Contributed by Robert G. Roeder, September 12, 1995 ...... Dynlacht, B. D., Hoey, T. & Tjian, R. (1991) Cell 66, 563-576. 30. Takada, R., Nakatani, Y., Hoffmann, ...
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11864-11868, December 1995 Biochemistry
Core promoter-specific function of a mutant transcription factor TFIID defective in TATA-box binding ERNEST MARTINEZ*, QIANG ZHOUtt, NOELLE D. L'ETOILEt, THOMAS OELGESCHLAGER*, ARNOLD J. BERKt, ROBERT G. ROEDER*§
AND
*Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021; and Department of Microbiology and Molecular Genetics, University of California, Los Angeles, CA 90024-1570
tMolecular Biology Institute and
Contributed by Robert G. Roeder, September 12, 1995
In conjunction with other general initiation ABSTRACT factors, the TATA box-binding protein (TBP) can direct basal transcription by RNA polymerase II from TATA-containing promoters, but its stable interaction with TBP-associated factors (TAFs) in the TFIID complex is required both for activator-dependent transcription and for basal transcription directed by an initiator element. We have generated a TATAbinding-defective TFIID complex containing an amino acid substitution in the DNA-binding surface of its TBP subunit. This mutated TFIID is defective in both basal and activated transcription from core promoters containing only a TATA box but supports transcription from initiator-containing promoters independently of the presence or absence of a TATA sequence. Our results show that a functional initiator element is needed to bypass the requirement for an active TATA DNA-binding surface in TFIID and imply that gene-specific transcription can be achieved by modulating distinct core promoter-specific TFIID functions-e.g., TBP-TATA versus TAF-initiator interactions.
addition, evidence suggests that TBP binding to DNA is not a rate-limiting step for the initial stages of TFIID recruitment to several initiator-dependent TATA-less promoters (17). Together, these observations raise the intriguing possibility that TBP-DNA interactions, especially as observed in the TBPTATA cocrystal structure (14, 15), may play an important role only on certain class II promoters. By using a mutant TFIID containing a TBP subunit defective in TATA DNA binding, we show that TBP-DNA interactions are largely dispensable for specific transcription of initiator-dependent TATA-less class II genes. Our results thus demonstrate that TBP. like TAFs, differentially contributes to basal transcription in different core promoter contexts.
MATERIALS AND METHODS Plasmids. The 70-kDa heat shock protein (Hsp7O) and terminal deoxynucleotidyltransferase (TdT) core promoter templates were respectively plasmids pHsp70(- 33/ +99)-CAT and pTdT(-41/+59) (17). Linear templates G5-TdT and G5-E1B were, respectively, plasmids pG5TdT(-41/+59) and pG5-ElB-CAT (19) cut with Nde I. pG5TdT(-41/+59) was obtained by digestion of pTdT(-41/+59) with EcoRI at a unique site, filling in the ends with Klenow DNA polymerase, and blunt-end ligation to the Pvu II-Xba I Klenow-filled DNA fragment containing five Gal 4 sites from pG5-ElB-CAT. T- I+, T+I+, and T+I are, respectively, plasmids pG5TdT(-41/+33), pG5TdT( -41TATA+/ +33), and pG5TdT (-41TATA+/Inr-+33) obtained as described in Fig. 5 and previously (17). The ,3-Pol template is plasmid p,BP10 (20). The G5,B-Pol template is plasmid pG5j3Pol(-41/+58)CAT and was obtained by replacing, in pG5-ElB-CAT, the Xba I-BamHI fragment containing the E1B TATAboxwith anXba I-BamHI PCR DNA fragment containing the human ,B-polymerase core promoter sequences from -41 to +58. PCR mutagenesis was used to replace the threonine-210 codon with a lysine codon in the gene encoding the influenza hemagglutinin (HA)-epitope-tagged human TBP in plasmid pLTReTBP (21), creating pLTReTBPT21OK. Cell Lines and Protein Purification. A HeLa cell line stably expressing mutant HA-TBPT2l0K (clone 12 cells) was established by retrovirus-mediated transformation (21) using the vector pLTReTBPT2l0K. Nuclear extracts from wild-type HATBP-expressing cells (LTRct3 cells) (21) and clone 12 cells were fractionated on Whatman phosphocellulose P11. The HA-tagged TFIIIB present in the P11 B fraction (0.1-0.3 M KCl) was immunoaffinity-purified on a monoclonal antibody 12CA5 resin as described (21). The P11 D fraction (0.5-0.85
