TFIIB Recognition Elements Control the TFIIA-NC2 Axis in Transcriptional Regulation

TFIIB recognizes DNA sequence-specific motifs that can flankthe TATA elements of the promoters of protein-encoding genes.The TFIIB recognition elements (BRE^u and BRE^d) can have positiveor negative effects on transcription in a promoter context-dependentmanner. Here we show that the BREs direct the selective recruitmentof TFIIA and NC2 to the promoter. We find that TFIIA preferentiallyassociates with BRE-containing promoters while NC2 is recruitedto promoters that lack consensus BREs. The functional relevanceof the BRE-dependent recruitment of TFIIA and NC2 was determinedby small interfering RNA-mediated knockdown of TFIIA and NC2,both of which elicited BRE-dependent effects on transcription.Our results confirm the established functional reciprocity ofTFIIA and NC2. However, our findings show that TFIIA assemblyat BRE-containing promoters results in reduced transcriptionalactivity, while NC2 acts as a positive factor at promoters thatlack functional BREs. Taken together, our results provide abasis for the selective recruitment of TFIIA and NC2 to thepromoter and give new insights into the functional relationshipbetween core promoter elements and general transcription factoractivity.

INTRODUCTION

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INTRODUCTION

Transcription of a gene by RNA polymerase II requires the assemblyof the general transcription factors at the promoter to forma preinitiation complex (PIC). The eukaryotic core promotercontains DNA sequence elements that direct the assembly of thegeneral transcription factors and provide a platform for therecruitment of the PIC (4, 29, 53, 59). These elements includethe TATA box, initiator, downstream promoter element (DPE),downstream core element, and motif 10 element (41). In addition,the general transcription factor TFIIB can recognize two distinctelements within the core promoter that flank the TATA box: theupstream and the downstream TFIIB recognition elements (BRE^uand BRE^d) (reviewed in reference 17).

Interaction with BRE^u is mediated by a helix-turn-helix withinthe second direct repeat of TFIIB, and interaction with BRE^dis mediated through a recognition loop within the first directrepeat (16, 39, 60). These interactions serve to increase theaffinity of TFIIB in forming a complex with TATA-binding protein(TBP) at the promoter. Each of the BREs is potentially presentin up to 30% of human TATA-containing promoters, but the extentof their functional influence remains to be determined (16,25, 39). BREs can also function at TATA-less promoters, buttheir identification in this context is less reliable and thustheir potential genome-wide prevalence is difficult to estimate.The function of BREs in transcription appears to be complex.The BREs can act as both positive and negative elements (8,16, 19, 39). Moreover, activator proteins can modulate the interactionbetween TFIIB and BRE^u (19).

At the adenovirus major late (AdML) promoter the BREs act asnegative elements (8, 16, 19). The reason for this is not known,but candidates for involvement include the general transcriptionrepressors Mot1 and NC2. Mot1 acts to prevent the interactionbetween TBP and the TATA box, but NC2 can assemble with TBPat the promoter and precludes the assembly of TFIIB or TFIIA,thus preventing formation of the PIC (reviewed in references50 and 59). NC2 is composed of two subunits, ${alpha}$ and β, alsoknown as Dr1 and DRAP, respectively (27, 33, 34, 36, 48). TFIIAcan compete with NC2 for association with TBP at the promoter,and this mutually exclusive assembly is underpinned by geneticstudies of Saccharomyces cerevisiae that show a direct relationshipbetween TFIIA and NC2 (64). In addition, assembly of NC2 withTBP at the TATA box can mobilize the complex to translocatealong the DNA (52). NC2 also inhibits transcription by RNA polymeraseIII (pol III), but not by pol I (35, 62). A few studies overthe last few years have unhinged the apparently establishedreciprocity between NC2 and TFIIA. Several reports have suggesteda positive function for NC2 (5, 7, 11, 22, 63). In addition,the transcription-regulatory properties of NC2 exhibit corepromoter-specific activities (1, 45, 63). How the positive activitiesof NC2 reconcile with the reciprocal nature of NC2 and TFIIAin directing transcription has not been determined.

TFIIA is composed of three subunits, ${alpha}$ , β, and ${gamma}$ (reviewedin reference 30), with the ${alpha}$ and β subunits derived froma single gene (15, 43, 49, 57, 66). Although TFIIA was originallycharacterized as a general transcription factor, many in vitrotranscription systems do not require TFIIA, and TFIIA is perhapsbetter described as a general cofactor that enhances transcriptionactivation. Indeed, transcriptional activator proteins can stimulatethe assembly of a TFIIA-TFIID complex at the promoter (9, 37,40, 42, 44, 55).

In this study we have analyzed the role of the BREs in the negativeregulation of the AdML promoter in living cells. We find thatthe BREs direct the mutually exclusive assembly of TFIIA andNC2. Moreover, TFIIA and NC2 correlate with low and high promoteractivities, respectively. Use of small interfering RNA (siRNA)to specifically deplete TFIIA and NC2 in living cells confirmsa positive role for NC2 in transcription and, surprisingly,a transcriptional dampening role for TFIIA. We provide evidencethat this involves overlapping recognition of the core promoterby TFIIA with the BREs.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Plasmids, site-directed mutagenesis, and cloning. G5ML containing nucleotides –50 to +22 from the AdML promoterdownstream of five GAL4 DNA-binding sites and the plasmid drivingexpression of GAL4 region II activation domain linked to theGAL4 DNA-binding domain have been described before (19). AllAdML promoter derivatives were produced with the QuikChangekit (Stratagene). G5ML and derivatives were subcloned into thereporter vector pGL3 basic (Promega).

