the Sub9 construct, mutant in RBP bind- that observed in vivo. ing, were similar to those of the wild-type, the addition of increasing amounts of RBP was ineffectual in this Repression as a function of transcription preinitiation case. This result established optimal conditions of acti- subcomplex formation vated pIX transcription, optimal conditions of RBP-me- diated repression, and verified the requirement of the The establishment of this reconstituted transcription as- RBP site in mediating pIX repression in vitro. say validated the interpretation of the results we ob- Next, we compared the levels of Sp1-activated pIX tained in vivo. In addition, this assay allowed us to ex- transcription and susceptibility to repression from the plore two aspects of RBP repression at the molecular RBP/Sp1 and RBP center constructs relative to wild-type level: First, the stage of transcription preinitiation com- and Sub9 cases (Fig. 4). All of these constructs gave rise plex formation in which RBP repression is functional; to similar levels of Sp1-activated transcription. Similar and second, the components within the complex that to the results obtained in vivo, RBP repressed pIX tran- may be susceptible to repression. To this end, we first scription from the wild-type (lanes 1–4) and RBP center examined whether RBP-mediated repression in vitro was (lanes 13–16) constructs, but not from the Sub9 (lanes dependent on the order of RBP addition during transcrip- 5–8) or RBP/Sp1 (lanes 9–12) constructs. In addition, the tion preinitiation complex formation. When RBP is orientation of the RBP site immediately upstream of the added concomitantly with all of the transcription factors TATA box (RBP reverse) was inconsequential to repres- required for preinitiation complex formation, pIX repres- sion in vitro (lanes 17–20) and in vivo (data not shown). sion is apparent (Fig. 5a, lanes 1–3). However, if RBP is Although repression in the RBP center case is likely at- added subsequent to preinitiation complex formation, tributable to RBP-mediated occlusion of Sp1 binding, as repression was no longer observed (lane 4). This result opposed to the wild-type case, relief from repression in suggested that stable preinitiation complexes formed in the RBP/Sp1 case cannot be attributed to Sp1 blocking of the absence of RBP can thwart repression. RBP binding (see previous section). Therefore, repression We next sought to identify the minimal transcription is achieved dependent on both the presence and position components that can resist the repressive activity of RBP of the RBP site, as Sub9 that lacks the RBP site is unaf- when a subcomplex containing these components is pre-

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Transcription repression by RBP through TFIIA and TFIID

Figure 3. RBP represses pIX transcription in a highly purified in vitro transcription system. (a) Results of a transcription reac- tion performed in vitro with the wild-type pIX promoter. Transcription reactions con- tained two templates: the wild-type pIX promoter fused to a transcription unit con- taining a 112-nucleotide U-less cassette (pIX) and an internal control consisting of the adenovirus major late promoter fused to a transcription unit containing a 50- nucleotide U-less cassette (ML; see d and Materials and Methods). The ML promoter does not contain an Sp1- or RBP-binding site. (Arrows) pIX and ML transcription products. Basal and Sp1-activated pIX tran- scription are shown in lanes 1 and 2, re- spectively. Sp1 activation depends on the presence of cofactors TFIIA (lane 3) and PC4 (lane 4). (b) Purification scheme for re- combinant RBP protein. After induction, GST–RBP fusion protein was purified with GST beads, treated with bovine thrombin, and purified further with Mono S (FPLC) and Benzamidine Sepharose. The protein was concentrated on Mono S (SMART sys- tem, Pharmacia). (c) Silver-stain analysis of purified protein fractions after separation by SDS-PAGE. Fractions containing recombinant RBP as a function of purification as well as native RBP protein purified from HeLa cells are indicated at the top of the lanes (see Materials and Methods). (MWM) Protein molecular weight markers. (d) Schematic representation of the templates used in the in vitro transcription reactions and described in a. (Right) Results of transcription reactions performed with increasing amounts of recombinant RBP (rRBP) and pIX template con- taining the wild-type (lanes 1–6) or Sub9 mutant promoter (lanes 7–10) as indicated at the bottom of the lanes. The amounts of rRBP used are indicated at the top of the lanes. Repression is not detected in the Sub9 case in which the RBP-binding site is mutated. Both rRBP and native RBP have similar DNA-binding and repression activities.

formed prior to RBP addition. Binding of TFIID to the with additional candidate transcription factors for resis- TATA motif nucleates transcription preinitiation com- tance to repression upon subsequent addition of RBP. In plex formation (Buratowski et al. 1989; Orphanides et al. all cases, Sp1 and the candidate transcription factors 1996; Roeder 1996). Therefore, we tested subcomplexes were preincubated with pIX and control templates, and, formed in the presence of Sp1 with either TFIID alone or then, the remaining factors required for optimal pIX

Figure 4. The position of the RBP-binding site determines RBP-mediated repression in vitro. (Top) pIX promoter and start site for pIX transcription (+1). (Bottom) pIX pro- moters fused to the transcriptional unit containing a 112-nucleotide U-less cas- sette (see Materials and Methods). The lev- els of transcription from each construct were compared under the following condi- tions: in the absence of Sp1 (basal), in the presence of Sp1, and in the presence of Sp1 and increasing amounts of RBP repressor as indicated at the top of the lanes. Although the levels of Sp1-activated transcription are similar in all cases, susceptibility to RBP-mediated repression depends on the position of the RBP site. The levels of Sp1- activated transcription from the wild-type (lanes 1–4), RBP center (lanes 13–16), and RBP reverse (lanes 17–20) constructs were decreased in the presence of increasing amounts of RBP. The addition of RBP had no detectable affect on Sp1-activated transcription from Sub9 (lanes 5–8) and RBP/Sp1 (lanes 9–12) constructs. The pattern of pIX repression dependent on the position of the RBP-binding site is consistent with the results obtained from these constructs in vivo (Fig. 1b).

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Olave et al.

Figure 5. RBP mediates repression at the early stages of preinitiation complex formation. (a) Conditions used to obtain single round transcription with linearized templates in these assays (see Materials and Methods). In all cases, Sp1 and PC4 were added together with the general transcription factors (GTFs). The levels of pIX transcription obtained during preinitiation complex formation are shown as a function of time of addition of RBP repressor (R), or time of addition of TFIIA (IIA) or TFIIH (IIH) in the absence of or presence of RBP repressor as indicated at the top of the lanes. (Vertical arrows) Preincubation times of 30 min before the addition of NTPs. pIX and ML transcripts are indicated on the left. Preinitiation complexes formed in the absence of RBP are protected from repression. Coaddition of RBP during complex formation results in repression. Repression is apparent on later addition of RBP as a function of the presence of TFIIA, but not TFIIH. (b) Conditions of single round transcription as in a but in this case, subcomplexes were preformed for 30 min with specific candidate transcription factors as indicated: TFIID (D), TFIIA (A), and/or TFIIB (B). In all cases, Sp1 and PC4 were added together with the candidate transcription factors. The levels of pIX transcription obtained during preinitiation complex formation are shown as a function of the presence of the specific candidate transcription factors and time of addition of RBP repressor. Arrows at the top of the lanes indicate preincubation times of 30 min before addition of NTPs. pIX and ML transcripts are indicated on the left. Subcomplexes containing TFIID are partially resistant to the later addition of RBP, but are made completely resistant when preformed in the presence of TFIIA, but not TFIIB.