The general class II transcription initiation factor TFIID is a multisubunit complex composed of the TATA box-binding protein (TBP) and several TBP-associated factors (TAFs) (1). For TATA-containing class II genes, the binding of TBP to the TATA element recruits TFIID to the promoter and represents the first step in preinitiation complex assembly (2-4) that can be influenced by upstream activators (5-13). Although TBP, in the absence of TAFs, suffices to direct basal TATA-dependent transcription, TAFs are required for the stimulation by upstream activators (for review, see ref. 1). Since specific TBPTATA interactions dramatically distort DNA (14, 15), TBP may also control the architecture, and hence the function, of the preinitiation complex on TATA-containing promoters. In contrast to the situation in TATA-containing promoters, the mechanisms of transcription initiation at promoters that lack a TATA box, and in particular the role of TBP in transcription initiation at TATA-less genes, are still poorly characterized. Earlier studies showed that an initiator DNA sequence can function as an independent core promoter element which specifies the position of the transcription start site in TATAless promoters and that it also can stimulate basal transcription from TATA-containing promoters when located about 25 bp downstream of a TATA box (reviewed in ref. 16). Recently, TAFs (in association with TBP) in the human TFIID complex have been found to be essential not only for activatordependent transcription but also for the basal (activatorindependent) transcription function of an initiator element, regardless of the presence or absence of a TATA box (17). This novel initiator-dependent function of TAFs in basal transcription was subsequently confirmed for Drosophila TFIID as well, albeit in the context of a TATA-containing promoter (18). In
Abbreviations: TBP, TATA box-binding protein; TAF, TBP-associated factor; Hsp, heat shock protein; TdT, terminal deoxynucleotidyltransferase; HA, hemagglutinin; EMSA, electrophoretic mobility-shift assay. tPresent address: Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139. §To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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inactivation of TFIID in HeLa nuclear extracts, RNA polymerase II transcription reactions, and primer extension analyses were performed as described (17, 25). EMSA was performed (10) with a 200-bp PCR probe containing the E1B TATA box from pG5-EIB-CAT (19) and with equal amounts of each TFIID complex as determined by silver staining and Western blotting.
RESULTS AND DISCUSSION
T21 0
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208 R T T A L I F214
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AI
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FIG. 1. Structure of Arabidopsis thaliana TBP a-carbon chain bound to a TATA element (15). The DNA filling the interior concave surface is not shown. Threonine-70, corresponding to threonine-210 (T210) in human TBP, is shown as a side chain and in space-filling mode. The position of the threonine-210 -> lysine substitution (T210K) in the first repeat (arrow) of the human TBP core domain (residues 160-339) is schematically indicated below the structure.
