©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Transcriptional Activation in Vitro by NF-B/Rel Proteins (*)

(Received for publication, July 25, 1994; and in revised form, November 3, 1994)

Rongtuan Lin (1)(§) Dirk Gewert (2) John Hiscott (1)(¶)

From the  (1)Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, Jewish General Hospital and the Departments of Microbiology and Immunology and Medicine, McGill University, Montreal, Quebec H3T 1E2, Canada and the (2)Department of Cell Biology, Wellcome Foundation Limited, Langley Court, Beckenham, Kent BR3 3BS, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Distinct NF-kappaB subunit combinations contribute to the specificity of NF-kappaB-mediated transcriptional activation and to the induction of multiple cytokine genes including interferon-beta (IFN-beta). To evaluate the regulatory influence of different homo- and heterodimers, NF-kappaB subunits were analyzed for transcriptional activity in vitro using test templates containing two types of NF-kappaB recognition elements (the human immunodeficiency virus type 1 enhancer and the IFN-beta-positive regulatory domain II (PRDII)) as well as IFN-beta PRDIII-PRDI-PRDII linked to the -56 minimal promoter of rabbit beta-globin. Recombinant NF-kappaB subunits (p50, p65, c-Rel, p52, and IkappaBalpha) and interferon regulatory factor 1 were produced from either Escherichia coli or baculovirus expression systems. Transcriptional analysis in vitro demonstrated that 1) various dimeric complexes of NF-kappaB differentially stimulated transcription through the human immunodeficiency virus enhancer or PRDII up to 20-fold; 2) recombinant IkappaBalpha specifically inhibited NF-kappaB-dependent transcription in vitro; and 3) different NF-kappaB complexes and interferon regulatory factor 1 cooperated to stimulate transcription in vitro through the PRDIII-PRDI-PRDII virus-inducible regulatory domains of the IFN-beta promoter. These results demonstrate the role of NF-kappaB protein dimerization in differential transcriptional activation in vitro and emphasize the role of cooperativity between transcription factor families as an additional regulatory level to maintain transcriptional specificity.


INTRODUCTION

Transcriptional activation of eukaryotic gene expression in response to developmental or environmental signals requires the assembly of multiple DNA-binding proteins with specific DNA regulatory domains in a stereospecific nucleoprotein complex (reviewed in Refs. 1 and 2). Changes in the specific combination of transcription factors associated with a particular gene by modification, de novo synthesis, or protein-protein interaction may account for developmental or temporal regulation of transcription. Protein-protein interactions between transcription factors also confer a level of transcriptional specificity that would not be achieved by individual proteins(1, 2) .

Two broad groups of transcription factors, general transcription factors and upstream activators, are involved in the accurate transcription of mRNA by the enzyme RNA polymerase II. The general transcription factors including the entire TFIID (^1)complex, which consists of TBP and the TBP-associated factors, and RNA polymerase II are required for transcriptional regulation by upstream activators(1) . TBP and TFIIB are targets for the direct interaction with upstream activator proteins (3, 4, 5) . Mutations in basic residues of TFIIB that crucially affect the interaction with acidic activators fail to bind activator proteins such as VP16 and do not respond to activation, but still function in basal transcription. These observations suggest that interaction between an acidic activator and TFIIB is required for transcriptional activation (6) .

Modulation of cellular transcription can also be produced as a consequence of virus infection. The type 1 interferon genes (IFN-alpha and IFN-beta) have served as a paradigm to examine the transcriptional mechanisms controlling virus-inducible gene expression(2, 7, 8) . The IFN-beta promoter contains multiple regulatory domains that are targets for transcription factors involved in inducible expression of the IFN-beta gene(2, 7, 9, 10) . The positive regulatory domains I and III (PRDI and PRDIII) interact with interferon regulatory factors 1 and 2 (IRF-1 and IRF-2)(8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) , while PRDII interacts with subunits of the NF-kappaB/Rel family of transcription factors(23, 24, 25, 26, 27, 28) . PRDIV interacts with ATF-2 and c-jun; in vivo synergism between PRDIV and PRDII is provided by the binding of the high mobility group I/Y proteins to the minor groove of DNA within AT-rich sites in PRDIV and PRDII(29, 30) .

NF-kappaB/Rel proteins participate in the activation of numerous genes involved in inflammatory reactions and immune regulatory functions (reviewed in (31, 32, 33) ). The DNA-binding NF-kappaB family members share a Rel homology domain that is responsible for DNA binding, nuclear localization, and protein dimerization. DNA-binding members of NF-kappaB/Rel include p50 (NFKB1)(34, 35, 36) , p65 (RelA)(37, 38) , c-Rel (39, 40) , p52 (NFKB2, lyt-10)(41, 42, 43) , RelB (I-Rel)(44, 45) , and dorsal(46) . p50 and p52 are synthesized as precursors of p105 and p100, respectively, that are proteolytically processed to generate active DNA-binding p50 and p52(34, 35, 36, 41, 42, 43) . Recently, the acidic activation domains of both c-Rel and v-Rel were shown to interact with TBP and TFIIB in vitro and in vivo(5) ; p65 also interacted with TBP and TFIIB in vitro, demonstrating that NF-kappaB/Rel activators stimulate transcription via direct interaction with basal factors.