transcription were added. Each of the candidates was This result strongly suggested that the presence of TFIIA tested in sets of three with respect to RBP addition: In rendered a subcomplex completely resistant to later RBP the complete absence of RBP to gauge optimal pIX tran- addition. scription under these conditions; in the presence of RBP added during preincubation; or in the presence of RBP Coaddition of RBP and TFIIA is required added subsequently along with the remaining factors. for functional repression Figure 5b shows the results of this analysis. The levels of Sp1-activated pIX transcription in the Because subcomplex containing Sp1 and TFIID could be absence of RBP were similar in all of the preincubation rendered as resistant to RBP as the complete complex as trials (Fig. 5b, cf. lane 1 with lanes 2, 5, 8, and 11). Similar long as TFIIA was present, we next tested whether RBP to the results obtained with the complete complex, all of was specifically targeting TFIIA during preinitiation the subsets of candidate factors examined were suscep- complex formation. The TFIIA can be added tible to repression when RBP was added during complex at any time during transcription preinitiation complex formation (cf. lanes 3, 6, 9, and 12). Subcomplex contain- formation without an effect on activated transcription ing Sp1 and TFIID alone was partially resistant to later (Fig. 5a, cf. lanes 2 and 5). In this case, we tested whether RBP addition (cf. lanes 3–4, see below). On the other or not subcomplexes devoid of TFIIA are resistant to the hand, preincubation with Sp1, TFIID, and TFIIA was subsequent addition of RBP when TFIIA is also added completely resistant to later addition of RBP, similar to later with RBP. As a control, we similarly tested TFIIH, the results shown above when the complex was pre- which is required for transcription (lane 9) and is the last formed with all of the factors (lanes 5–7). This result factor that assembles into the preinitiation complex (cf. suggested that stable complex formed in the presence of lanes 1 and 7). As described above, complex containing TFIIA and Sp1–TFIID was able to impede the repressive all the factors is susceptible to concomitant, but not sub- activity of RBP. This finding was specific to TFIIA as sequent, RBP addition (lanes 1–4). However, when a preformed complex containing Sp1, TFIID, and TFIIB similar complex is formed in the absence of TFIIA, the was as equally susceptible to later RBP addition as was later addition of RBP with TFIIA now renders the com- Sp1–TFIID alone (lanes 8–10). On the other hand, when plex susceptible to repression (lane 6). This is not the TFIIA was present, the Sp1–TFIID–TFIIB subcomplex case with TFIIH, however. Complex formed with all the was now made resistant to RBP repression (lanes 11–13). factors including TFIIA, but in the absence of TFIIH, is

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Transcription repression by RBP through TFIIA and TFIID resistant to RBP when RBP is added later with TFIIH (lanes 7–9). Therefore, not only does the presence of TFIIA render a subcomplex resistant to RBP as shown in the previous section, but the omission of TFIIA during preinitiation complex formation renders the complex susceptible to repression when complemented later for TFIIA in the presence of RBP. This result strongly sug- gests that RBP is functionally targeting TFIIA in medi- ating repression of activated pIX transcription.

RBP, Sp1, TFIID, and TFIIA co-bind the pIX promoter Thus far, our results show that the complex must con- tain TFIIA to fully resist repression and that TFIID alone renders the complex partially resistant to RBP activity. The functional relevance of this finding to repression is dependent on the ability of RBP to co-bind with Sp1– TFIID and with Sp1–TFIID–TFIIA. The footprinting re- sults shown in Figure 2a demonstrated that RBP binding immediately upstream of the TATA box in the pIX pro- moter does not occlude TFIID or Sp1 binding and, there- fore, repression does not entail overlapping DNA-bind- ing sites for these three factors. Previous studies showed that TFIIA can extend the DNA coverage of TFIID by several nucleotides upstream Figure 6. RBP-mediated repression does not affect TFIIA bind- of the TATA box at some promoters (Van Dyke et al. ing and preinitiation complex formation does not inhibit RBP 1988; Buratowski et al. 1989; Cortes et al. 1992). If such binding. (a) Schematic representation of the experimental de- an extension were relevant at the pIX promoter, TFIIA sign employing immobilized pIX template. Transcription pre- initiation complexes were bound to the pIX promoter immobi- binding may overlap with that of RBP, which binds im- lized on magnetic beads. RBP was added at time 0 or after 40 mediately upstream of the TATA box. Repression may min of preincubation time. Reactions were then incubated for then entail RBP-mediated occlusion of TFIIA binding. an additional 40 min, immobilized templates were washed, and, This possibility would be consistent with RBP targeting then, aliquots were tested for levels of pIX transcription (b), or of TFIIA in the subcomplex experiments shown above. for proteins bound to the pIX template by use of Western blot For example, stable complexes formed in the presence of analyses (c); (for details, see Materials and Methods). (b) (Lanes TFIIA may occlude RBP binding upon its later addition, 3–6) Results of in vitro transcription assays using conditions for while complexes formed in the presence of RBP may single round transcription with aliquots of immobilized tem- result in RBP occlusion of TFIIA binding and hence, re- plates prepared as described in a. The order of addition of spe- pression. Given this possibility, we tested for co-binding cific factors is indicated on top of each lane. (Vertical arrows) Preincubation times of 30 min before the addition of NTPs. of RBP, Sp1, TFIID, and TFIIA. We did not observe any Preinitiation complexes formed on immobilized templates in detectable difference in footprinting analyses using the absence of TFIID do not give rise to detectable levels of TFIID in the absence or presence of TFIIA on the pIX transcription (lanes 5, 6). Susceptibility to repression is apparent promoter. Therefore, we could not use this assay to test on coaddition of RBP, but resistance to repression is apparent for co-binding of TFIIA with the other factors including upon later addition of RBP (lanes 3, 4). These results are similar RBP. Instead, we employed two additional assays. to those shown in Fig. 4a and repeated here in lanes 1 and 2 with First, we tested directly for the presence of RBP with nonimmobilized pIX template. (c) Western blot analyses of pro- TFIIA at the pIX promoter under conditions that produce teins bound to the immobilized pIX templates prepared as de- a transcriptionally competent complex that is resistant scribed in a. (Lanes 1–3) Controls for the presence of TFIIA, to RBP repression. Figure 6a shows a schematic repre- TFIIB, and RBP, respectively. The protein specificity of each antibody used is shown at left. Similar levels of either TFIIA or sentation of the experiment performed. The pIX tem- RBP are detected on immobilized pIX templates irrespective of plate was immobilized on beads under complete tran- the time of addition of RBP during complex formation (lanes 4, scription conditions in the presence of RBP added either 5). The level of RBP protein detected is not affected by the concomitant with or subsequent to the other factors. absence of TFIID during complex formation (lane 6); however, Then, aliquots of the beads were analyzed independently TFIIA is not detectable in the absence of TFIID (lanes 6,7). De- for the levels of pIX transcription attained and for the tection of RBP is dependent on its addition (lane 7). presence of candidate transcription factors (TFIIA, RBP, and TFIIB as control, see Materials and Methods). We observed that a preformed complex on an immo- lanes 1–4). In addition, transcription under these condi- bilized pIX template exhibited similar resistance to sub- tions exhibited the expected requirement for TFIID (cf. sequent, but not concomitant, RBP addition, as in the lanes 3–6). Although the complete complex was suscep- case of the nonimmobilized, control template (Fig. 6b, cf. tible to repression dependent on the order of RBP addi-