Cell lines. Human embryonic kidney 293T cells were cultured in completemedium (Sigma) as described previously (6, 47). The HEK293 stablecell lines were generated using the Flp-In system (Invitrogen).Briefly, the system uses an engineered 293 cell line derivativethat contains a single copy of the Flp recombinase targetingsite (FRT) under the control of a zeocin selection marker. TheAdML promoter (wild type or AdMLmBRE^ud derivative) linked toluciferase was cloned into pcDNA5/FRT, replacing the cytomegaloviruspromoter within the plasmid. The resulting plasmids (pcDNA5/FRT-AdML-lucand pcDNA5/FRT-AdMLmBRE-luc) were transfected into the zeocin-resistantHEK 293 cells along with the plasmid pOG44 (Invitrogen), whichexpresses the recombinase. Stable cell lines were obtained fromsingle colonies through selection with hygromycin b.

Transfection, luciferase assays, and immunoblotting. Human embryonic kidney 293T cells were cultured in completemedium (Sigma) as described previously (6, 47). The HEK 293Tcells were transfected with Lipofectamine 2000 transfectionreagent (Invitrogen). Cells were harvested at 48 h posttransfection,and the luciferase activity was determined by using the luciferaseand β-galactosidase dual-light detection system (AppliedBiosystems) as described previously (28). Meanwhile, 20 µlof cell lysate for each transfection was used for immunoblotting,where all primary antibodies for NC2β (ab50783), TFIIA ${alpha}$ /β(ab28176), TFIIA ${gamma}$ (ab37846), TFIIB (ab12094), the pol II C-terminaldomain (ab24758), TBP (ab818), and Mot1 (ab5268) were purchasedfrom Abcam. Secondary antibodies were from Jackson ImmunoResearch.Detection in Western blots was performed by enhanced chemiluminescence(GE Healthcare).

ChIP assays. Chromatin immunoprecipitation (ChIP) assays were performed accordingto the published method (54) with modifications. Briefly, cellswere cross-linked with 1% formaldehyde-phosphate-buffered salinefor 10 min at room temperature and quenched by the additionof glycine to a final concentration of 250 mM. The cells werewashed twice with cold phosphate-buffered saline and harvested.For the ChIP assays of the AdML promoter and derivatives, thecells were Dounce homogenized. Cell lysates were then transferredto microtubes, and the nuclei were collected by brief centrifugationand then resuspended in lysis buffer (50 mM Tris-Cl, pH 8.0,10 mM EDTA, 1% sodium dodecyl sulfate). Samples were then sonicatedfor 10 min at high power with a Diagenode sonicator. After sonication,the samples were centrifuged in a benchtop microcentrifuge at4°C, and the supernatants were diluted with ChIP dilutionbuffer (0.01% sodium dodecyl sulfate, 1.1% Triton X-100, 1.2mM EDTA, 16.7 mM Tris-Cl, pH 8.0, 167 mM NaCl). For immunoprecipitation,the samples were precleared using preblocked protein G-Sepharose(Sigma) with 1 mg/ml bovine serum albumin and 0.1 mg/ml salmonsperm DNA. Two micrograms of each antibody was added to thesamples, and samples were incubated overnight at 4°C, followedby the addition of 20 µl preblocked protein G-Sepharoseto the samples and incubation for 1 h at room temperature. Theremainder of the procedure was followed according to the methoddescribed previously (54) except that the washing step was prolongedto 0.5 to 1 h for each wash and that the DNA was purified witha Qiagen DNA purification kit. For endogenous promoter ChIPassays, the cells were directly lysed in nucleus lysis bufferafter harvesting, followed by sonication. All other steps werethe same as those used for the AdML promoter ChIP assays. Theprimers used in the PCRs were as follows: AdML forward, 5'-CTAGCAAAATAGGCTGTCCC-3';reverse, 5'-TCCAGCGGATAGAATGGC-3'; Bcl2 forward, 5'-TTCCTGCATCTCATGCCAAG-3';reverse, 5'-GGATGTACTTCATCACTATCTC-3'; insulin-like growth factorII (IGFII) forward, 5'-CTCTTGGCTCGGGTTGCGG-3'; reverse, 5'-GCAGGCGTGGGCCAGGAGG-3'.

The primers used for real time PCR were as follows: forward,5'-GAGGATCCGTGTTCCTGAAG-3'; reverse, 5'-GAGTCAGTGAGCGAGGAA-3'.Real time PCR was performed with CYBR green master mix (Eurogentec)and a Chromo 4 thermal cycler machine (MJ Research). The programused for PCR was 94°C for 45 s, 60°C for 30 s, and 72°Cfor 30 s for a total of 35 to 40 cycles. The enrichment of targetDNA (relative occupancy) is expressed as the ratio of the amountof promoter DNA bound to the protein of interest compared tononpromoter DNA. The input DNA was derived from 1/10 of thevolume of sample used for immunoprecipitation of the targetprotein. All experiments were performed at least in triplicate.