The intracellular localization and post-translational activity of NF-kappaB/Rel proteins are regulated by the ankyrin repeat-containing IkappaB proteins (IkappaBalpha, IkappaB, bcl-3, p105, and p100) (reviewed in (47, 48, 49, 50) ). The in vitro DNA binding activity of NF-kappaB complexes can be inhibited or dissociated by IkappaBalpha addition(51, 52) ; in some cases, IkappaBalpha addition can enhance DNA binding activity, depending on the NF-kappaB subunits(53) .

Different NF-kappaB dimers bind to variant NF-kappaB sites (consensus sequence, 5`-GGGANNYYCC-3`) (54) present in the promoter regions of many genes (reviewed in (31) and (33) ). Virtually every homo- and heterodimer combination has been identified: p50 and p52 homodimers were detected in unstimulated T cells and macrophages(55, 56) ; p50/c-Rel and p50/p65 heterodimers were constitutively present in B cells(33) ; p50/p65, p50/c-Rel, p65/p65, and p52/c-Rel complexes were present in stimulated T cells(57, 58) ; and p65/c-Rel heterodimers were purified from HeLa cells(59) . These observations indicate that various homo- or heterodimeric NF-kappaB complexes exist in different cell types and contribute to differential regulation of NF-kappaB-dependent gene expression(33) .

Qualitative changes in NF-kappaB heterodimer formation also occur as a function of time after virus induction or cellular differentiation(17, 57, 58, 60) . Previously, we demonstrated a temporal shift in the composition of NF-kappaB subunits in association with PRDII that correlated with a virus-induced degradation and de novo resynthesis of IkappaBalpha(17) . In this study, recombinant NF-kappaB subunits and IRF-1 proteins were produced from either Escherichia coli or baculovirus expression systems and were used to examine NF-kappaB-dependent and IFN-beta promoter-mediated transcription in vitro. These experiments demonstrated that 1) various dimeric complexes of NF-kappaB differentially stimulated transcription through two distinct NF-kappaB elements; 2) recombinant IkappaBalpha specifically inhibited NF-kappaB-dependent activation; and 3) different NF-kappaB heterodimers and IRF-1 cooperated to activate transcription in vitro through the PRDIII-PRDI-PRDII virus-inducible regulatory domains of interferon-beta.


MATERIALS AND METHODS

Plasmid Construction

Plasmids for the expression of NF-kappaB-glutathione S-transferase fusion proteins were produced by subcloning different NF-kappaB cDNAs into pGEX2T or pGEX3X vectors (Pharmacia Biotech Inc.). For p50, a 1480-bp StuI-XbaI fragment from KBF-1 (encoding amino acids 10-502 of p105) (36) was filled in with Klenow enzyme and subcloned into the EcoRI site (filled in with Klenow enzyme) of pGEX2T. For p52, a 1503-bp HindIII-EcoRI fragment from p52-containing pBluescript SK(43) was filled in with Klenow enzyme and subcloned into the SmaI site of pGEX3X. For p50/p65 chimera, the cDNA encoding the transactivation domain of p65 was obtained by polymerase chain reaction amplification with the p65 cDNA clone (cloned into pBluescript) (38) using a specific primer corresponding to positions 1270-1290 (5`-TAGCTCTAGACATGGTATCTGCTCTGGCCC-3`) and the KS primer; the XbaI-XhoI fragment of the polymerase chain reaction product was cloned into the XbaI-XhoI fragment of the pSVK3/p105 construct; and a 2286-bp StuI fragment from the resulting plasmid (pSVK3/p50-p65) was subcloned into the SmaI site of pGEX3X. The amplification of IkappaBalpha cDNA (49) and the plasmid for producing recombinant IkappaBalpha protein were described previously(61) .

Vectors for production of recombinant baculoviruses were generated by subcloning different cDNAs into the pAcH6N1 vector. For p65, a 2232-bp BamHI fragment (38) from pGEX3X/p65 was inserted into the BglII site of pAcH6N1. For p50/p65 chimera, a 2286-bp StuI fragment from pSVK3/p50-p65 was subcloned into the BglII site (filled in with Klenow enzyme) of pAcH6N1. For c-Rel, a 2169-bp BamHI-EcoRI fragment (the EcoRI site was filled in with Klenow enzyme) (39) from pGEX2T/c-Rel was subcloned into the BamHI-BglII fragment (the BglII site was filled in with Klenow enzyme) of pAcH6N1. For IRF-2, a 1.4-kilobase XbaI-XhoI fragment from CMV-BL/IRF-2 (18) was inserted into the XbaI-XhoI fragment of pSVK3, and a 1.3-kilobase Cfr10I-KpnI fragment (the Cfr10I site was filled in with Klenow enzyme) from the resulting plasmid was subcloned into the BglII-KpnI fragment (the BglII site was filled in with Klenow enzyme) of pAcH6N1. IRF-1 (22) was similarly subcloned into the pAcH6N1 vector.