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Olave et al. tion, similar levels of RBP, TFIIA, and TFIIB were found to be associated with the immobilized pIX template, irrespective of the order of RBP addition (Fig. 6c, lanes 4, 5). The detection of these factors was specific, as shown in the case of complexes formed in the absence of TFIID. Because TFIID tethers the preinitiation complex to the promoter, the omission of TFIID resulted in un- detectable levels of TFIIB and TFIIA, as expected (lanes 6, 7). On the other hand, because RBP binds the pIX pro- moter independently, the omission of TFIID had no af- fect on the levels of RBP found complexed to the pIX promoter (cf. lanes 4–6). Because the levels of RBP detected in the case of the complete complex were un- affected by the order of RBP addition, the inability of RBP to repress at later times appears not to be attribut- able to its occlusion by stable preinitiation complex for- mation. Moreover, because the levels of TFIIA detected in the presence of TFIID were similar when RBP was added together with TFIIA or at a later time, these re- sults also show that TFIIA is apparently not occluded during complex formation in the presence of RBP. There- fore, complex susceptibility or resistance to repression appears not to correlate with TFIIA or RBP occlusion, Figure 7. RBP binding does not occlude TFIIA. (a,b) Results of respectively. 2+ Second, we assayed directly for the presence of RBP, Mg –agarose gel-shift assays performed with radiolabeled probe containing the wild-type pIX promoter (nucleotides −60 to Sp1, TFIID, and TFIIA at the pIX promoter using agarose +140 relative to the start site for pIX transcription) and the gel retardation assays and radiolabeled probe containing proteins indicated at the top of each lane; the addition of spe- the pIX promoter region (Fig. 7; see Materials and Meth- cific antibodies is also indicated. See Materials and Methods for ods). To optimize visualization of complexes containing details. (S) Sp1; (R) RBP; (D) TFIID; and (A) TFIIA. Specific oli- the multiple factors, conditions of limiting probe were gonucleotide competitors are Sp1 consensus site (S oligo); employed. The controls for this experiment are con- TATA consensus (TATA oligo). Antibodies used are antibody tained in panel b. First, complex containing Sp1 and RBP specific to RBP (␣-RBP); antibody specific to the ␥-subunit of migrated slower than complex containing Sp1 alone (cf. TFIIA (␣-IIA␥). lanes 2 and 3) and more diffusely than complex contain- ing RBP alone (cf. lanes 1 and 3). This complex contained both proteins as evidenced first by its complete super- The observed supershift of this complex with anti-TFIIA shifting with antibody specific to RBP (cf. lanes 3 and 6). antibody was specific as a complex formed in the pres- Second, the mobility of the complex reverted to that of ence of Sp1, TFIID, and RBP, without TFIIA, was unaf- RBP alone in the presence of excess Sp1 oligonucleotide fected by the addition of anti-TFIIA antibody (Fig. 7b, (cf. lanes 1, 3, and 4), but not in the presence of excess lanes 7, 8). TATA oligonucleotide (cf. lanes 3 and 5). This result Having established these conditions, we next analyzed confirms that RBP and Sp1 can co-bind the pIX promoter complex formation in the presence of all four proteins: under these conditions. Sp1, TFIID, TFIIA, and RBP (Fig. 7a, lane 10). The pres- Next, we tested for co-binding of Sp1, TFIIA, and ence of RBP during this complex formation resulted in a TFIID. Among these factors, only TFIIA does not contain species that migrated only slightly more slowly than a specific DNA-binding activity; the association of TFIIA complex formed in its absence (cf. lanes 5 and 10). On the with the promoter is completely dependent on the pres- other hand, the presence of Sp1 during this complex for- ence of TFIID (as shown above in Fig. 6c). Although a mation substantially decreased its mobility relative to complex containing only TFIID and TFIIA was not stably the complex formed in its absence (cf. lanes 10 and 14). formed under these assay conditions, a complex formed That this complex did indeed contain TFIIA, RBP, and in the presence of these two factors and Sp1 gave rise to Sp1 was confirmed by its reactivity with antibody spe- a supershifted complex relative to Sp1 alone (Fig. 7a, cf. cific to TFIIA (lanes 10, 11) or to antibody specific to RBP lanes 1–5). The dependence of this complex on Sp1 was (lanes 10, 12) and to its inhibition by excess Sp1 oligo- evident by its inhibition with excess Sp1 oligonucleotide nucleotide such that the species remaining migrated (cf. lanes 5 and 6). The presence of TFIIA was evidenced similar to a complex containing only RBP, TFIID, and by reactivity of this complex with antibody specific to TFIIA (cf. lanes 10, 13, and 14). This result shows that all TFIIA (cf. lanes 5 and 7). The presence of TFIID was four proteins can coexist on the pIX promoter. Together, required to obtain this complex as evidenced by the lack the results shown in Figures 6 and 7 establish that the of reactivity to antibody specific to TFIIA when the com- presence of RBP does not occlude TFIIA binding, thereby plex was formed with only Sp1 and TFIIA (lanes 8, 9). eliminating this possible mechanism of repression.

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Transcription repression by RBP through TFIIA and TFIID

RBP interacts specifically with TFIIA and with TFIID TBP component, however (Fig. 8a). Next, we examined

whether GST–RBP targets one or more of the TAFIIs. On the basis of our results thus far, TFIIA, TFIID, and/or The candidate TAFIIs examined in this section were Sp1 were likely targets for RBP in repression. Because derived from Drosophila, whereas the functional assays RBP binding does not disrupt nucleation of these factors performed in the previous sections utilized human at the pIX promoter, we next tested whether RBP medi- TFIID. Nonetheless, substitution of dTFIID in the tran- ates repression by direct interaction with any of these scription reaction performed in vitro gave rise to similar candidates. We found that RBP inhibited both Sp1-acti- levels of Sp1-activated pIX transcription, as expected, vated pIX transcription (see above) as well as GAL4–Sp1- and similar levels of RBP-mediated repression (data not activated transcription from a pIX template containing shown). Therefore, functional repression is not depen- two GAL4 sites in lieu of the Sp1 site (data not shown). dent on the source of TFIID. Furthermore, the highly GST–RBP did not exhibit detectable interaction with purified preparation of TFIID that was used in the func- GAL4–Sp1 activator in GST pull-down experiments tional transcription assays presented above, contained (data not shown). In addition, the coactivator PC4 that detectable levels of all the human homologs of the was added with Sp1 during complex formation and in dTAFIIs identified (data not shown). functional transcription assays did not show detectable Of the several candidate dTAFIIs examined, GST–RBP interaction with GST–RBP (data not shown). On the interacted only with dTAFII110 (Fig. 9a, lanes 7–9). This other hand, GST–RBP did interact with both human interaction was specific as GST alone was ineffectual. TFIID and recombinant TFIIA (Fig. 8b,c). This interac- Next, we verified this interaction using Far Western tion was specific, as GST alone was ineffectual. More- analyses (Fig. 9b,c). RBP showed specific interaction over, GST-RBP did not exhibit detectable interaction with dTAFII110; no interaction was detectable between with TFIIB (Fig. 8d; see below). As shown above, the RBP and hTBP, hTFIIB, dTAFII150, the BSA control, or presence of TFIID during subcomplex formation exhib- the molecular weight markers. These results demon- ited partial resistance to subsequent RBP addition, and strate that the interaction between RBP and TFIID this partial resistance was increased in the presence of shown above specifically involves the dTAFII110 com- TFIIA, but not TFIIB. Interestingly, these experiments ponent of TFIID. demonstrate that RBP interacts specifically with the The results in this section showed that RBP interacts same factors required for subcomplex resistance to re- with TFIIA and TFIID, that the interaction between RBP pression. and TFIIA involves the ␣,␤ subunits (see below), and that Because TFIIA and TFIID contain several subunits, we the interaction between RBP and TFIID directly involves tested which specific components of these factors inter- dTAFII110. Previous results showed that dTAFII110 in- act with RBP. TFIIA consists of the ␣, ␤, and ␥ subunits; teracts with the activation domains of Sp1 (Hoey et al. ␣, ␤, and ␥ are required for activation, whereas ␤ and ␥ 1993; Gill et al. 1994), the only activator required for are sufficient for antirepression (Sun et al. 1994; Ma et al. optimal pIX expression. Interestingly, previous results 1996; for review, see Orphanides et al. 1996). The recom- showed that the interaction between TFIIA ␣,␤ and binant form of the ␣,␤ subunits of TFIIA (TFIIA ␣,␤) in- TFIID also directly involves dTAFII110, as well as TBP teracted with GST–RBP and not with GST alone (data (Yokomori et al. 1993). Therefore, RBP interacts with the not shown, see below). Our results with the ␥ subunit TAF component of TFIID that mediates its interaction alone were not conclusive as TFIIA ␥ exhibited similar with Sp1 and with TFIIA. Sp1 has been reported to in- low levels of interaction with GST–RBP as with GST teract specifically with the amino terminus of dTAFII110 alone (data not shown). (Hoey et al. 1993). However, the region of dTAFII110