M KCl) was loaded on a Whatman DE52 column, and the 0.1-0.3 M KCl fraction was used to immunopurify HA-tagged TFIID as above. Recombinant hexahistidine-tagged human TBP was expressed in bacteria and purified on Ni2+_ nitrilotriacetate (NTA) agarose as described (22) and then fractionated on heparin-Sepharose with step elution at 0.2-0.5 M KCl. Bacterially expressed Flag-tagged Gal4-VP16 was purified as reported (23). In Vitro Transcription and Electrophoretic Mobility-Shift Assay (EMSA). RNA polymerase III transcription reactions using pVA1 as template (24) contained 18 ,ug of TBP-immunodepleted P11 B fraction, 6 ,ug of P11 C fraction (0.3-0.6 M KCl), and 3 ,A (3 ng of TBP) of HA-TFIIIB complex. Heat
B
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To address directly the contribution of TBP-DNA interactions in the context of a complete TFIID complex during transcription of different class II genes, we have introduced a point mutation (T210K) that converts threonine-210 in the first repeat of the human TBP core domain to lysine (Fig. 1). The corresponding mutation in Saccharomyces cerevisiae TBP (Ti 12K) is dominant negative, prevents TBP from binding to a TATA element in vitro (26), and impairs transcription of TATA-containing class II but not TATA-less class I or class III genes (27). Threonine-210 directly contacts the DNA sugarphosphate backbone near the middle of the lower strand of the TATA element (14, 15). Thus, the larger lysine side chain in the mutant probably sterically interferes with stable DNA binding. A gene encoding a HA epitope-tagged human TBP carrying the T210K mutation was stably introduced into human HeLa cells. Nuclear extracts were prepared from cells expressing wild-type HA-TBP and mutant HA-TBPT2l0K and were used to immunopurify, respectively, the wild-type and mutant epitope-tagged TBP-TAF complexes TFIID and TFIIIB. We first ascertained that the human HA-TBPT2l0K mutant protein expressed in HeLa cells is correctly folded by showing that (i) the mutant TFIIIB complex can restore RNA polymerase III transcription of the tRNA-like adenovirus VAI gene in a reconstituted TBP-dependent transcription system to levels similar to those obtained with wild-type TFIIIB (Fig. 2A, compare lanes 2 and 3), (ii) all the major TAFs can associate with TBPT210K into a stable TFIID complex (Fig. 2B), and (iii) TBPT2L0K binds TFIIA and TFIIB like wild-type TBP (data not
shown). We verified that this mutation impairs TATA element binding by human TBP in association with TAFs in a stable TFIID complex by using an EMSA. Wild-type (Fig. 2C, lanes 2 and 3) but not mutant (lanes 4 and 5) TFIID generated a TFIID wt
TFIID mt 0 1e 2 3' 0 1e 2 3°
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FIG. 2. TFIIIB and TFIID complexes containing TBPT210K, a mutant protein defective in TATA-box binding. (A) RNA polymerase III transcription of the adenovirus VAI gene was analyzed in a TBP-dependent system in the absence (lane 1) or presence of equal amounts of either wild-type (wt; lane 3) or mutant T210K (mt; lane 2) HA-tagged TFIIIB complex. VAI RNA is indicated. (B) Subunit composition of immunoaffinity-purified TFIID complexes containing wild-type HA-TBP (TFIID wt) and mutant HA-TBPT210K (TFIID mt). A silver-stained 6-20% gradient SDS/polyacrylamide gel is shown. Lanes 0, 8 ,ul of the last buffer wash of the resin before TFIID elution; lanes 10, 2°, and 30, 8 ,ul each of consecutive TFIID elutions with the HA peptide. TAFs and their molecular mass (in kilodaltons) are indicated. A star indicates weakly stained or substoichiometric TAFs. (C) EMSA with increasing amounts of wild-type (wt; lanes 2 and 3) and T210K mutant (mt; lanes 4 and 5) TFIID complexes. A probe containing the adenovirus E1B TATA box was used. Arrow indicates the specific protein-DNA complex.
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Proc. Natl. Acad. Sci. USA 92
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FIG. 4. Activator-dependent function of mutant TFIID(TBPo210K) on the TATA-less ,B-polymerase promoter. (A) Basal transcription from the human ,B-polymerase core promoter (region -41 to +62) was analyzed as in Fig. 2, in the absence (lane 2) or presence of equivalent amounts (4 ng of TBP) of either wild-type (wt; lane 3) or mutant (mt,
lane 4) TFIID. Control (C; lane 1), as in Fig. 2, shows the position of
TFIID TFIID wt mt TBPwt mt TBP .