In vitro transcription templates HS-RbetaG, PRDII times 2, and PRDI times 2 were a kind gift from N. MacDonald and C. Weissmann and were described previously(8) ; the IFN-beta reference template p901 was also described previously ( (12) and (23) ; refer also to Fig. 3). Test transcription templates containing the insert sequences of the HIV-1 enhancer, the HIV-1 enhancer mutant, and the PRDIII-PRDI-PRDII elements (P512) (see Fig. 3) were synthesized chemically and flanked by a 5`-ClaI-compatible overhang (CG) on the top strand and a 5`-HindIII-compatible overhang (TCGA) on the bottom strand. These sequences were inserted into the ClaI-HindIII fragment at position -56 of PRDII-RbetaG to replace the PRDII sequence. For construction of the reference template DeltaRbetaG, HS-RbetaG was digested with SalI, filled in with Klenow enzyme, and self-ligated to block one of two AccI sites. The resulting plasmid was digested with AccI, treated with exonuclease III, and ligated, yielding an internal reference plasmid with a 22-bp deletion (residues 704-725). An upstream SV40 enhancer sequence was removed by digestion with HindIII and EcoRI, fill in with Klenow enzyme, and self-ligated. All constructions were verified by sequencing.


Figure 3: DNA templates for in vitro transcription analysis. The construction of HIV-1 enhancer, HIV-1 enhancer mutant, and PRDIII-PRDI-PRDII templates and the internal reference template DeltaRbetaG is described under ``Materials and Methods.'' The insert sequences were cloned upstream of the minimal rabbit beta-globin promoter (position -56) and structural sequences of the rabbit beta-globin gene(8) . A 5`-end-labeled 452-bp BamHI-PstI fragment derived from the RbetaG cDNA was used as probe for S1 nuclease mapping analysis; the 408-bp readthrough transcript, the 352-bp correctly initiated transcript from the test gene, and the 180-bp transcript from the reference gene are illustrated schematically. Hatchedboxes, TATA sequence; shaded boxes, RbetaG exon sequences; arrows, transcription start site. To eliminate readthrough from the reference template, DeltaRbetaG was linearized with BglII. The IFN-beta cDNA deleted to position -56 (p901) was used as an internal reference gene in some experiments(11, 12) .



Expression and Purification of Recombinant Proteins

Glutathione S-transferase fusion proteins were isolated from E. coli strain DH5alpha (Life Technologies, Inc.) following 3 h of induction with 1 mM isopropyl-1-thio-beta-D-galactopyranoside (Pharmacia Biotech Inc.) at 37 °C. Bacterial extracts in phosphate-buffered saline containing 1% Triton X-100 were incubated with glutathione-Sepharose beads (Pharmacia Biotech Inc.) for 20 min at room temperature. After washing three times with phosphate-buffered saline, fusion proteins were enzymatically cleaved by thrombin (Sigma) in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl(2), and 2.5 mM CaCl(2) for 2-4 h at room temperature. The final cleaved protein products were eluted from the beads in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl(2) and stored with the addition of 1 mM dithiothreitol and protease inhibitors. Recombinant p52 protein was eluted with 15 mM glutathione as a glutathione S-transferase fusion protein without cleavage.

For the production of polyhistidine-tagged protein, recombinant baculoviruses were prepared by using a BaculoGold transfection kit as recommended by the manufacturer (Pharmingen). Sf9 cells were infected with recombinant baculoviruses and cultured for 4 days at 28 °C. Infected cells were harvested, washed with phosphate-buffered saline, and lysed in binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl (pH 7.9)). Recombinant proteins were purified by using rapid affinity purification with His-Bind metal chelation resin under nondenaturing conditions as recommended by the manufacturer (Novagen, pET system manual) (76).

Electrophoretic Mobility Shift Assay

HIV oligonucleotide (5`-AGGGACTTTCCGCTGGGGACTTTCC-3`), the PRDII (P2) probe (5`-GGGAAATTCCGGGAAATTCC-3`), and the PRDI (P1) probe (5`-GGGAGAAGTGAAAGTG-3`) were labeled with T(4) polynucleotide kinase and [-P]ATP. The binding mixture (20 µl) contained 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, 0.5% Nonidet P-40, 10 mg/ml bovine serum albumin, and 62.5 µg/ml poly(dI-dC). After 20 min of incubation with labeled probe at room temperature, the mixture was loaded on a 5% polyacrylamide gel prepared in 0.5 times Tris borate/EDTA and electrophoresed at 200 V for 2-3 h.

In Vitro Transcription Assay

The in vitro transcription reactions were carried out with nuclear extracts from HeLa cells (HeLaScribe, Promega) essentially as described previously (11, 12, 23) . Transcription reactions (25 µl) contain 10 mM HEPES (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 8.5% glycerol, 10 units of RNasin (Pharmacia Biotech Inc.), 4 units of HeLa nuclear extract, the indicated amount of purified recombinant proteins, 250 ng of each test and reference template, 8 mM MgCl(2), and 0.4 mM NTP. To generate NF-kappaB heterodimers for the in vitro transcription assays, an equal molar ratio of individual NF-kappaB subunits was mixed in renaturation buffer, and heterodimers were allowed to form for 1 h at 37 °C. RNA from the transcription reaction was harvested after 1 h of incubation at 30 °C by a small-scale guanidinium isothiocyanate procedure(62) . The test RbetaG transcripts and the reference transcripts from DeltaRbetaG and p901 were quantitated by S1 nuclease mapping as described previously ((12) ; refer to Fig. 3).