siRNA knockdown, RNA isolation, and RT-PCR. Double-stranded siRNA oligonucleotides for TFIIA ${alpha}$ β, TFIIA ${gamma}$ ,NC2 ${alpha}$ , and NC2β were purchased from Eurofins Biotech. Thesecond TFIIA ${alpha}$ β siRNA (2) was obtained from Santa Cruz Biotechnology.Sequences of the siRNAs were as follows: NC2 ${alpha}$ , 5'-AGAGGAACGAAGAAGAUUACTT-3';NC2β, 5'-UUGGAGAGCUAAGUAAGUATT-3'; TFIIA ${alpha}$ β, 5'-GUACUGAUGGAACUAAAAATT-3';TFIIA ${gamma}$ , 5'-ACAGAUCACCCCCCAACUUTT-3'. siRNAs (10 pmol) were transfectedwith Lipofectamine 2000 (Invitrogen) either alone or in combinationwith plasmid DNA. Forty-eight hours after transfection, cellswere harvested and used for either luciferase assays, Westernblotting, or the preparation of RNA. Total RNA was preparedwith the RNeasy minikit (Qiagen). For reverse transcription-PCR(RT-PCR), cDNAs were synthesized from 3 µg of total RNAfor each sample with the Access reverse transcription kit (Promega)and subjected to PCR with primers to amplify the RNA. Primersused for RT-PCR were as follows: IGFII forward, 5'-GAGTGCTGTTTCCGCAGCTGTG-3';reverse, 5'-CTGAACGCCTCGAGCTCCTTG-3'; P21 forward, 5'-GAACTTCGACTTTGTCACCG-3';reverse, 5'-GGTAGAAATCTGTCATGCTG-3'; Egr1 forward, 5'-CGCAAGAGGCATACCAAGATCC-3';reverse, 5'-CAGCTGAGGAAGGGAAGCTG-3'; BCL2 forward, 5'-GTGGTGGAGGAGCTCTTCAG-3';reverse, 5'-GCAGAGTCTTCAGAGACAGCC-3'; 18S forward, 5'-GTAACCCGTTGAACCCCATT-3';reverse, 5'-CCATCCAATCGGTAGTAGCG-3'.

Proteins and band shift. Promoter DNA for wild-type AdML and AdML derivatives was labeledwith [ ${alpha}$ -³²P]dATP (GE Healthcare). Purified native TFIIA was obtainedfrom Protein One Inc. NC2 protein was prepared from Escherichiacoli BL21 coexpressing NC2 ${alpha}$ and NC2β from a bicistronicplasmid (described in reference 44). Ni-nitrilotriacetic acid-agarosechromatography was used to purify the NC2 via a His tag at theN terminus of NC2 ${alpha}$ . Recombinant human TBP and TFIIB were purifiedas described before (16, 26). Band shift assays were performedas described previously (26).

RESULTS

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RESULTS

The AdML promoter contains both a BRE^u and BRE^d that flank theTATA element (Fig. 1A). Based on previous functional data wegenerated mutant derivatives of this promoter that lack a functionalBRE^u (AdMLmBRE^u), BRE^d (AdMLmBRE^d), both BREs (AdMLmBRE^ud),or the TATA element (AdMLmTATA) (16, 19, 39). The promoter derivatives,driving luciferase expression and controlled by five GAL4 DNA-bindingsites, were transfected into human embryonic kidney 293T cellsalong with a plasmid driving expression of GAL4-RII (GAL4 residues1 to 147, linked to the region II activation domain of GAL4)and a β-galactosidase internal control. Cells were harvested48 h later, and luciferase activity was measured and presentedgraphically in Fig. 1B. Consistent with our previous resultsand those of others, mutation of either BRE^u or BRE^d causedan increase in promoter activity (8, 16, 19). Mutation of bothBREs within the AdML caused a further increase in promoter activity,and mutation of the TATA element resulted in a significant reductionin promoter activity. Thus, the BREs within the AdML promoteract as negative elements either alone or in combination.

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FIG. 1. Differential recruitment of TFIIA and NC2 at the AdML promoter is dependent upon the BREs. (A) The promoter sequence of the wild-type (wt) AdML promoter is shown at top, with the TATA element underlined and BRE^u and BRE^d shaded at the bases that match their consensus sequences. (B) The promoter derivatives in panel A, linked to five GAL4 DNA-binding sites and driving luciferase transcription, were transfected into embryonic kidney 293T cells along with a vector driving expression of GAL4 activation domain II linked to the GAL4 DNA-binding domain. Forty-eight hours later luciferase activity was measured and normalized to β-galactosidase activity and is presented graphically. Each bar represents three independent experiments with standard deviation. (C) 293T cells were transfected as in panel B and subjected to ChIP with antibodies against TBP, TFIIB, and pol II (left) and TFIIA ${alpha}$ /β or NC2β (right). The results are presented as enrichment of specific promoter DNA over nonpromoter DNA in the immunoprecipitate. Each bar represents at least three independent experiments with standard deviation. A gel-based version of these data, including the analysis of additional factors, is shown in Fig. S1 in the supplemental material. IgG, immunoglobulin G.

To determine the mechanisms that underlie the negative functionof the BREs within the AdML promoter, we next assessed the patternof assembly of key general transcription factors at the AdMLpromoter derivatives in living cells. The AdML promoter derivativeswere transfected as described above, and then ChIP was performedwith the antibodies indicated in Fig. 1C. TBP assembled at thepromoters in a BRE-independent manner but showed a significantreduction in assembly at the AdMLmTATA derivative (Fig. 1C,left). TFIIB and pol II assembled at the promoter derivativesto levels that reflect their transcriptional potency, exhibitinga greater level of assembly at the AdML promoter derivativeswith defective BREs (Fig. 1C, left). NC2β also showed anincrease in assembly at the AdML promoters with mutant BREs(Fig. 1C, right), as did NC2 ${alpha}$ (see Fig. S1 in the supplementalmaterial). TFIIA showed robust assembly at the wild-type AdMLpromoter but lower levels of assembly when either or both ofthe BREs were mutated (Fig. 1C, right). Taken together, thesedata show a strong correlation between the BREs and TFIIA/NC2assembly. Specifically, the presence of the BREs within theAdML promoter enhances TFIIA assembly, and the converse is observedwith NC2 (Fig. 1C, right). It was unexpected, however, thatTFIIA assembly should correlate with the repressive functionof the BREs while NC2 associates with the more active promoterderivatives.