G5-TdT-i_ (TATA-)
specific transcripts (,B-Pol arrow). (B) Activator-dependent transcrip-
tion from a template containing five Gal4 sites upstream of the 13-polymerase core promoter (region -41 to +59) was performed as above, in the presence of either bovine serum albumin (0.5 jig; lanes 1 and 5), hexahistidine-tagged human TBP (4 ng of TBP; lanes 2 and 6), wild-type TFIID (wt, 1 ng of TBP; lanes 3 and 7), or mutant TFIID (mt, 1 ng of TBPT21OK; lanes 4 and 8) in the absence (lanes 1-4) or presence (lanes 5-8) of 1 pmol of Flag-tagged Gal4-VP16. Position of correctly initiated transcripts is shown (G513-Pol arrow and arrowhead). Transcription in the absence of activator was not detected with this template and at the low level of. TFIID employed; the weak nonspecific band in lanes 1-6 is located above the position of the specific transcripts and is resistant to low concentrations of a-amanitin
(data not shown). G5-El B -_-
were obtained in DNase I footprinting assays in which wildtype but not mutant TFIID interacted with a TATA-
(TATA ) 1
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FIG. 3. Mutant TFIID(TBPT2I0K) supports basal and activatormediated transcription from TATA-less but not TATA-dependent promoters. (A) Transcription from a supercoiled human Hsp7O core promoter was analyzed in normal (control, C; lane 1) and TFIIDinactivated (lanes 2-9) HeLa nuclear extracts. Reactions (lanes 2-9) were complemented with either bovine serum albumin (2 jig; lane 2), HA peptide (4 jig; lane 6), or increasing amounts of wild-type (wt) TFIID (4, 6, and 8 ng of TBP; lanes 3-5) or mutant (mt) TFIID (2, 3, and 4 ng of TBPT21oK; lanes 7-9). Specific transcripts (Hsp7O arrow) were detected by primer extension. (B) Transcription from a supercoiled TATA-less mouse TdT core promoter was analyzed as in A. Specific transcripts are indicated (TdT arrow). A bracket indicates weak initiations upstream of the main + 1 start site. (C) Transcription from linear templates containing five Gal4 binding sites upstream of the TdT core promoter (G5-TdT) or the ElB TATA box (G5-ElB) was analyzed in a TFIID-inactivated nuclear extract complemented with either bovine serum albumin (1 jig; lanes 1 and 8), wild-type TFIID (2.5 ng of TBP; lanes 2 and 5), mutant TFIID (2.5 ng of TBPT2lOK; lanes 3 and 6), or recombinant human hexahistidine-tagged TBP (20 ng of TBP; lanes 4 and 7). Reactions were performed in the absence (lanes 1-4) or presence (lanes 5-8) of a 1 pmol of Flag-tagged Gal4-VP16 (Gal-VP16). Specific transcripts are indicated by arrows for G5-TdT and G5-EIB. The bracket is as in B.
protein-DNA complex with a DNA probe containing the TATA box of the adenovirus E1B promoter. Similar results
containing promoter DNA fragment (T+I+ in Fig. 5) both in the absence and in the presence of TFIIA (data not shown). We next examined the ability of mutant TFIID to support transcription from TATA-containing and TATA-less class II promoters in a TFIID-inactivated nuclear extract (Fig. 3). Consistent with its deficiency in TATA-box binding, the mutant TFIID complex did not support basal transcription from the TATA-dependent natural human Hsp7O core promoter (Fig. 3A, lanes 7-9) and the synthetic G5-E1B promoter that contains the adenovirus ElB TATA box (Fig. 3C, lane 3). These results demonstrate that TFIID interactions with the general transcription machinery cannot compensate for the DNA-binding deficiency of mutant TBPT21oK on these two TATA-containing promoters. In contrast, mutant TFIID directed specific basal transcription from the TATA-less mouse TdT core promoter to levels similar to those obtained with equivalent amounts of wild-type TFIID (Fig. 3B, lanes 3-5 and 7-9; Fig. 3C, lanes 2 and 3). Moreover, transcription initiation directed by mutant TFIID was more specific for the + 1 position of the TdT initiator element (Fig. 3 B and C, TdT arrow) as compared with transcription directed by wild-type TFIID, which also directed weak initiation from TdT promoter positions just upstream of the main + 1 initiation site (Fig. 3 B and C, brackets) and from flanking plasmid sequences farther upstream (data not shown). Because upstream activators can influence the binding of TFIID to TATA-containing promoters (5-13), we tested
Biochemistry: Martinez et A. wt TT-
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Proc. Natl. Acad. Sci. USA 92 (1995)
2
INR
T TII i TATA FIG. 5. The activity of mutant TFIID(TBPT210K) is independent of the -30 DNA sequence but requires a functional initiator element. Transcription activities mediated by TFIID(TBPT2l0K) in the presence of Gal4-VP16 of the templates T-I+, T+I+, and T+I- are compared. T-I+ contains five Gal4 sites upstream of the natural TATA-less initiator (INR)-containing region (-41 to +33) of the TdT promoter. T+I+ differs from T-I+ by the substitution of nucleotides in the natural TdT -30 region to create a consensus TATAAAA element. T+I- differs from T+I+ by six base-pair substitutions in the initiator element that abolish initiator activity (17). Transcription reaction mixtures (as in Fig. 2) contained 1 pmol of Flag-tagged Gal4-VP16 and either bovine serum albumin (0.5 ,tg; lanes 1, 4, and 7) or equal amounts (1.5 ng of TBP) of wild-type TFIID (wt, lanes 2, 5, and 8) or mutant TFIID (mt, lanes 3, 6, and 9). Specific transcripts (arrow) were detected by primer extension using the same end-labeled primer for all three constructs.