The complex TFIID consists of TBP and the TAFIIs (for interaction with TFIIA ␣,␤ has not been reported. There- review, see Burley and Roeder 1996). GST–RBP interac- fore, to identify factor interactions involving dTAFII110 tion with TFIID is not mediated by interaction with the that may be impeded in the presence of RBP, we exam-

Figure 8. RBP interacts specifically with TFIIA and TFIID. The results of GST pull- down assays analyzed by Western blot with an antibody specific to the protein indi- cated at the top of a–d. GST pull-down as- says were performed with GST or GST–RBP as indicated at the top of a–d and the pro- tein indicated at the top as follows: (a) hTBP, human recombinant TBP; (b) eT- FIID, epitope-tagged TFIID; (c) rTFIIA, re- combinant TFIIA; and (d) rTFIIB, recombi- nant TFIIB. (Lanes 1) An aliquot of the pro- tein specified as a positive control for the Western analysis. (M) Protein molecular weight markers right.

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Olave et al.

TFIIA ␣,␤ interact with the same region of dTAFII110, that is, the carboxyl terminus, which is distinct from that reported for interaction with Sp1, that is, the amino terminus.

RBP interaction with TFIIA inhibits dTAFII110 interaction The results in the previous section showed that both RBP and TFIIA ␣,␤ interact specifically with the car-

boxyl terminus of dTAFII110 (C-TAFII110, 666–921). To determine the functional relevance of these interactions with respect to repression, we tested whether complexes containing GST–RBP and TFIIA ␣,␤ retain the ability of

either protein to interact with C-TAFII110 or if subse- quent C-TAFII110 interaction is now impeded (Fig. 10b). The levels of C-TAFII110 binding to GST–RBP were ex- amined as a function of preincubation of GST–RBP with increasing amounts of either TFIIA ␣,␤ or TFIIB. The

levels of C-TAFII110 binding to GST–RBP were found to markedly decrease as a function of increasing TFIIA ␣,␤ preincubation (cf. lanes 2–6). This was not the case with TFIIB, however, which does not interact with RBP (Fig. Figure 9. RBP interacts specifically with dTAFII110. (a) GST 35 pull-down assays with S-radiolabeled TAF candidates pre- 8). The levels of C-TAFII110 binding to GST–RBP were pared by in vitro and GST or GST–RBP as indicated unaffected by increasing levels of TFIIB, comparable to at the top of the lanes. The first lane of each set of three shows those used for TFIIA ␣,␤ (lanes 8–12). the radiolabeled TAFII examined (10% of input) and the second A similar result was obtained with GST–RBP in the and third lanes show the results from GST pull-down assays presence of a high level of either TFIIA ␣,␤ or TFIIB and with this TAFII and either GST or GST–RBP, respectively. (b) subsequent addition of increasing amounts of C- ∼ Far Western analysis with 0.5 µg of protein indicated at the top TAF 110 (Fig. 10c). The levels of C-TAF 110 interaction and biotinylated rRBP. (c) SDS-PAGE analysis of ∼0.5 µg of pro- II II with GST–RBP were consistently reduced in the case of tein indicated at the top and visualized by Coomassie blue staining. (MWM) Protein molecular weight markers, the sizes of preincubation with TFIIA ␣,␤, relative to the control which are indicated right. case using TFIIB (cf. lanes 2–6 with lanes 8–12). These results showed that interaction between GST–RBP and

TFIIA ␣,␤ specifically inhibits subsequent C-TAFII110 interaction with either GST–RBP or TFIIA ␣,␤. ined discrete regions of dTAFII110 for interaction with RBP and for interaction with TFIIA ␣,␤. Activator-induced resistance to repression Distinct from Sp1, RBP and TFIIA ␣,␤ interact Our results thus far have shown that RBP targets TFIIA specifically with the carboxyl terminus of dTAFII110 and TFIID by direct interaction, but that RBP, TFIIA, and TFIID can co-bind the pIX promoter. The interaction be- Figure 10a shows the results of GST pull-down assays tween TFIID and TFIIA presumably mediates activation with either GST–RBP or a GST-fusion protein contain- by altering TFIID conformation. This conformational ing the ␣,␤ subunits of TFIIA (GST–IIA ␣,␤) and candi- change may be induced/stabilized in the presence of an date truncation mutants of dTAFII110. Both GST–RBP activator (Lee et al. 1992; Lieberman and Berk 1994; Chi and GST–IIA ␣,␤ exhibited interaction with full-length et al. 1995; Chi and Carey 1996; Oelgeschla¨ger et al. dTAFII110, as expected (amino acids 1–921, lanes 1–3 1996), but prevented/destabilized in the presence of RBP and lanes 13–15, respectively). Both GST–RBP and GST– repressor, which interacts with both TFIID and TFIIA.