RESULTS

Expression and Purification of NF-kappaB and IRF Proteins

To obtain relatively large amounts of protein for in vitro studies, the E. coli glutathione S-transferase gene fusion system and baculovirus expression system were used to express NF-kappaB and IRF proteins. The cDNAs encoding the NF-kappaB subunits p50, p52, and IkappaBalpha were expressed as glutathione S-transferase fusion proteins (Fig. 1A) and purified from bacterial lysates by affinity chromatography using glutathione-Sepharose 4B. Purified proteins were analyzed by SDS-polyacrylamide gel electrophoresis, and the purity was estimated from Coomassie-stained gels at 60, 80, and 60% for p50, p52, and IkappaBalpha, respectively (Fig. 1C, lanes 1-3).


Figure 1: Expression of recombinant NF-kappaB and IRF proteins. A, schematic representation of recombinant p50, p52, and IkappaBalpha proteins expressed as glutathione S-transferase (GST) fusion proteins in the E. coli glutathione S-transferase gene fusion system. p50 and IkappaBalpha were enzymatically cleaved with thrombin to release glutathione S-transferase. The p50/p65 fusion protein (see below) was also produced a a glutathione S-transferase-linked protein (data not shown). B, p65, c-Rel, p50/p65 fusion, IRF-1, and IRF-2 recombinant proteins expressed as polyhistidine-tagged proteins in the baculovirus system. All proteins contain 12-21 polyhistidine-linked amino acids at their N-terminal ends (illustrated as checkeredboxes). The boxes represent different protein domains: dark grayboxes, Rel homology domain; boxes with diagonal widewhite stripes, transactivation domain; boxes with wavy lines, ankyrin domain; ( ANK ); box with verticalstripes, glutathione S-transferase domain; boxes with diagonalnarrowblackstripes, IRF-binding domain; light gray boxes, IRF-1 transactivation domain; NLS, nuclear localization sequence. C, Coomassie-stained SDS-polyacrylamide gel showing the purified recombinant proteins (indicated above the lanes). The molecular masses of the size markers are indicated in lane9. aa, amino acids.



The p65, c-Rel, p50/p65 fusion, IRF-1, and IRF-2 proteins were expressed in baculovirus as polyhistidine-tagged proteins (Fig. 1B). These polyhistidine-containing recombinant proteins were isolated from insect cell lysates under native elution conditions by a rapid affinity purification using His-Bind metal chelation resin. The purified recombinant proteins were analyzed by SDS-polyacrylamide gel electrophoresis, and their apparent molecular masses were slightly higher than predicted: 85, 85, 75, 50, and 48 kDa for p50/p65, c-Rel, p65, IRF-1, and IRF-2, respectively. The purity was estimated at >80% for p50/p65, p65, and IRF-1; 60% for IRF-2; and only 10% for c-Rel (Fig. 1C, lanes 4-8).

The DNA binding activity of the recombinant NF-kappaB proteins was assessed in vitro by electrophoretic mobility shift assay using the HIV-1 enhancer and P2 probes. All recombinant NF-kappaB subunits were able to bind to either the HIV-1 enhancer or P2 probe (Fig. 2, lanes1, 5, 13, and 17; data for the P2 probe not shown). The binding of p65 generated one major and one minor DNA-protein complex (Fig. 2, lane5) due to a truncated N-terminal p65 (as shown in Fig. 2, p65 contained 25-30% degraded product). When p50 and p65 were preincubated to form heterodimers, two distinct DNA-protein complexes were formed, reflecting the involvement of full-length and truncated p65 (Fig. 2, lane9). Heterodimers consisting of p50/c-Rel, p65/c-Rel, p52/p65, and p52/c-Rel also formed DNA-protein complexes in binding assays; the binding of recombinant NF-kappaB protein to both probes was specific since the DNA binding activity of recombinant NF-kappaB subunits could be competed with excess HIV-1 kappaB probe, but not with the HIV-1 mutant probe (data not shown).


Figure 2: Binding of NF-kappaB subunits to HIV-1 enhancer probe and effect of IkappaBalpha on DNA binding. Recombinant proteins of p50 (1 ng), p65 (1.3 ng), c-Rel (1.5 ng), and p52 (20 ng) were individually assayed for specific binding to the HIV probe (lanes1, 5, 13, and 17). Heterodimers of p50 (0.5 ng) and p65 (0.65 ng), prepared as described under ``Materials and Methods,'' were also analyzed for DNA binding (lane9). Binding assays with recombinant proteins were performed in the presence of increasing amounts of recombinant IkappaBalpha (1, 3.3, and 10 ng, as indicated by the triangles above the lanes).



Recombinant IkappaBalpha, preincubated with the NF-kappaB proteins before probe addition, affected the DNA binding activity of all recombinant NF-kappaB subunits. IkappaBalpha completely inhibited the binding activity of p65 and the p50/p65 heterodimer (Fig. 2, lanes6-8 and 10-12); after IkappaBalpha inhibition of p50/p65 complex formation, a complex comigrating with the p50 dimer was formed, indicating the specificity of IkappaBalpha inhibition for p50/p65 (lanes 10-12). IkappaBalpha also inhibited formation of the c-RelbulletDNA complex (Fig. 2, lanes 14-16). Surprisingly, the DNA binding activity of p50 and p52 was stimulated by recombinant IkappaBalpha (Fig. 2, lanes 2-4 and 18-20); DNA binding of the p50/p65 chimeric protein, like p50, was also enhanced by the addition of IkappaBalpha to the reaction (data not shown).