T
TXN
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whether a functional TATA-binding domain in TFIID is required for the response to upstream activators in different core promoter contexts. We first analyzed activation by the chimeric Gal4-VP16 activator from templates containing five Gal4 sites upstream of the EiB TATA box (G5-E1B) and of the TATA-less TdT core promoter (G5-TdT) in the presence of either the wild-type or the TATA-binding-deficient TFIID (Fig. 3C). Whereas wild-type TFIID supported activation by Gal4-VP16 from both templates (lane 2 versus lane 5), mutant TFIID mediated activation only of the G5-TdT promoter and to a level comparable to that of wild-type TFIID (lanes 5 and 6). These results demonstrate that, as for basal transcription, activation by Gal4-VP16 from the G5-E1B template requires a functional TATA-binding domain within TFIID. In contrast, neither a TATA box nor a functional TATA-binding domain is needed for TFIID to stimulate basal and Gal4-VP16 activator-dependent transcription from the TdT promoter. Equivalent results were obtained with Gal4-Spl activator (data not shown). In addition, because mutant TFIID is required for basal and activated transcription from both supercoiled (Fig. 3B) and linear (Fig. 3C) TdT templates, the mechanisms involved differ from the recently described TBP-independent basal RNA polymerase II transcription that requires the use of supercoiled promoters (28). We analyzed similarly the activity of mutant TFIID on the TATA-less human f3-polymerase gene promoter (Fig. 4). In contrast to the results obtained with the TdT promoter, the mutant TFIID did not support basal transcription from the ,B-polymerase core promoter (Fig. 4A, lane 4). This suggests that nonspecific TBP-DNA interactions contribute to the formation of a productive basal transcription initiation complex on the f3-polymerase promoter. Interestingly, however, on a template with five Gal4 sites located upstream of the f3-polymerase core promoter, wild-type and mutant TFIID
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FIG. 6. Summary and model for core promoter-specific function of TATA-binding-defective TFIID. Line 1: On a TATA-containing initiator-less promoter, wild-type TFIID (TBP wt) binds to the TATA element and interacts both with upstream activators, through TAFs (21, 23, 29-33) and/or additional cofactors (33), and with components of the general transcription machinery-i.e., general transcription factors (GTF) and RNA polymerase II (POL), to direct specific transcription (TXN). Depending on the promoter, TAFs may or may not interact (schematized with a double-headed arrow) with the initiatorless region downstream of the TATA box (10, 33, 34) in a manner that can be influenced by upstream activators (8-10). This is consistent with the ability of TAFs to directly contact two DNA regions downstream of the initiator in some genes (35). Line 2: On this initiatorless promoter the TATAbinding-defective mutant TFIID (TBP mt) does not support transcription, suggesting that interactions of mutant TFIID with activators and the rest of the transcription machinery are not sufficient for its functional recruitment to this type of promoter. Lines 3 and 4: In the presence of a functional initiator element, TATA-bindingdefective TFIID directs a similar level of transcription in the presence (line 4) or absence (line 3) of a consensus TATA element. This indicates that specific TBP mt-DNA interactions are largely dispensable (symbolized by a TBP mt that does not touch the promoter) and are most likely compensated by the concerted interactions of different TAFs with activators, the initiator, components of the general transcription machinery (GTF and POL), and possibly other factors that recognize this complex and/or the initiator element (X?); the latter may include previously described initiator-binding proteins (reviewed in ref. 17). The present data do not exclude the possibility of nonspecific low-affinity contacts between TBP mt and DNA in the preinitiation complex. Line 5: In the presence of wild-type TFIID (TBP wt), TATA and initiator elements synergize to give maximal levels of transcription.