Our results so far provide a correlation between TFIIA and NC2assembly and the presence of the BREs. Furthermore, TFIIA assemblyand that of NC2 are associated with low and high promoter activities,respectively. To assess the functional relevance of the recruitmentevents in Fig. 1C, we next determined the contribution of TFIIAand NC2 to BRE-dependent transcription at the AdML promoter.293T cells were transfected with siRNA targeting NC2 ${alpha}$ along withthe AdML derivative promoters, a plasmid driving expressionof GAL4-RII, and a β-galactosidase internal control. Forty-eighthours later cells were harvested, samples were taken for immunoblotting,and the remainder was used to measure luciferase activity (Fig.2A). The immunoblot confirmed that NC2 ${alpha}$ was reduced in cellsthat were transfected with the NC2 ${alpha}$ siRNA. The graph in Fig.2A shows luciferase activity of the AdML promoter derivativesin the presence of either control siRNA or NC2 ${alpha}$ siRNA. At thewild-type AdML promoter NC2 ${alpha}$ knockdown did not have a significanteffect on promoter activity. However, knockdown of NC2 ${alpha}$ causedsignificant reductions in the activities of the AdML promoterderivatives in which the BREs were individually or both mutated(2.2-, 2.7-, and 3-fold). The mutant TATA AdML derivative alsoshowed a reduction in activity in the presence of NC2 ${alpha}$ siRNA(1.9-fold). Taken together the data show that NC2 ${alpha}$ does not playa crucial role in transcription of the wild-type AdML promoterbut has a positive role when either or both of the BREs aremutated. We next assessed the effect of an siRNA directed againstNC2β in the same way (Fig. 2B). As we observed with NC2 ${alpha}$ ,knockdown of NC2β by siRNA had a negligible effect on thewild-type AdML promoter but significantly reduced the activitiesof the AdML derivative promoters that contain the mutant BREs(1.5-, 1.6-, and 1.9-fold). Western blotting confirmed thatthe expression of NC2β was reduced by the siRNA. A secondsiRNA targeting NC2β, NC2β (2) siRNA, produced comparableresults (Fig. 2C). Taken together, the data show that ablationof either NC2 ${alpha}$ or NC2β does not significantly affect transcriptionat the wild-type AdML promoter. However, when either or bothof the BREs are ablated, NC2 ${alpha}$ and NC2β have a positive effecton transcription. These data are consistent with our ChIP analysis(Fig. 1C); NC2 ${alpha}$ or NC2β ablation elicits the greatest effecton the same AdML derivatives that also show greater NC2 recruitmentin vivo.

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FIG. 2. NC2 acts as a positive transcription factor when the BREs in the AdML promoter are rendered nonfunctional. (A) 293T cells were transfected as for Fig. 1B except that either control siRNA or siRNA targeting NC2 ${alpha}$ was included. Forty-eight hours later cells were harvested and used to monitor luciferase activity and also for Western blotting with anti-NC2 ${alpha}$ antibodies or, as a control, anti-β-tubulin antibodies. Note that the immunoblot analysis concerns the whole cell population and therefore underestimates the extent of NC2 ${alpha}$ knockdown. The graph shows the relative luciferase activity of the AdML promoter derivatives in the presence of control (Ctrl) or NC2 ${alpha}$ siRNA. The numbers above the bars indicate the changes in activity that are caused by the NC2 ${alpha}$ siRNA. wt, wild type. (B) Same as panel A, except that siRNA targeting NC2β was used. (C) Same as panel A, except that a different siRNA targeting NC2β, NC2β (2) siRNA was used and only the wild-type AdML and AdMLmBRE^ud reporters were tested. Each bar represents three independent experiments with standard deviation.

We next used siRNA to target the ${alpha}$ and β subunits of TFIIAand monitored the activity of the AdML promoter derivativesas described above (Fig. 3A). Western blotting confirmed thatTFIIA ${alpha}$ and particularly TFIIAβ expression was knocked downby the siRNA. At the wild-type AdML promoter, knockdown of TFIIA ${alpha}$ and TFIIAβ (TFIIA ${alpha}$ /β) caused a 2.1-fold increase inpromoter activity. At the AdML derivatives harboring individualor combined mutant BREs, however, the effect of TFIIA ${alpha}$ /βsiRNA was reduced (1.4- to 1.5-fold). The TFIIA ${alpha}$ /β siRNAalso had little effect on the AdML promoter derivative witha defective TATA element. A second siRNA targeting TFIIA ${alpha}$ /β,TFIIA ${alpha}$ /β (2) siRNA, produced comparable results (Fig. 3B).siRNA targeting TFIIA ${gamma}$ also elicited a positive effect on transcriptionat the wild-type AdML promoter (2-fold) but had a reduced effecton transcription at the AdML derivatives with individual (1.3-and 1.7-fold) or combined (1.1-fold) defective BREs (Fig. 3C).Also, as observed with the TFIIA ${alpha}$ /β siRNA, the TFIIA ${gamma}$ siRNAdid not significantly affect transcription at the AdML promoterderivative with a defective TATA element. Again, these resultsare consistent with the ChIP data of Fig. 1C; TFIIA recruitmentis greatest at the wild-type AdML promoter, at which TFIIA ablationby siRNA also has the greatest effect. Moreover, consistentwith the observation that the extent of TFIIA recruitment correlateswith the least-active TATA-containing promoter, TFIIA knockdownhas a positive effect on transcription at this same promoter.These results suggest that TFIIA has a dampening effect on transcriptionat the AdML promoter that is dependent upon the presence ofthe BREs.