Activation of NF-kappaB-dependent Transcription by Different Subunit Combinations

The combinatorial association of distinct NF-kappaB subunits contributes to the specificity of NF-kappaB-mediated transcriptional activation(63) . To evaluate the regulatory influence of different homo- and heterodimers, NF-kappaB subunits were analyzed for transcriptional activity in vitro using test templates containing either the HIV-1 enhancer or two copies of IFN-beta PRDII linked to the -56 minimal promoter and structural sequences of the rabbit beta-globin gene. The same -56 RbetaG template containing a HIV-1 enhancer mutant served as a control template (Fig. 3). Two templates were used as internal reference genes: DeltaRbetaG, which is the HS-RbetaG template without insert and with a 22-bp deletion (Fig. 3), and p901, which contains the human IFN-beta structural gene and promoter deleted to position -56(11) . After in vitro transcription, total RNA was extracted from the reaction mixture, and specific transcripts were quantified by S1 nuclease protection. The structures of the DNA templates and the probes used to detect readthrough and correctly initiated transcripts by S1 nuclease protection are shown in Fig. 3.

The HIV enhancer template was stimulated 13- or 15-fold by homodimers of p50 or p65, respectively (Fig. 4, lanes8 and 9), whereas the same proteins were unable to stimulate transcription in vitro using the HIV enhancer mutant template (lanes 1-3). The p50/p65 fusion protein links the N-terminal DNA-binding region of p50 (amino acids 10-502) to the C-terminal transactivation domain of p65 (amino acids 397-550). This fusion protein also stimulated NF-kappaB-dependent transcription in vitro 15-fold from the wild type, but not from the HIV-1 enhancer mutant template (Fig. 4, lanes4, 5, 10, and 11). The p50/p65 fusion proteins were purified either from E. coli (Fig. 4, lanes5 and 11) or from baculovirus (lanes4 and 10). Preincubation of p50 and p65 to permit heterodimer formation also resulted in a dramatic 17-fold increase in NF-kappaB-dependent transcription in vitro (Fig. 4, compare lanes6 and 12). In contrast to the results obtained with the HIV-1 enhancer template, the same template containing two copies of the PRDII element was not stimulated by p50 homodimers (Fig. 4, lane14), in agreement with previous results(23, 24) . Homodimers of p65 or the p50/p65 fusion protein resulted in 3.4- and 4.4-fold increases, respectively, in PRDII-dependent transcription (Fig. 4, lanes 15-17), which is significantly lower than the induction observed with the HIV enhancer test template (Fig. 4, lanes 9-11). Formation of p50/p65 heterodimers stimulated PRDII-dependent transcription by 4.2-fold (Fig. 4, lane18).


Figure 4: Transcriptional activation by recombinant NF-kappaB in vitro. The transcriptional activity of recombinant NF-kappaB subunits was measured using HeLa cell nuclear extracts in vitro. Transcription reactions (25 µl) contained 250 ng of HIV-1 enhancer mutant-RbetaG (lanes 1-6), HIV-1 enhancer-RbetaG (lanes 7-12), or PRDII-RbetaG (lanes 13-18) test genes and 250 ng of p901 internal reference gene(11, 12) . Homo- or heterodimers of NF-kappaB (200 fmol) were added to the reactions as indicated below the lanes (+). Bands corresponding to the S1 nuclease mapping product for readthrough (R.T.) transcripts and correctly initiated (C.I.) transcripts are indicated. LaneP, probes; laneM, size markers (pAT153 digested by HaeIII). RTL, relative transcription level, which was calculated as the ratio of the test correctly initiated transcript level divided by the reference correctly initiated transcript level, as determined by laser densitometric scanning of appropriately exposed autoradiographic films. The transcript ratio obtained in the control reaction (without the addition of protein) was taken as a value of 1. All other relative transcription levels were expressed relative to the control.



The effects of other NF-kappaB subunit combinations on transcriptional activation in vitro were also examined; in some reactions, recombinant IkappaBalpha was included to evaluate the effect of the inhibitor on transcriptional activity (Fig. 5). The NF-kappaB subunits p52 and c-Rel were unable to stimulate transcription from the HIV-1 enhancer in vitro (Fig. 5, lanes5 and 7), in contrast to the inducibility by p50 and p65 (Fig. 4, lanes8 and 9). Interestingly, the p50/p65 fusion protein stimulated transcription 10-fold (Fig. 5, lane3); the addition of IkappaBalpha to the reaction containing the p50/p65 fusion protein reduced transcriptional induction to <2-fold. The effect of IkappaBalpha on relative transcript levels was specific to NF-kappaB-dependent transcription since IkappaBalpha addition did not affect the basal level of transcription from the HIV-1 test template or from the reference template (Fig. 5, lanes1 and 2). Differential transcriptional induction by NF-kappaB heterodimer combinations was also sensitive to IkappaBalpha-mediated inhibition. For example, p50/p65 and p50/c-Rel heterodimeric complexes enhanced NF-kappaB-dependent transcription 15- and 17-fold, respectively (Fig. 5, lanes9 and 11), and the strong induction by both heterodimers was blocked by coincubation with IkappaBalpha (lanes10 and 12). Heterodimer combinations consisting of p52/p65, p52/c-Rel, and p65/c-Rel also differentially stimulated NF-kappaB-dependent transcription 7-, 6.5-, and 3.3-fold, respectively (Fig. 5, lanes13, 15, and 17). Coincubation with IkappaBalpha inhibited transcriptional induction in each case (Fig. 5, lanes14, 16, and 18). Differential stimulation of transcription in vitro by NF-kappaB heterodimer combinations was completely dependent upon a functional NF-kappaB site since the heterodimer combinations had no stimulatory effect on transcription in vitro from the HIV-1 enhancer mutant template (data not shown).