IIA ␣,␤ interacted with regions of dTAFII110 spanning Consistent with this, we showed in the previous section amino acids 1–684 (lanes 4–6 and lanes 16–18, respec- that RBP interaction with TFIIA ␣,␤ disrupts interaction tively) and the carboxyl terminus of dTAFII110 spanning with the relevant domain of dTAFII110. We also showed amino acids 666–921 (lanes 10–12 and lanes 22–24, re- in previous sections that transcription preinitiation spectively). The interactions were specific as GST alone complexes were resistant to repression upon later addi- was ineffectual. On the other hand, neither GST–RBP tion of RBP, as long as TFIIA was present during complex nor GST–IIA ␣,␤ exhibited detectable interaction with formation. Even though later addition of TFIIA did not the amino terminus of dTAFII110 (1–307), which has affect the levels of Sp1 activation, later addition of TFIIA been reported to interact with Sp1 (lanes 7–9 and lanes with RBP now rendered the complex susceptible to re- 19–21, respectively). This result showed that RBP and pression. This suggests that the presence of TFIIA during

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Transcription repression by RBP through TFIIA and TFIID

Figure 10. Both RBP and TFIIA ␣,␤ inter- act specifically with the carboxy-terminal

domain of dTAFII110, which is inhibited from interaction when RBP complexes with TFIIA ␣,␤.(a) GST pull-down assays 35 with different S-labeled dTAFII110 trun- cated proteins translated in vitro, as indi- cated at the top of the lanes. Analyses us- ing either GST or GST–RBP (lanes 1–12) and either GST or GST–IIA␣␤ (lanes 13– 24) are shown. The first lane of each set of three shows 10% of the input radiolabeled

truncated dTAFII110 examined, the sec- ond lane shows the pull-down result with GST, and the third lane shows the pull- down result with either GST–RBP or GST–IIA␣␤, as indicated. (b) GST pull- down experiments with GST–RBP in the absence or presence of increasing amounts of either TFIIA ␣,␤ or TFIIB, as indicated, and subsequent addition of 3 µl of 35S-la-

beled dTAFII110 truncation protein con- taining amino acids 666–921, as indicated. A Western blot analysis of TFIIA ␣,␤ in- teraction with GST–RBP as a function of the addition of increasing amounts of TFIIA ␣,␤ is also shown. (+) 10% of the input protein. (c) Similar analysis as per- formed in b, but with the highest level of TFIIA ␣, ␤ or TFIIB shown in b and in- 35 creasing amounts of S-labeled dTAFII110 truncated protein containing amino acids 666–921, as indicated. A Western blot analysis of TFIIA ␣,␤ interaction with GST–RBP under these conditions is also shown. complex formation with the other factors alters the na- Therefore, the reduced levels of activated pIX transcrip- ture of the complex with respect to RBP-mediated re- tion observed upon later Sp1 addition is not consistent pression. Therefore, we tested the functional relevance with Sp1 occlusion. Instead, complexes that are pre- of RBP-mediated disruption of dTAFII110/TFIIA ␣,␤ in- formed in the presence of TFIID and TFIIA, in the ab- teraction during preinitiation complex formation, this sence of activator, appear to be refractory to optimal ac- time as a function of Sp1 activator addition. tivation when Sp1 is added later. We first examined the levels of activated pIX transcrip- As shown in a previous section and again here, preini- tion obtained as a function of time of Sp1 addition during tiation complexes formed in the presence of Sp1 were preinitiation complex formation, in the absence of RBP susceptible to repression upon concomitant, but not sub- (Fig. 11). The addition of Sp1 after the transcription com- sequent, RBP addition (lanes 1–4). However, a different plex was formed gave rise to activated pIX transcription; result was obtained during conditions of suboptimal Sp1 however, the levels were markedly reduced relative to activation resulting from later Sp1 addition. In this case, those obtained when Sp1 was present during preinitia- the complexes that gave rise to suboptimal activation tion complex formation (cf. lanes 1, 2, and 5). Experi- were now susceptible to repression when RBP was added ments involving preinitiation complex formation in the subsequent to Sp1 (cf. lanes 5 and 6). This result strongly studies presented here were performed under conditions suggests that the complexes containing TFIID and TFIIA of excess amounts of the tested transcription factors. In that were preformed in the absence of Sp1 are not only addition, the studies in the previous sections showed refractory to activation but are now susceptible to re- that Sp1, TFIID, and TFIIA co-bind the pIX promoter. pression. Furthermore, the later addition of Sp1 cannot

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Olave et al.

component of TFIID. This same TAFII has been shown to interact with the activation domains of Sp1 (Hoey et al. 1993; Gill et al. 1994). The possibility existed that RBP

may compete with Sp1 for TAFII110. However, RBP also represses GAL4–VP16-mediated activation in vivo and in vitro (Dou et al. 1994; data not shown). VP16 interac-

tion with TFIID involves dTAFII40, not dTAFII110 (Go- odrich et al. 1993). Therefore, the interaction between TFIID and TFIIA was the more likely target for RBP in repression. In fact, the studies presented here show that both RBP and the ␣,␤ subunits of TFIIA interact specifi-

cally with the carboxyl terminus of dTAFII110 as op- posed to Sp1 which interacts with the amino terminus (Hoey et al. 1993). Furthermore, interaction between RBP and TFIIA ␣,␤ disrupts interaction with the relevant

domain of dTAFII110. Although our results do not elimi- nate the possibility that RBP interaction with dTAFII110 and with TFIIA may also result in disruption of Sp1 and Figure 11. Sp1 and RBP target the same step in complex for- TAFII110 interaction, the direct target of RBP appears to mation. The levels of pIX transcription obtained during preini- be interaction between TFIIA and dTAF 110. tiation complex formation are shown as a function of time of II TFIIA has been directly implicated in mediating tran- addition of Sp1 and RBP as indicated at the top of the lanes. Conditions of single round transcription were used. (Vertical scription activation in two ways (for review, see Burley arrows) Preincubation times of 30 min each before addition of and Roeder 1996; Orphanides et al. 1996). First, interac- NTPs. (S) Sp1; (R) RBP. Later addition of Sp1 gives rise to re- tion between TFIIA and TBP precludes TBP targeting by duced levels of activated pIX transcription. Sp1 is required dur- other repressors (Meisterernst and Roeder 1991; In- ing complex formation to inhibit repression by RBP. ostroza et al. 1992; Auble and Hahn 1993; Merino et al. 1993). Second, interaction between TFIIA and TFIID sta- bilizes TFIID promoter interaction and is believed to in- restore complex resistance to subsequent RBP-mediated duce conformational changes in the presence of an acti- repression. Therefore, resistance to repression is depen- vator that are conducive to transcription activation (Li- dent on the presence of Sp1 along with the two targets of eberman and Berk 1994; Chi et al. 1995; Kobayashi et al. RBP: TFIIA and TFIID. Our results are consistent with 1995; Chi and Carey 1996; Oelgeschla¨ger et al. 1996; RBP interaction with TFIIA and TFIID to destabilize Lieberman et al. 1997). The documented roles of TFIIA conformational changes that are normally stabilized in in activated transcription suggested several possible the presence of Sp1 and thereby facilitate activated tran- means by which RBP may be repressive. RBP interaction scription. with TFIIA may impede activation by exposing TBP to other repressors. Because the reconstituted transcription reaction used here contains recombinant factors or Discussion highly purified native factors, we believe the presence of The studies presented here show that RBP is distinct in other putative repressors is unlikely. On the other hand, mediating repression with respect to the previously re- RBP interaction with TFIIA may completely disrupt ported repressors. Although RBP binds very closely to TFIIA interaction with TFIID. However, because TFIIA transcription factors that are crucial for activated tran- is tethered to the promoter by interaction with TFIID, scription, occlusion of adjacent factor binding has been and our studies show that RBP binding to the pIX pro- eliminated as a possible repressive mechanism at the pIX moter does not dislodge TFIIA, we believe this possibil- promoter (Dou et al. 1994; this study). RBP does not in- ity is also unlikely. Instead, our results strongly support teract with components of the basal transcriptional ma- that RBP interaction with TFIIA specifically disrupts in- chinery such as TBP or TFIIB, nor does it functionally teraction with the TAFII110 component of TFIID. This interact with the Sp1 activator. Instead, RBP evolved to would not necessitate TFIIA occlusion from the pro- silence activated transcription by interacting directly moter as TFIIA also interacts with TFIID via the TBP with two transcription coactivators. Our findings re- component that is not targeted by RBP. ported here are the first example of a transcription re- TFIIA interaction with TFIID has been shown to alter pressor directly targeting, by interaction, TFIIA and a TFIID conformation, which may mediate activated tran-