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FIG. 3. (A) BRE-dependent function for TFIIA at the AdML promoter. 293T cells were transfected as for Fig. 1B except that either control siRNA or siRNA targeting TFIIA ${alpha}$ /β was included. Forty-eight hours later cells were harvested and used to monitor luciferase activity and also for Western blotting with anti-TFIIA ${alpha}$ /β antibodies or, as a control, anti-β-tubulin antibodies. Note that the immunoblot analysis concerns the whole cell population and therefore underestimates the extent of TFIIA ${alpha}$ /β knockdown. The graph shows the relative luciferase activity of the AdML promoter derivatives in the presence of control (Ctrl) or TFIIA ${alpha}$ /β siRNA. The numbers above the bars indicate the changes in activity that are caused by the TFIIA ${alpha}$ /β siRNA. The difference in changes between the wild-type (wt) AdML reporter and AdMLmBRE^ud was subjected to Student's t test and was found to be significant (P < 0.05). (B) Same as panel A, except that a different siRNA targeting TFIIA ${alpha}$ /β, TFIIA ${alpha}$ /β (2) siRNA, was used and only the wild-type AdML and AdMLmBRE^ud reporters were tested. (C) Same as panel A, except that siRNA targeting TFIIA ${gamma}$ was used. Each bar represents three independent experiments with standard deviation.

Our studies so far have used transiently transfected AdML promoterderivatives. We were interested in determining if the effectsthat we observed would also occur at AdML promoter derivativesthat were stably integrated within the genome. For this analysiswe used the Flp-In system, which employs a 293 cell line derivativecontaining a single FRT within the genome. Plasmids containingthe wild-type AdML promoter or mutant BRE^ud derivative linkedto luciferase and flanked by FRT sequences were generated andused to create 293 cell line derivatives that contain a singlecopy of the AdML promoter (or BRE^ud mutant derivative) integratedat a defined location. Three independent cell lines each forthe wild-type AdML promoter or BRE^ud mutant were derived, andthe activities of the promoters were compared by luciferaseassay (Fig. 4A). Consistent with our transient transfectiondata, all three cell line derivatives containing the AdMLmBRE^udpromoter exhibited significantly greater luciferase activitythan the cell lines generated with the wild-type AdML promoter.

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FIG. 4. Analysis of TFIIA and NC2 function at stably integrated AdML derivatives. (A) Three independent cell lines were generated for both the wild-type (wt) AdML promoter and mutant BRE^ud derivative using the Flp-In system. Luciferase activity for each of the cell lines was assessed and is presented graphically as the mean average of three independent experiments with standard deviation. (B) The wild-type AdML and AdMLmBRE^ud stable cell lines were used for ChIP analysis to assess the promoter occupancy of TFIIA ${alpha}$ /β and NC2β (left) and TFIIB and pol II (right). ChIP was performed as for Fig. 1C except that detection was by real-time PCR, and the results are presented graphically. Immunoglobulin G (IgG) is used as a negative control antibody in the reaction. The results are presented as enrichment of precipitation of specific promoter DNA over nonpromoter DNA. Each bar represents at least three independent experiments with standard deviation. (C) The wild-type AdML and AdMLmBRE^ud stable cell lines were transfected with either TFIIA ${alpha}$ /β, NC2β, or a control siRNA. Luciferase activity was assessed and is presented graphically as the mean of three independent experiments with standard deviation. The difference in changes between the wild-type AdML and AdMLmBRE^ud was subjected to Student's t test and found to be significant (P < 0.05). At the right is an immunoblot showing successful depletion of TFIIA ${alpha}$ and NC2β expression by the siRNA.

We next performed ChIP by quantitative PCR with the stable AdMLand AdMLmBRE^ud cell lines to assess the occupancy of the stablyintegrated promoters by TFIIA ${alpha}$ /β and NC2β (Fig. 4B).The results are in agreement with our ChIP analysis of the episomalwild-type AdML promoter and AdMLmBRE^ud derivative (Fig. 1C).Specifically, TFIIA preferentially associates with the integratedwild-type AdML promoter and NC2 preferentially associates withthe integrated AdMLmBRE^ud derivative. Moreover, as observedwith ChIP of the episomal AdML derivatives, TFIIB and pol IIshowed significantly higher levels of occupancy at the moreactive AdMLmBRE^ud promoter than at the wild-type AdML promoter.We further analyzed the stable cell lines using siRNA to targeteither TFIIA ${alpha}$ /β or NC2β expression (Fig. 4C). As before,the wild-type AdML promoter was more sensitive to TFIIA depletion,showing enhanced transcriptional function in response to theTFIIA ${alpha}$ /β siRNA. Conversely, the AdMLmBRE^ud derivative wasmore sensitive to NC2 depletion, showing reduced transcriptionfunction in response to NC2β siRNA. Thus, the suppressiveeffect of TFIIA and positive effect of NC2 on BRE-dependenttranscription are equivalent at both episomal and stably integratedAdML promoter derivatives.

We next sought to confirm and extend our data using a seriesof natural promoters that contain different combinations ofand levels of conformity to the consensus sequences of the BREs(Fig. 5A). Plasmids containing these promoters driving luciferaseexpression were transfected into 293T cells along with eitherNC2 ${alpha}$ or NC2β siRNA. Forty-eight hours later the cells wereharvested. NC2 ${alpha}$ and NC2β expression was monitored by Westernblotting, and luciferase activity was measured and is presentedgraphically (Fig. 5B). To aid interpretation, the effects ofeach of the NC2 siRNAs are presented relative to that of thecontrol siRNA at each promoter, which has been set at an arbitraryvalue of 10. siRNA-mediated knockdown of NC2 caused a decreasein activity of the BCL2, p21, podocalyxin, and Egr1 promoters.At the BCL2 and Egr1 promoters, however, we note that the NC2 ${alpha}$ siRNA was consistently less effective than the NC2β siRNA.The IGFII promoter was refractory to both the NC2 ${alpha}$ and NC2βsiRNAs; significantly, this promoter contains the sequence mostsimilar to the consensus BRE sequences of all the promoterstested. Indeed, we have reported previously that the IGFII promoterBRE^u is contacted by TFIIB (20). In addition, band shift analysisconfirmed that the BRE sequences of the IGFII promoter providedgreater functionality in TFIIB recognition than the correspondingsequences of the BCL2 promoter (see Fig. S2 in the supplementalmaterial).