Figure 5: Transcription in vitro by NF-kappaB combinations and IkappaBalpha inhibition. In vitro transcription reactions were performed as described for Fig. 4, except that 250 ng of linearized DeltaRbetaG internal reference template was used to replace the p901 template. The test template was the HIV-1 enhancer template. Recombinant IkappaBalpha (50 fmol) and different NF-kappaB complexes (200 fmol) were added as indicated below the lanes (+). See Fig. 4legend for definitions of abbreviations.



The capacity of NF-kappaB heterodimer combinations to modulate PRDII-dependent transcriptional activation was also evaluated in vitro, and the cumulative results of these studies are summarized graphically in Fig. 6. The inhibitory effects of IkappaBalpha on HIV-1 enhancer-mediated transcriptional activity are also summarized. Heterodimers of p50/c-Rel resulted in a 10-fold stimulation of PRDII-dependent transcription, whereas p52/c-Rel, p65/c-Rel, and p52/p65 heterodimers stimulated transcription to a lesser extent: 6.0-, 7-, and 3-fold, respectively (Fig. 6B). The addition of IkappaBalpha to the transcription reactions containing either homodimers (Fig. 6A) or heterodimers (Fig. 6B) inhibited NF-kappaB-specific stimulation of transcription in all cases. These results demonstrate the differential inducibility of PRDII and the HIV-1 enhancer element by distinct NF-kappaB heterodimer combinations in an in vitro transcription assay and also illustrate the sensitivity of transcriptional activation in vitro to the presence of IkappaBalpha.


Figure 6: Transcriptional activity by NF-kappaB subunit combinations. The cumulative results of in vitro transcription reactions using different NF-kappaB combinations are summarized for the HIV-1 enhancer template (black bars) and the PRDII template (shaded bars). The effect of IkappaBalpha addition (50 fmol) on HIV-1 enhancer-dependent transcription is also shown (cross-hatched bars). The bar graphs show the relative transcript levels normalized to the internal reference template and represent the average of duplicate measurement with <20% variation. A, transcriptional effect of NF-kappaB homodimers and inhibition by IkappaBalpha; B, transcriptional effect of NF-kappaB heterodimer combinations and inhibition by IkappaBalpha.



Activation of IFN-beta Enhancer PRDIII-PRDI-PRDII by NF-kappaB and IRF-1

The efficient induction of the human IFN-beta promoter requires the synergistic interaction of at least the IRF and NF-kappaB proteins(7, 13, 17, 20) . A test template containing an insert of PRDIII-PRDI-PRDII at position -56 in RbetaG was used to investigate transcriptional cooperation between the various NF-kappaB complexes and IRF-1. Certain NF-kappaB homodimers alone were able to variably stimulate transcription through PRDIII-PRDI-PRDII. Homodimers of p50, p65, or the p50/p65 fusion protein resulted in 2-, 4-, and 2.5-fold increases in relative transcript levels, respectively (Fig. 7, lanes 2-4), whereas p52 or c-Rel homodimers decreased relative transcript levels slightly (lanes5 and 6). NF-kappaB heterodimer combinations of p50/p65, p50/c-Rel, p52/p65, and p65/c-Rel stimulated transcription 2.6-4.5-fold through the IFN-beta enhancer (Fig. 7, lanes 7-9 and 11), with the exception of the p52/c-Rel heterodimer, which had no effect (lane10). Recombinant IRF-1 alone resulted in a 3-fold increase in test gene transcription (Fig. 7, lane12). Other experiments using a PRDI template demonstrated that IRF-1 specifically stimulated transcription 2-5-fold through PRDI (data not shown).


Figure 7: Cooperation between NF-kappaB and IRF-1 in PRDIII-PRDI-PRDII-dependent transcription in vitro. The test template contained one copy of PRDIII-PRDI-PRDII (positions -94 to -55) of the IFN-beta promoter linked to the -56 promoter of RbetaG. Homo- or heterodimers of NF-kappaB (200 fmol) and/or IRF-1 (200 fmol) was used in the transcription reactions as indicated below the lanes. The DeltaRbetaG internal reference template was used as control in the in vitro transcription assay. See Fig. 4legend for definitions of abbreviations.