TAFII component of TFIID. As shown here, repression scription (Chi et al. 1995; Oelgeschla¨ger et al. 1996). This can be relieved when stable preinitiation complexes are phenomenon appears to be the most likely target for RBP formed before addition of RBP repressor. The resistant in repression. RBP interaction with TFIIA and TFIID subcomplexes are Sp1–TFIID–TFIIA dependent. may impede the induction of conformational changes in TFIID has been shown to be required for activated TFIID, which, in turn, may subvert activated transcrip- transcription in vitro by use of highly purified transcrip- tion. Our results showed that transcription preinitiation tion factors. RBP interacts specifically with the TAFII110 subcomplexes are completely resistant to repression

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Transcription repression by RBP through TFIIA and TFIID when RBP is added subsequent to Sp1–TFIIA–TFIID. When subcomplexes are formed in the presence of TFIID and Sp1 activator, repression is achieved only when RBP is added along with TFIIA. However, complexes formed in the presence of TFIIA and TFIID, but in the absence of activator, are not sufficient to thwart repression. Our results also show that formation of TFIIA–TFIID com- plexes in the presence of Sp1 is required not only for optimal levels of activation, but also to render TFIIA– TFIID resistant to RBP. In the absence of repressor, later addition of Sp1 re- sulted in markedly reduced levels of activated transcrip- tion. That optimal activation requires the coaddition of Sp1 during subcomplex formation is consistent with ac- tivator-stabilized/induced TFIIA–TFIID interactions that facilitate increased transcription. The nature of the TFIIA–TFIID complexes formed before Sp1 addition cor- relates with reduced accessibility to Sp1 and, therefore, with increased accessibility to RBP, even though RBP Figure 12. Diagram of the molecular basis for RBP repression was added after the activator. Therefore, activator-in- showing competition between RBP and Sp1 for the TFIIA–TFIID duced complexes are required for protection from RBP subcomplexes. RBP interacts with TFIIA and with the dTAF 110 component of TFIID. This interaction disrupts repression. Our findings showed that RBP and the ␣,␤ II TFIID–TFIIA interactions that involve dTAF 110 but does not subunits of TFIIA both interact with the carboxyl termi- II dislodge TFIIA, which may still interact with the TBP compo- nus of TAFII110 and that RBP interaction with TFIIA ␣,␤ nent of TFIID. Disruption of TFIID–TFIIA interactions by RBP disrupts interaction with the relevant domain of is presumed to inhibit Sp1-induced conformational changes in TAFII110. Taken together, these results strongly suggest TFIID mediated by TFIIA that are required for increased tran- that the TFIIA–TFIID interaction in the presence of Sp1 scription. On the other hand, Sp1-induced conformational is stabilized from interaction with RBP, but in the ab- changes in TFIID–TFIIA during complex formation provide pro- sence of Sp1, RBP disrupts TFIIA–TFIID interactions tection from subsequent RBP interaction. that are required for activated transcription. A model depicting this mechanism of repression is shown in Fig- ure 12. with Sp1 for TFIIA–TFIID as a function of DNA folding, Our results described here also showed that the posi- which may bring RBP in closer proximity to its targets. tion of the single RBP site in the pIX promoter is deter- Previous studies have shown that GAL4–RBP represses minant to repression, but that this effect is not attribut- transcription irrespective of the position of multiple able to overlap with other transcription factor binding GAL4-binding sites within an artificial promoter con- sites. If the RBP site is situated upstream of the Sp1 struct (Hsieh and Hayward 1995). Because our results consensus site, repression is inoperative in vivo and in show that RBP interacts with TFIID and TFIIA, the pres- vitro. In the case of simple promoters like pIX and E1B, ence of multiple RBP repressor molecules, as in the the distance of the single Sp1 site from the TATA box is GAL4 case, may effectively increase the chances of RBP crucial to activation (Wu and Berk 1988). Stable com- interaction with these targeted factors with resultant re- plexes between Sp1 and TFIID–TFIIA appear to be re- pression irrespective of position. However, our results stricted by the separation between activator and coacti- described here show that there are restrictions to the vators in these simple promoters. RBP may only disrupt position of a single RBP site, as exists in the natural pIX Sp1 interaction with TFIID–TFIIA efficiently when situ- promoter, with respect to pIX repression in vivo and in ated close to the coactivators or alternatively, stable vitro. complexes between RBP and TFIID–TFIIA may also be The disparate role of RBP in repression versus activa- restricted by distance. This restriction on repression in tion in mammalian and Drosophila cells, respectively, the natural case may ultimately reveal an important has yet to be clarified. However, RBP/Su(H) is targeted facet to activated transcription, which is targeted by by a number of viral and Drosophila proteins that modu- RBP. late its activity (see introductory section). Therefore, the Although the position of RBP immediately upstream possibility exists that an as-yet-unidentified endogenous of Sp1 relieved repression, this was not attributable to factor present in Drosophila cells may also modulate Sp1-mediated occlusion of RBP binding. Also, reposi- RBP activity and result in transcription activation. This tioning of the RBP site 20 nucleotides upstream of Sp1 possibility is more likely than RBP conversion to a re- relieved repression. Although we have not examined an pressor by a possible endogenous factor present in mam- RBP site positioned further upstream of Sp1 for repres- malian cells, as purified RBP protein functions as a re- sion in this study, the possibility exists that repression pressor in transcription assays performed in vitro with will be restored if the RBP site is positioned at greater partially purified (Dou et al. 1994) and highly purified distances. In this case, RBP may effectively compete (this study) factors.

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Olave et al.

Transcriptional repressors have been studied exten- nucleotide U-less cassette that were analyzed in transcription sively in prokaryotes, thereby providing a strong prece- assays performed in vitro were derived as follows. DNA frag- dent for the more recently appreciated role of repressors ments containing each of the pIX promoters were generated by in eukaryotes. These previous studies revealed that al- PCR with the appropriate pIX plasmids described above as tem- most all stages of the transcription process can be tar- plates and the primers delineated below. Then, the PCR-derived pIX promoter fragments were inserted into plasmid pMLP-U112 geted by different repressors; indeed, eukaryotic repres- after digestion of the fragments and plasmid with EcoRI sors have been documented to target specific activators and ApaI. The specific 5Ј primers used to generate the PCR and specific components of the basal transcription ma- products were as follows; for pIX/wt, Sub9, and RBP reverse, chinery (for review, see Herschbach and Johnson 1993; 5Ј-TAGATGGAATTCTGGGCGTGGCTTAAGGT-3Ј; for RBP/ Johnson 1995). In this report, we show yet another stage SP1, 5Ј-TAGATGGAATTCGGTGGGAAGAAGTGGGCG-3Ј; in the transcription process to be targeted by RBP—that and for RBP center, 5Ј-TAGATGGAATTCTGGGCGTGGCT- involving TFIIA and TFIID-mediated activation. Future TATGGGA-3Ј; the EcoRI site is shown in bold print. The 3Ј studies of the molecular basis of repression may serve to primer used in all cases was as follows: 5Ј-GTGCTAGGGCCC- provide further insight into the activation process itself. GTTGGTGTGCAAAACTACATAAGACCC-3Ј; the ApaI site is shown in bold. The pMLP-U50 template used as internal control contains the adenovirus major late core promoter se- quences fused to a 50-nucleotide U-less cassette. Materials and methods