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FIG. 5. Natural promoters exhibit BRE-dependent TFIIA and NC2 function. (A) The core promoter regions of five natural promoters are shown, with the TATA element underlined. The BRE^u and BRE^d regions are shown, and bases that conform to the consensus sequences are shaded. (B) The five promoters indicated, linked to luciferase, were transfected into 293T cells along with control (Ctrl) siRNA, NC2 ${alpha}$ siRNA, or NC2β siRNA. Forty-eight hours later the cells were harvested and used to monitor luciferase activity and also for Western blotting with anti-NC2 ${alpha}$ antibodies, anti NC2β antibodies, or, as a control, anti-β-tubulin antibodies. Luciferase activity is shown graphically, and each bar represents three independent experiments with standard deviation. To aid comparison of the effects of the siRNAs on the different promoters, the activity of each promoter in the presence of control siRNA was set to 10. (C) The five promoters indicated, linked to luciferase, were transfected into 293T cells as for panel B, but along with control siRNA, TFIIA ${alpha}$ /β siRNA, or TFIIA ${gamma}$ siRNA. Forty-eight hours later the cells were harvested and used to monitor luciferase activity and also for Western blotting with anti-TFIIA ${alpha}$ /β antibodies, anti TFIIA ${gamma}$ antibodies, or, as a control, anti-β-tubulin antibodies. Luciferase activity is shown graphically, and each bar represents three independent experiments with standard deviation. To aid comparison of the effects of the siRNAs on the different promoters, the activity of each promoter in the presence of control siRNA was set to 1. (D) Summary of the effects of NC2 and TFIIA siRNAs on the activities of the natural promoters. For each promoter the BRE^u and BRE^d match to the consensus (out of 7 bases) is shown. The transcriptional effects of the siRNAs are denoted by upward (stimulation)- and downward (repression)-pointing arrowheads. The number of arrowheads in each case is proportional to the change in luciferase activity. The BCL2 and IGFII promoters are discussed further in the text.

We next tested the effects of TFIIA ablation on the naturalpromoters (Fig. 5C). All of the promoters elicited a positiveeffect in response to TFIIA ${alpha}$ /β and TFIIA ${gamma}$ siRNA. However,the greatest effect was observed with the IGFII promoter whilethe smallest effect was observed with the BCL2 promoter. Althoughthe effects of NC2 and TFIIA siRNA at the natural promotersare more complex than those observed with the AdML mutant derivatives(in which targeted mutations of the BREs were performed basedon previous functional data), it is clear that the IGFII promoter,which contains the sequence most similar to the consensus BREsequences, is least dependent on NC2 for activity and is suppressedto the greatest degree by TFIIA (see summary diagram in Fig.5D). All the other promoters show some degree of dependenceon NC2 for full activity, while TFIIA is least inhibitory atthe BCL2 promoter.

We next analyzed the effect of TFIIA and NC2 siRNA on the expressionof the endogenous genes that correspond to the reporter plasmidsused in Fig. 5. 293T cells were transfected with control, NC2 ${alpha}$ ,NC2β, TFIIA ${alpha}$ /β, or TFIIA ${gamma}$ siRNA, and 48 h later thecells were harvested and used to prepare whole-cell extractsor total RNA. RT-PCR was used to analyze the expression levelsof IGFII, p21, Egr1, BCL2, and, as a control, pol I-transcribed18S rRNA (Fig. 6A). Western blots of the associated whole-cellextracts with antibodies against several general transcriptionfactors are shown. The endogenous podocalyxin gene is not expressedin 293 cells. Both TFIIA ${alpha}$ /β and TFIIA ${gamma}$ siRNA caused an increasein Egr1 and IGFII mRNA. Significantly, the Egr1 and IGFII promoterswere found to be the most responsive to TFIIA siRNA when thereporters were tested (Fig. 5C). The NC2β siRNA causeda reduction in the abundance of BCL2, Egr1, and p21 mRNA buthad no effect on the level of IGFII mRNA. NC2 ${alpha}$ siRNA also hada negative effect on BCL2 and p21 promoter activity, but notEgr1 or IGFII promoter activity. Taken together, the resultsof the analysis of the endogenous genes confirm that IGFII isactively suppressed by TFIIA but is not dependent upon NC2 foractivity. Conversely, the BCL2 promoter is dependent on NC2for full activity but is not suppressed by TFIIA.

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FIG. 6. Endogenous promoters exhibit a BRE-dependent transcriptional requirement for and recruitment of TFIIA and NC2. (A) 293T cells were transfected with either a control (Ctrl) siRNA or the siRNAs indicated at the top. Forty-eight hours later the cells were harvested and both total RNA and whole-cell protein lysates were prepared. BCL2, Egr1, p21, and IGFII mRNAs were detected by RT-PCR, and assurance that the reactions were performed in the linear range was determined by a dilution profile of the RNA sample obtained from cells treated with control siRNA. Below, Western blots of the whole-cell lysates with the antibodies indicated at the right are shown. (B) 293T cells were subjected to ChIP with the antibodies indicated at the top. The core promoter regions of the BCL2 and IGFII genes were amplified by PCR, and amplification of the 18S coding region served as a negative control (bottom). Assurance that the reactions were performed in the linear range was determined by a dilution profile of the input chromatin prior to PCR. IgG, immunoglobulin G.

We next performed ChIP analysis on the endogenous BCL2 and IGFIIcore promoters to assess the relative assembly of the generaltranscription factors, including TFIIA and NC2 (Fig. 6B). TheBCL2 core promoter showed robust assembly of NC2 ${alpha}$ and NC2β,but not TFIIA, consistent with its poor BRE sequences and alsothe effects of the NC2 and TFIIA siRNAs. In addition, the IGFIIpromoter showed robust TFIIA assembly but low levels of NC2 ${alpha}$ and NC2β assembly, consistent with the strong BRE consensussequences and the effects of the NC2 and TFIIA siRNAs.