Differential stimulation of the PRDIII-PRDI-PRDII-dependent template was observed when distinct NF-kappaB complexes were used together with IRF-1 in vitro. p50, p65, or p50/p65 fusion homodimers together with IRF-1 increased test gene transcription 4.2-, 6.5-, and 6.9-fold, respectively (Fig. 7, lanes 13-15), whereas the p52 homodimer alone slightly inhibited IRF-1 stimulation (lane16). c-Rel completely inhibited the transcriptional activation in vitro, including the stimulation by IRF-1 (Fig. 7, lane17). Strikingly, the combination of the NF-kappaB heterodimeric complex p50/p65 and IRF-1 stimulated PRDIII-PRDI-PRDII-dependent transcription 10-fold; other heterodimer combinations (p50/c-Rel, p52/p65, p52/c-Rel, or p65/c-Rel) incubated together with IRF-1 stimulated transcriptional activation 4-8-fold (Fig. 7, lanes 18-22). In all cases, heterodimer combinations stimulated transcription in vitro more strongly in the presence of IRF-1 protein than in its absence (Fig. 7, compare, for example, lanes7 and 18, 8 and 19, 9 and 20, 10 and 21, and 11 and 22). These experiments demonstrate transcriptional cooperation between NF-kappaB heterodimeric complexes and IRF-1 in the in vitro activation of the IFN-beta PRDII-PRDI-PRDII virus-inducible enhancer element.


DISCUSSION

In this study, we used recombinant NF-kappaB and IRF proteins to evaluate transcriptional specificity and cooperativity in vitro in the activation of interferon-beta regulatory domains or the HIV-1 enhancer. We demonstrate that 1) distinct combinations of NF-kappaB subunits contributed to the specificity of transcriptional activation in vitro either through the HIV-1 enhancer or IFN-beta PRDII; 2) IkappaBalpha protein specifically inhibited NF-kappaB-dependent DNA transcription in vitro; and 3) homo- and heterodimeric NF-kappaB combinations together with IRF-1 cooperated to stimulate interferon-beta PRDIII-PRDI-PRDII-dependent transcription in vitro.

Transcriptional Activation by NF-kappaB p50 and p65

In previous studies, purified or recombinant NF-kappaB proteins stimulated transcription in vitro 3-15-fold using test genes containing different NF-kappaB motifs (23, 24) or the HIV-1 enhancer element(64, 65) . These studies revealed an unexpected complexity of NF-kappaB-mediated gene regulation in that p50 homodimers specifically stimulated transcription from the immunoglobulin kappa-site (equivalent to the HIV-1 enhancer site), but not from the PRDII site. On the other hand, heterodimers of p50/p65 stimulated transcription from all NF-kappaB-binding sites examined(23, 64) . We have extended these previous studies to demonstrate differential activation of NF-kappaB-dependent transcription using several physiologically relevant NF-kappaB heterodimer combinations. We also generated a chimeric p50/p65 fusion protein that mimicked the p50/p65 heterodimer and stimulated transcription from both the HIV-1 enhancer template and PRDII.

Transcriptional Activation by p52 Homo- and Heterodimers

Homodimers of p52 alone did not stimulate transcription through the HIV-1 enhancer and only weakly activated transcription through PRDII. On the other hand, p52/p65 or p52/c-Rel heterodimers stimulated transcription of the HIV-1 enhancer 7-fold. Interestingly, the combination of p52/c-Rel induced PRDII 6-fold and was a stronger activator of PRDII-dependent transcription than either c-Rel or p52 alone. These results are consistent with previous experiments indicating that p52 acted essentially as a DNA-binding partner with little intrinsic transcriptional activity, whereas association with p65 or c-Rel resulted in formation of trans-activating heterodimers in vivo and in vitro(53, 63, 66) .

Transcriptional Activation by c-Rel Homo- and Heterodimers

c-Rel protein alone was unable to activate transcription from NF-kappaB-dependent templates, indicating that c-Rel homodimers poorly activated transcription. Interestingly, however, c-Rel acted in combination with p50, p52, or p65 to stimulate NF-kappaB-dependent transcription. In this study, the combination of p50 and c-Rel was the strongest activating heterodimer combination, efficiently activating both PRDII- and HIV enhancer-dependent transcription 10- and 17-fold, respectively. The p65/c-Rel combination was unique in its ability to stimulate PRDII-dependent transcription more strongly than the HIV-1 enhancer-driven transcription. The presence of p65/c-Rel as a stable complex in HeLa cells was confirmed recently by purification studies, demonstrating the unique involvement of this heterodimer in the stimulation of urokinase gene transcription via a rel-related binding element(59) .

Transcriptional Inhibition by IkappaBalpha

The addition of IkappaBalpha to the in vitro transcription reactions effectively inhibited transcription by p65 or c-Rel homodimers or by NF-kappaB heterodimers containing p65 or c-Rel. In vitro DNA binding studies also demonstrated that IkappaBalpha inhibited the DNA binding activity of homodimers of p65 or c-Rel as well as heterodimeric complexes containing p65 or c-Rel subunits(51, 52, 53) . Surprisingly, the activation of the HIV enhancer by p50 homodimers or of PRDII by p52 was also inhibited by IkappaBalpha; it remains unclear why IkappaBalpha inhibited transcriptional activity in vitro by p50 and p52 since IkappaBalpha stimulated p50 and p52 DNA binding activity (Fig. 2)(53) . One explanation consistent with these results and the previous studies of Duckett et al.(53) is that IkappaBalpha may have a bifunctional effect on NF-kappaB-mediated transcription that involves 1) removal of p65 activator protein and 2) stimulation of p50 or p52 binding activity that has no activating potential in the presence of IkappaBalpha.