mediated similar levels of Gal4-VP16-dependent transcription (Fig. 4B, lanes 7 and 8; see also figure legend). This indicates
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that upstream activators can compensate for the TATAbinding deficiency of mutant TFIID on the 13-polymerase promoter but not on the G5-E1B promoter (Fig. 3C). Together, these results suggest that differences in core promoter sequences are responsible for the differential activity of mutant TFIID on the TATA-containing Hsp7O and E1B and TATA-less TdT ,3-polymerase templates. To identify the core elements that bypass the requirement for a functional TATA-binding surface in TFIID, we tested the contribution of the TdT -30 and initiator regions for Gal4VP16-activated transcription in the presence of the TATAbinding-deficient TFIID. Substituting a consensus TATA element for the natural -30 region of the TdT core promoter (T+I+) strongly stimulated activated transcription mediated by wild-type TFIID (Fig. 5; lane 2 versus lane 5) but did not affect transcription directed by mutant TFIID (lane 3 versus lane 6). This further confirms that mutant TFIID has no residual TATA DNA-binding activity and that the T210K mutation in TBP does not create a new DNA-binding surface specific for the natural TdT -30 region. Significantly, in the presence of a TATA box and of Gal4-VP16, mutation of the initiator element (T+I-) abolished transcription directed by mutant TFIID (lane 9 versus lane 6). These results demonstrate that the transcription activity of mutant TFIID is independent of the DNA sequence in the -30 region but requires a functional initiator element. Differences in TdT and ,B-polymerase initiator regions could thus explain the requirement of an activator for transcription mediated by mutant TFIID from the f3-polymerase promoter (see above). We have not been able to detect significant binding of wild-type or mutant TFIID to the TATA-less TdT promoter (T-I+) by .Nase I footprinting assays (data not shown). In contrast, wild-type (but not mutant) TFIID binds efficiently to the initiator-less T+I- template, producing a footprint that extends from the TATA element to sequences downstream of the initiation site (data not shown). Thus, the similar transcription activity of T-I+ and T+I- templates (Fig. 5) does not correlate with the differential affinity of TFIID for these promoters. This suggests that the function of the initiator may also involve its recognition by other components of the transcription machinery (see legend of Fig. 6). In conclusion, we have demonstrated that a functional TATA-binding domain in TFIID is required for the activity of TATA-containing initiatorless promoters but dispensable for specific transcription from promoters containing an initiator element (Fig. 6). Since TAFs are essential for basal initiator function (17, 18, 34) but dispensable for basal transcription from TATA-dependent promoters (2-4), TFIID thus contains at least two separable core promoter-specific functions-i.e., TBP-TATA and TAF-initiator interactions (see legend of Fig. 6)-that are variably required or dispensable depending on the core promoter structure. Our results thus suggest the possibility of selective gene regulation by modulating distinct core promoter-specific TFIID functions. This is consistent with the observed core promoter-specific activity of some activators and repressors (36-41). Finally, since TBP binding to the TATA element induces a sharp DNA bend (14, 15), our results raise the possibility either that core promoter bending is not essential for transcription from some promoters or that bending is achieved by a different mechanism and/or requires additional factors at TATA-less genes. We thank S. Burley for comments on the manuscript and J. Huang and X. Zhang for excellent technical assistance. This work was supported by National Institutes of Health grants to R.G.R. and A.J.B. and by general
(1995)
support from the Pew Trusts to the Rockefeller University. E.M. was supported by a postdoctoral fellowship from the Swiss National Science Foundation; Q.Z. and N.D.L. were supported by a National Institutes of Health grant to A.J.B., and T.O. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft.
1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14.
15. 16.
17. 18. 19. 20.
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23. 24. 25. 26. 27. 28. 29. 30. 31.
32. 33. 34. 35. 36.
37. 38.
39. 40. 41.
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