Our data so far suggest that the presence of BREs within a promoterresults in the preferred assembly of TFIIA instead of NC2 atthe promoter and elicits a dampening effect on transcription.In the absence of BREs (or with BREs having low similarity tothe high-affinity sequences), NC2 preferentially assembles overTFIIA and elicits a positive effect on transcription. We weretherefore interested to determine directly the effect of theBREs on the affinity of TFIIA and NC2 for promoter-bound TBPin the absence of other factors. The wild-type AdML promoterand mutant BRE derivative AdMLmBRE^ud were radiolabeled and incubatedwith recombinant human TBP along with TFIIA, and the complexeswere resolved by native electrophoresis (Fig. 7A). TFIIA-TBP-promotercomplexes formed more avidly with the wild-type AdML promoterthan with the AdML promoter derivative in which the BREs hadbeen rendered nonfunctional (AdMLmBRE^ud). We next performeda band shift reaction with purified recombinant NC2 and TBP(Fig. 7B). The NC2-TBP-AdML complexes resolved as two forms;thus, the presence of NC2 within the complexes was verifiedby supershifting the complex with anti-NC2 ${alpha}$ antibodies. Significantly,NC2-TBP-promoter complexes did not show any dependence on theBRE for their formation. Assembly of either TFIIA or NC2 atthe AdML promoter required the presence of TBP (Fig. 7C). Inaddition, consistent with previous studies, TFIIB shows reducedassembly at the AdMLmBRE^ud promoter derivative compared withthat at the wild-type AdML promoter (Fig. 7D). Thus, the BREsdrive formation of either TFIIA-TBP-promoter complexes or TFIIB-TBP-promotercomplexes, but not NC2-TBP-promoter complexes.

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FIG. 7. TFIIA assembly at the promoter is BRE dependent. (A) The wild-type AdML promoter and mutant derivative AdMLmBRE^ud were radiolabeled, and assembly of TBP and TFIIA was monitored by band shift analysis. Where indicated, 50 ng of human TBP (hTBP) was included in the reaction mixture. Either 50 ng or 250 ng of TFIIA was added. Free probe and TBP-TFIIA-promoter complexes are indicated at the right. (B) Same as panel A except that 10 ng and 50 ng of purified recombinant NC2 were included instead of TFIIA. Also, where indicated 500 ng of anti-NC2 ${alpha}$ antibody was included, and the supershifts are indicated. (C) TFIIA (50 ng) or NC2 (50 ng) was incubated with the radiolabeled AdML promoter either alone or along with hTBP (50 ng). Complexes were resolved as for panel A. TFIIA-TBP-AdML and NC2-TBP-AdML complexes and free probe are indicated. (D) Same as panel A except that 50 ng or 250 ng of recombinant human TFIIB was used instead of TFIIA.

DISCUSSION

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DISCUSSION

The BREs can elicit either positive or negative effects on transcription.While the positive effect can be explained by the increasedaffinity of TFIIB for the promoter, the molecular basis forthe negative effect is unclear. In this study we have shownthat the presence of BREs in the AdML promoter determines therelative assembly of TFIIA and NC2. Surprisingly, however, TFIIArecruitment correlated with reduced promoter activity whileNC2 recruitment correlated with high promoter activity. Treatmentof cells with siRNA to reduce endogenous TFIIA and NC2 levelsrevealed that TFIIA suppresses transcription at the AdML promoterin a BRE-specific manner and that NC2 provides a positive transcriptionalfunction in the absence of BREs. In vitro studies confirmedthat the BREs enhance the formation of a TFIIA-TBP-promotercomplex.

A positive role for NC2 in transcription has been observed before(5, 7, 11, 22, 63). How NC2 can act as a positive factor isnot yet known, but it is possible that it acts by stabilizingor "priming" TFIID at the promoter, before release and replacementby factors (e.g., TFIIB) that can drive PIC assembly forward.Such a mechanism has been described before for the positiveactivities of Mot1 (14, 23, 24, 32). Indeed, active transcriptioncomplexes that contain Mot1 also contain TFIIB and pol II, butnot TFIIA (24). We also note that RNA interference (RNAi)-mediatedknockdown of NC2 ${alpha}$ frequently elicited a less pronounced effecton transcription than RNAi-mediated knockdown of NC2β,particularly with the natural promoters. This is consistentwith previous reports of potentially independent activitiesfor NC2 ${alpha}$ or NC2β (13, 33, 65), although our ChIP analysissuggests that both components generally assemble at the promotertogether.

Recent genome-wide ChIP-on-chip experiments have found thatNC2 associates with 25% of promoters in the genome and generallycorrelates with high expression, reaffirming a positive functionfor NC2 in transcription (1). Furthermore, this same study showedthat association of NC2 with promoters shows an inverse correlationwith the BRE core promoter element. Our results are entirelyconsistent with these findings and further suggest that thisinverse correlation between the BRE and NC2 promoter occupancyarises from the binding of TFIIA to BRE-like sequences. Thus,our data raise the prospect of TFIIA recognition elements thatshare at least some similarity to the BREs. Indeed, previouscross-linking studies have shown that TFIIA contacts the corepromoter within both the BRE^u and BRE^d elements, and it is possiblethat TFIIA might share overlapping DNA sequence-specific recognitionwith TFIIB (12, 38). The crystal structures of yeast TFIIA complexedwith TBP at the AdML and CYC promoters revealed base contactupstream of the TATA element, including contact with a G 3 nucleotidesupstream of the TATA element (3, 21, 58). We note that a G 3nucleotides upstream of the TATA element forms a critical partof BRE^u and was replaced in our mutant AdML derivatives. Inaddition, several other residues in the above structure werein close proximity to the upstream DNA with potential to formdirect contact. The DNA fragment used in these studies did notextend sufficiently 3' of the TATA box to encompass the BRE^dregion. Our band shift data, revealing BRE-dependent enhancementof TFIIA-TBP-promoter complex formation, are therefore consistentwith previous studies and also with our ChIP data.