Transcriptional induction of IkappaBalpha mRNA by NF-kappaB itself, leading to increased IkappaBalpha levels and subsequent sequestration of NF-kappaBbulletIkappaBalpha complexes, clearly represents an important autoregulatory mechanism for down-regulation of NF-kappaB-induced gene expression(60, 67, 68, 69, 70, 71) . The ability of IkappaBalpha to dissociate NF-kappaBbulletDNA complexes under gel shift conditions (52) and the presence of IkappaBalpha in the nucleus when overexpressed (72) suggest that another function of newly produced IkappaBalpha may be to enter the nucleus and dissociate NF-kappaBbulletDNA transcriptional complexes(47, 48) . Since the concentration of IkappaBalpha required to completely inhibit NF-kappaB DNA binding or transcription was at least four times lower than the concentration of NF-kappaB, it would appear that inhibition by IkappaBalpha is not due solely to a stoichiometric formation of NF-kappaBbulletIkappaBalpha complexes.

Cooperation between IRF-1 and NF-kappaB in the Activation of PRDIII-PRDI-PRDII

The activation of IFN-beta gene transcription depends on synergistic interactions among NF-kappaB, IRF-1, and other transcription factors that bind to distinct regulatory domains in the promoter(7) . During the induction of IFN-beta gene expression, alterations in the composition of NF-kappaB subunits associated with PRDII occur as a function of time after virus infection. Formation of the PRDII-specific complexes preceded the onset of detectable IFN-beta transcription in Sendai virus-infected cells, and temporal shift in the composition of NF-kappaB subunits in association with PRDII was observed after infection(17) . The main NF-kappaB proteins in association with PRDII during the early phase of IFN-beta induction were p65, p50, and c-Rel. Heterodimers of p50/p65, p50/c-Rel, and p65/c-Rel were induced to bind to PRDII of the IFN-beta promoter early after Sendai virus infection of human cells(17) . Consistent with these in vivo results, the present studies demonstrate that p65/p50, p50/c-Rel, and p65/c-Rel heterodimers were also the strongest activators of PRDII or IFN-beta transcription in vitro. These data indicate that fluctuations in NF-kappaB heterodimer formation may directly influence IFN transcriptional activation.

Transcriptional cooperativity between NF-kappaB complexes and IRF-1 was also examined using the PRDIII-PRDI-PRDII template. Both NF-kappaB heterodimers and IRF-1 activated the transcription from the test promoter to differing degrees; in all cases, however, transcriptional activity in vitro from the PRDIII-PRDI-PRDII template was increased by the simultaneous incubation with NF-kappaB complexes and IRF-1. In cotransfection experiments, overexpression of IRF-1 with p50 and p65 activated transcription from the intact IFN-beta promoter in a synergistic manner(17) . The in vitro transcription assay demonstrated additive but not synergistic activation by NF-kappaB proteins and IRF-1, suggesting that other proteins required for proper regulation of IFN-beta transcription in vivo are absent from in vitro systems. While the exact nature of these factors remains unclear, previous studies showed that HIV-1 promoter activation by p50 or by p50 + p65 was absolutely dependent on the cofactor fraction USA(61, 73) . Another cofactor, the chromatin-associated high mobility group I/Y proteins, was also shown to be involved in the activation of NF-kappaB-dependent IFN-beta gene expression. High mobility group I/Y protein does not act as an activator of transcription, but interacts with AT-rich DNA sequences through the minor groove to facilitate the activity and/or binding of NF-kappaB to PRDII(29, 30) . While it may be expected that high mobility group I/Y proteins are abundant in nuclear extracts, it is possible that the proper stereospecific arrangement of transcription factors and cofactors may not be achieved in vitro. Transcription from a target promoter may also be regulated by histone H1-mediated repression. Transcriptional activators in part modulate transcription by counteracting histone H1-mediated repression (antirepression); for example, the GAL4-VP16 fusion protein stimulated transcription 2-fold in the absence of histone H1 (true activation) and 20-fold in the presence of histone H1, i.e. 10-fold antirepression(74, 75) . These limitations notwithstanding, these in vitro transcription studies emphasize the role of protein-protein dimerization as a distinct level of control that permits functional diversification of a limited number of components. Cooperativity between transcription factors such as NF-kappaB and IRF-1 maintains an additional level of specificity that would not be achieved by individual proteins.


FOOTNOTES

*
This work was supported in part by grants (to J. H.) from the Medical Research Council of Canada and the National Cancer Institute of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Medical Research Council postdoctoral fellowship.

Recipient of a Medical Research Council scientist award. To whom correspondence should be addressed: Lady Davis Inst. for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8260 (ext. 5265); Fax: 514-340-7576.

(^1)
The abbreviations used are: TF, transcription factor; TBP, transcription factor-binding protein; IFN, interferon; PRD, positive regulatory domain; IRF, interferon regulatory factor; bp, base pair; HIV, human immunodeficiency virus.


ACKNOWLEDGEMENTS

We thank Drs. Craig Rosen and Steven Ruben for plasmids containing RelA (p65) and DeltaRelA (p65Delta), Dr. Gary Nabel for the NFKB2 clone, Dr. Alain Israel for the KBF-1 clone (NFKB1), Dr. Nancy Rice for the c-Rel cDNA, and Dr. T. Taniguchi for the IRF-1 and IRF-2 expression plasmids. We also thank Normand Pepin for technical assistance with the preparation of expression plasmids and Mary Wiebe for typing and editing the manuscript.


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