Transient expression assays and RNA analyses Purification of native and recombinant RBP Undifferentiated F9 cells were grown in DMEM containing 10% Purification of native RBP from HeLa cell nuclear extract was defined calf serum (Hyclone) on gelatin-treated 100-mm tissue performed as described previously (Dou et al. 1994). Induction of culture plates. Cells were transfected by use of the calcium- GST–RBP fusion protein and its purification with GST beads phosphate precipitation technique as described previously (Ba- was performed as described previously (Yeung et al. 1994). Once biss and Vales 1991). Each transfection was performed with 10 GST–RBP was bound to the beads, 40 NIH units of thrombin µg of construct containing the pIX gene, 10 µg of CMV/RBP (Calbiochem) was added and the sample incubated 4 hr at room expression vector, 10 µg of carrier pGEM-1, and 10 µg of SV/ temperature to release RBP from the GST moiety following pro- dl17 internal control for transfection efficiency (Kannabiran et cedures described for the GST-Gene Fusion System (Pharma- al. 1997). The isolation of RNA and analysis by RNase T2 pro- cia). RBP protein (1.6 mg) was dialyzed at 4°C against buffer C tection with an antisense probe that spans the start site for pIX (20 mM Tris-HCl at pH 7.9, 0.2 mM EDTA, 10 mM ␤-mercapto- transcription were as described previously (Babiss and Vales ethanol, 10% glycerol) with the addition of 80 mM KCl and then 1991). In this case, template for SP6 probe isolation was digested loaded onto a Mono S HR 5/5 column (FPLC, Pharmacia) equili- with AflII so that the probe spans nucleotides 3535–3785 of brated in buffer C. Protein was eluted with a linear gradient adenovirus type 5. The start site for pIX transcription is nucleo- between 80 and 1000 mM KCl in the same buffer. RBP elutes tide 3580 so that pIX mRNA is expected to protect a fragment of with 0.3 M KCl. Fractions were pooled (1.3 mg), dialyzed at 4°C 205 nucleotides. The SV/dl17 internal control contains the against buffer E (50 mM Tris-HCl at pH 8.0, 500 mM NaCl), and SV40 promoter fused to an internal portion of the pIX gene loaded onto a Benzamidine–Sepharose column equilibrated in contained within nucleotides 3560–5640 SV/dl17-derived the same buffer. The flowthrough was collected and reloaded mRNA is expected to protect a fragment of 225 nucleotides. five times onto the column before dialysis at 4°C against buffer Transcripts that initiate within vector sequences upstream of C containing 80 mM KCl and 0.2 mM PMSF. Finally, RBP (0.7 the pIX promoter utilize the adenovirus E1B acceptor splice site mg, 9.5 ml) was concentrated on a Mono S column (SMART at nucleotide 3595 and protect a fragment of 190 nucleotides. System, Pharmacia) equilibrated in the same buffer. RBP was step eluted with buffer C containing 1000 mM KCl, dialyzed in buffer C containing 100 mM KCl, and stored frozen at −80°C. Derivation of plasmid constructs The pIX constructs used in transient expression assays were In vitro transcription assays derived as follows. Oligonucleotides containing the pIX pro- moter with a repositioned RBP site as delineated below were Transcription assays (20-µl reactions) were performed as indi- ligated into HindIII- and BamHI-digested plasmid DNA con- cated previously (Ma et al. 1996) with two DNA templates (100 taining −20 nucleotides upstream of the pIX cap sites as de- ng each) as indicated in the figures. Briefly, transcription factors scribed previously for the wild-type and Sub9 constructs (Babiss used in these assays were: epitope-tagged holo-TFIID (eTFIID, 5 and Vales 1991). The DNA sequences of the oligonucleotides ng of TBP determined by quantitative Western blots, Zhou et al. were as follows: RBP/Sp1, 5Ј-AGCTTGGTGGGAAAGAA- 1992), rTFIIA (␣,␤ and ␥ subunits, 75 ng), rTFIIB (20 ng), RNA GTGGGCGTGGCTTAAGGGCTCGAGCTCATATATAA-3Ј; polymerase II (alkyl–Superose fraction, 40 ng), rTFIIF (20 ng), RBP center, 5Ј-AGCTTGGGCGTGGCTTATGGGAAAGAA- rTFIIE (15 ng), and TFIIH (phenyl–Superose fraction, 65 ng). Ac- GATCTATATAA-3Ј; Sp1/RBP, 5Ј-AGCTTGGGCGTGGCT- tivation reactions additionally contained rPC4 (30 ng) and rSp1 GGGAAAGAAGAGCTCATATATAA-3Ј; RBP reverse, 5Ј- AG- (0.5 fpu; Promega). For RBP-mediated repression, the amounts of CTTGGGCGTGGCTTAAGGGTTCTTTCCCATATATAA-3Ј; RBP added to the reactions are indicated in each figure. Factors the core RBP site is shown in bold, the core Sp1 site is under- and templates were preincubated for 30 min at 30°C and nucleo- lined, and the linker substitution of the normal RBP site is tides were added and incubated for another 45 min at 30°C. The shown in italics. The opposite strands contained a BamHI RNA products were separated by electrophoresis on 8% dena- linker for ligation. The Sub9 construct used here contained an turing polyacrylamide gels. XhoI linker substitution of the RBP site as delineated in RBP/ Single round transcription conditions were established by Sp1 above. pulse and chase procedures with nucleotide starvation. Briefly, The analogous pIX promoter constructs containing the 112 after preincubation of the factors with the template as described