Our experiments revealed the surprising finding that reductionof cellular TFIIA enhances transcription at the wild-type AdMLpromoter. Moreover, BRE-directed recruitment of TFIIA at theAdML promoter correlated with reduced promoter activity. A previousstudy found that a highly pure TFIIA fraction repressed transcriptionin vitro at the AdML promoter, but not at TATA-less promoters(2). Indeed, we also observed that transcription at the AdMLderivative that lacks a TATA element was not affected by theTFIIA siRNAs. However, the TATA-less podocalyxin promoter wasrepressed by TFIIA, suggesting that the repressive functionof TFIIA is dependent on multiple factors. Indeed, Stargellet al. (56) have reported core promoter-dependent effects ofTFIIA. It will therefore be important to test the other corepromoter sequence elements (including the initiator, DPE, downstreamcore element, and the motif 10 element) with regard to TFIIAand NC2 function. Recent work by Hsu et al. (31) employed RNAiin drosophila cells to assess the role of several general transcriptionfactors at DPE-containing versus TATA-containing core promoters.In addition to a positive function for NC2, they uncovered DPE-and TATA-dependent roles for both TBP and NC2. In addition,they found that depletion of TFIIA decreases the activity ofa hybrid core promoter containing the AdML TATA region linkedto the Ant P2 or E74B initiator. It is therefore likely thatthe core promoter context of the BREs, particularly in relationto the initiator, plays a role in determining the net contributionof TFIIA to transcriptional activity. Indeed, we have previouslyreported that the BREs can exert a positive or negative effecton transcription that is dependent upon the promoter context(16). Moreover, promoter-specific activities for TFIIA havealso been observed in other studies (10, 36, 40, 42, 46, 56,61). It should also be noted that, while yeast TFIIB can alsocontact BRE^d sequences (16), contact with BRE^u does not appearto be conserved (39), suggesting potential differences in theactivities of TFIIA with respect to the BREs.

It is generally assumed that TFIIA and NC2 act in a reciprocalmanner, with mutually exclusive recruitment to the promoterand effects on transcription. However, as stated previously,this has been difficult to reconcile with the numerous reportsthat NC2 can have a positive effect on transcription. Our findingsprovide an explanation that maintains the reciprocity betweenTFIIA and NC2, in that TFIIA and NC2 show mutually exclusiveeffects on transcription in a BRE-dependent manner. We suggestthat, at promoters with BRE-like sequences, TFIIA assemblesin preference over NC2. We did not find BRE-dependent effectsin the formation of NC2-TBP-AdML promoter complexes in vitro(Fig. 7), suggesting that enhanced assembly of NC2 at non-BREpromoters in vivo is due to reduced competition from TFIIA-promoterrecognition.

However, it is important to note that the BRE^u and BRE^d consensussequences are derived from binding site selection studies andtherefore represent the highest-affinity sequences. Physiologicallyrelevant sequences will likely fall across a broad spectrum,and it is likely that many BREs might be functional for TFIIBbinding but not act as affinity sites for TFIIA. In such casesTFIIA and NC2 might elicit more-competitive effects and thereforerespond to ablation of both TFIIA and NC2 expression. Indeed,this is likely the case at some of the natural promoters usedin this study.

It is well established that TFIIA can stabilize TFIID-promotercontacts, leading to productive PIC formation and transcription.Our data suggest that this pathway of PIC formation dominatesat BRE-containing promoters. An alternative pathway employsNC2 to stabilize TFIID-promoter complexes. Although both pathwaysare productive, the NC2 pathway appears to be more efficientin producing transcriptionally active complexes. Thus, eliminationof TFIIA by siRNA or diminished TFIIA-promoter recognition (byBRE mutation) leads to the more efficient NC2 pathway. Our previouswork on mammalian cells and the work of others on yeast suggestedthat TFIIB most likely assembles at the promoter as a complexwith pol II (18, 51). It is possible that ablation of TFIIAby RNAi might release the BREs for recognition by the TFIIB-polII machinery, thereby enhancing transcription. TFIIA and NC2,by mutually exclusive assembly with TFIID at the promoter, couldtherefore provide a commitment step that can either precludeBRE function or enhance it. Thus, TFIIA might "shield" the BREsfrom TFIIB recognition and weaken the promoter. Conversely,if NC2 "primes" the promoter-TFIID complex but is subsequentlyreleased, this would allow recognition of the BREs by TFIIB,thereby eliciting a positive effect on transcription. Furtherstudies will shed light on the TFIIB-TFIIA-NC2 dynamic in livingcells.

ACKNOWLEDGMENTS

We thank Bob White and members of the Oelgeschläger andRoberts labs for comments on the manuscript. We also thank DanielHaber for the podocalyxin promoter reporter, Andrew Ward forthe IGFII promoter reporter, Neil Perkins for the BCL2/p21 promoterreporters, Andy Sharrocks for the Egr1 promoter reporter, andStephen Taylor for help with the Flp-In system.

This work was funded by the Wellcome Trust (061207/Z/00/Z/CH/TG/dr).S.G.E.R. is a Wellcome Trust Senior Research Fellow.

The authors have no competing financial interests.

FOOTNOTES

* Corresponding author. Mailing address: Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. Phone: 0161 275 5758. Fax: 0161 275 5082. E-mail: stefan.roberts{at}manchester.ac.uk

Published ahead of print on 29 December 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

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