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Transcription repression by RBP through TFIIA and TFIID above, transcription reactions were pulsed by addition of cold constructs as described previously (Dou et al. 1994). The follow- ATP and GTP (0.1 mM final) plus [␣-32P]CTP (0.625 µM). After 2 ing amounts of protein were used: eTFIID (15 µl, containing 5 ng min of incubation, the reactions were chased for 30 min by the of TBP per microliter), rRBP (43 ng), and rSp1 (2 fpu; Promega). addition of cold CTP (0.1 mM) and the products separated as Binding reactions were performed as described above and were above. incubated for 1.5 hr at 30°C. DNase I (BRL, 10 U/µl) digestions were performed with 0.01 units for 30 sec. The reaction was stopped, phenol–chloroform extracted, ethanol precipitated Immobilized templates with carrier RNA, dried, resuspended in formamide loading Formation of transcription competent complexes on immobi- buffer, heated for 2 min at 90°C, and resolved on a 6% denatur- lized templates was performed as described previously (Zawel et ing polyacrylamide gel. al. 1995) with the following modifications: The immobilized template used was a HindIII–NdeI fragment containing the pIX Protein–protein interactions performed in vitro promoter (∼400 bp) derived from the pIX U112 plasmid. The template was biotinylated by Klenow with biotin– Approximately 20 µl of glutathione–agarose beads containing 2 dATP at the NdeI site at 220 bp upstream of the transcription µg of either GST or GST–RBP fusion protein were mixed with initiation site. The amount of DNA and factors used for each 20 µl of eTFIID (containing 5 ng of TBP/µL, determined by reaction was equivalent to seven standard transcription reac- quantitative Western blot analysis), 0.35 µg of rTFIIA, 0.5 µg of tions described above. Biotinylated DNA (0.7 µg; 0.382 pmole) rTFIIB, or 10 µl of hTBP (induced Escherichia coli extract; the was bound to 100 µg of streptavidin-coated magnetic beads ac- equivalent of 2 µg of hTBP was used). Incubation was performed cording to the manufacturer (Dynabeads M280, Dynal, Inc.). for 1 hr at 4°C in a total volume of 0.3 ml with buffer C (20 mM The DNA-bound beads were washed twice with transcription Tris-HCl pH 7.9, 0.2 mM EDTA, 0.1 mM PMSF, 10% glycerol) buffer (Zawel et al. 1995) containing 5 µg/µl of BSA, then incu- containing 100 mM KCl and 0.2% NP-40. The beads were then bated for 20 min in the same buffer and finally washed three washed four times with buffer C containing 500 mM KCl and times with buffer without BSA. Transcription factors were 0.4% NP-40. The bound proteins were then eluted with SDS- added to the template in the presence of 0.4 µg of pBR322 as PAGE loading buffer, resolved by SDS-PAGE, and analyzed by competitor DNA. Bound complexes were washed three times Western blotting. eTFIID interaction was scored by use of with transcription buffer containing 0.05% sarkosyl, and a mag- 12CA5 mAb, TFIIA with 7C12/E2 mAb, and hTBP and TFIIB netic stand (Promega) was used to concentrate the beads to the with affinity-purified antibodies. wall of the tube. The same washing procedure was repeated For assays with proteins translated in vitro (candidate omitting Sarkosyl. Finally, the amount of beads equivalent to dTAFIIs, Fig. 9), equal amounts of protein were used as deter- two transcription reactions was assayed for transcription by use mined by fluorography. Positive lanes show 10% of the proteins of single round transcription as described above, and the re- translated in vitro that were used in the interaction studies. maining beads were used to analyze the factors bound to the Two micrograms of GST–RBP or GST–TFIIA ␣,␤ was used to template by Western blotting. analyze the interaction with different truncated forms of 35S- dTAFII110 (Fig. 10a). Beads were first preincubated for 1 hr at 4 °C with 50 µg of BSA in 0.1 ml of buffer C containing 0.1% Gel retardation and footprinting assays NP-40, and then 3 µl of the different truncated mutant proteins Reaction conditions for gel mobility shift assay were as de- was added and the sample incubated for another hour at 4°C. scribed previously (Maldonado et al. 1990). The probe was pre- The beads were then washed four times with 0.5 ml of buffer C pared as follows from a plasmid containing the wild-type pIX containing 500 mM KCl and 0.2% NP-40, and once with water. gene that was described previously (Dou et al. 1994). pIX plas- The bound proteins were eluted with SDS-PAGE loading buffer, mid DNA digested with NcoI was radiolabeled with Klenow in resolved by SDS-PAGE, the gels treated with 3ENHANCE the presence of all four [␣-32P]dNTPs followed by subsequent (NEN) following the manufacterer indications, dried, and ex- digestion with HindIII. The resultant radiolabeled 160-bp frag- posed overnight. ment contained the pIX promoter from nucleotide −60 to For competiton experiments (Fig. 10b,c), 2 µg of GST–RBP nucleotide +100 relative to the start site for pIX transcription. protein was incubated overnight at 4°C with the indicated Binding assays containing eTFIID were performed in 20 µl re- amounts of rTFIIA ␣,␤ or rTFIIB in 0.1 ml of buffer C containing actions with Mg2+-agarose gels as described previously (Lieber- 0.1% NP-40 and 50 µg of BSA. Beads were washed three times, man and Berk 1994). Incubations were performed at 30°C for 1 resuspended in 0.1 ml of the same buffer, 3 µl (Fig. 10b) or the 35 hr as described above using poly(A-U) (0.5 µg) as a nonspecific amounts indicated (Fig. 10c) of S-labeled dTAFII110 added, and competitor and 5 mM ZnCl2. The amounts of protein in the the reactions incubated for another hour at 4°C. The beads were reactions were as follows: eTFIID (5 µl, containing 25 ng of then treated as specified above. TBP), rTFIIA (75 ng), rSp1 (2 fpu; Promega), and rRBP (71 ng). Far Western analyses of protein–protein interactions were Anti-TFIIA␥ antibodies (1.8 µg) or protein-A-purified anti-GST– performed as described previously (Inostroza et al. 1992). RBP antibodies (1.7 µg) were added when indicated and the Briefly, ∼0.5 µg of each candidate protein was electrophoresed in sample incubated for an additional 30 min at 30°C. The com- two SDS-PAGE mini gels. One was stained with Coomassie petitor oligonucleotides Sp1 and TATA (20 ng) have been de- blue and the other blotted to PVDF membrane. Proteins in the scribed previously (Dou et al. 1994). Then, the complexes were membrane were denatured and renatured as described and then resolved in a 0.7% low EEO–agarose gel (Promega) containing incubated overnight with biotinylated-rRBP (1 µg/ ml). The 3% glycerol in 1× G buffer (5 mM Mg-acetate, 3% glycerol, 0.5× blots were then washed, incubated with streptavidin–alkaline TBE) after electrophoresis at 55 V in the same buffer for 6 hr at phosphatase for 1 hr, washed, and developed with NBT/BCIP as 4°C. described previously. Footprinting assays were performed in 40-µl reactions con- taining 2.5 fmoles of radiolabeled probe. The probe was an Nco- Acknowledgments I–NheI fragment (∼ 390 nucleotides) obtained from plasmid DNA containing either the wild-type or the RBP/Sp1 promoter We gratefully acknowledge the members of the laboratories of

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Olave et al.

D.R. and L.D.V. for their ideas and helpful discussions during Gill, G., E. Pascal, Z.H. Tseng, and R. Tjian. 1994. A glutamine- the progress of this work. We express special gratitude to Dr. rich hydrophobic patch in transcription factor Sp1 contacts

Jin-Long Chen and Dr. Ludger Klein-Hitpass for their generous the dTAFII110 component of the Drosophila TFIID complex donations of the dTAFII110 truncation mutants, which greatly and mediates transcriptional activation. Proc. Natl. Acad. expedited our research studies. This work was supported by Sci. 91: 192–196. grants from the National Science Foundation (MCB-9407333) Goodrich, J.A., T. Hoey, C.J. Thut, A. Admon, and R. and from the National Institutes of Health (GM54890) to L.D.V. Tjian.1993. Drosophila TAFII40 interacts with both a VP16 and by a grant from the National Institutes of Health (GM- activation domain and the basal transcription factor TFIIB. 48518) and by funding from the Howard Hughes Medical Insti- Cell 75: 519–530. tute to D.R. Grossman, S.R., E. Johannsen, X. Tong, R. Yalamanchili, and E. The publication costs of this article were defrayed in part by Kieff. 1994. The Epstein-Barr virus nuclear antigen 2 trans- payment of page charges. 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GENES & DEVELOPMENT 1637 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press

The mammalian transcriptional repressor RBP (CBF1) targets TFIID and TFIIA to prevent activated transcription

Ivan Olave, Danny Reinberg and Lynne D. Vales

Genes Dev. 1998, 12: Access the most recent version at doi:10.1101/gad.12.11.1621

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