Ikappa B-alpha Enhances Transactivation by the HOXB7 Homeodomain-containing Protein*

Alain ChariotDagger §, Frederic PrincenDagger , Jacques GielenDagger , Marie-Paule MervilleDagger , Guido Franzosoparallel , Keith Brownparallel , Ulrich Siebenlistparallel , and Vincent BoursDagger **

From the Dagger  Laboratory of Medical Chemistry and Medical Oncology, Pathology, University of Liege, Sart-Tilman, 4000 Liege, Belgium and the parallel  Laboratory of Immunoregulation, NIAID, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
Top
Abstract
Introduction
References

Combinatorial interactions between distinct transcription factors generate specificity in the controlled expression of target genes. In this report, we demonstrated that the HOXB7 homeodomain-containing protein, which plays a key role in development and differentiation, physically interacted in vitro with Ikappa B-alpha , an inhibitor of NF-kappa B activity. This interaction was mediated by the Ikappa B-alpha ankyrin repeats and C-terminal domain as well as by the HOXB7 N-terminal domain. In transient transfection experiments, Ikappa B-alpha markedly increased HOXB7-dependent transcription from a reporter plasmid containing a homeodomain consensus-binding sequence. This report therefore showed a novel function for Ikappa B-alpha , namely a positive regulation of transcriptional activation by homeodomain-containing proteins.

    INTRODUCTION
Top
Abstract
Introduction
References

Multiple transcription factors establish combinatorial interactions to achieve their in vivo specificity. These protein-protein interactions modulate the activating or repressing abilities of the complexes. The identification of all the partners interacting with a transcription factor is thus essential for the understanding of its biological functions.

Homeodomain-containing proteins are transcription factors that play a crucial role in the development of many species, including humans (1-4). They share a highly conserved 60-amino acid DNA-binding domain, the homeodomain, and control the expression of many target genes, most of which remain unknown (5). These proteins are encoded by 39 HOX genes, which are organized in four clusters (loci A, B, C, and D) located on chromosomes 7, 17, 12, and 2, respectively (6). Interestingly, their pattern of expression along the anteroposterior axis of the developing embryo is closely related to their chromosomal position on the cluster (7), defining a "spatial colinearity." Although homologous recombination experiments have clearly demonstrated their in vivo specificity, all the HOX gene products bind to very similar sequences in vitro (8-10). Their specificity may thus be achieved not only through DNA-protein interactions but also through protein-protein interactions with other transcription factors whose identities remain largely unknown. Among these partners, the extradenticle/Pbx homeodomain-containing proteins were the first to be identified as co-factors for HOX proteins (11). Interaction with the PBX protein requires the pentapeptide, a conserved domain located upstream of the DNA-binding domain of most HOX gene products and required for the interactions of HOX proteins with other peptides (11), as well as the HOX cooperativity motif, a sequence C-terminal to the Pbx homeodomain (12). Because AbdB-like HOX proteins do not harbor any pentapeptide-like sequence, they cannot interact with Pbx proteins (13), thus suggesting that other partners might be involved. Indeed, a recent report has illustrated the existence of heterodimeric complexes between HOX and Meis1 proteins (13). Moreover, it is likely that other proteins yet to be identified also interact with HOX proteins and contribute to their biological function.

HOXB7 cDNA was initially isolated from an SV40-transformed human fibroblast cDNA library (14). The HOXB7 protein is involved in a variety of developmental processes, including hematopoietic differentiation and lymphoid development (15-17). Because of its expression in lymphoid and nonlymphoid cells, the HOXB7 protein might be involved in the regulation of a common transcriptional event rather than in lineage-specific gene expression (18). However, despite the demonstration of HOXB7 protein binding to DNA (19), little is known about its transcriptional properties and interacting partners in vivo. We first demonstrated that the HOXB7 protein as well as a naturally occurring mutant harboring a truncated C-terminal tail both transactivate from a HOX-binding consensus sequence in breast cancer cells (20) and physically interact with the coactivator CREB-binding protein.1

The NF-kappa B proteins form a family of transcription factors that play a central role in the cellular responses to stress, cytokines, and pathogens (22-24). Indeed, these transcription factors are activated in response to a variety of extracellular signals such as phorbol esters, tumor necrosis factor-alpha , interleukin-1, lipopolysaccharide, UV irradiation, viral infection, and growth factors (24, 25) and regulate a wide spectrum of immune and inflammatory responses (26). In unstimulated cells, NF-kappa B activity is inhibited by another class of proteins that includes Ikappa B-alpha (27, 28), Ikappa B-beta (29), Ikappa B-epsilon (30), p105, and p100. These inhibitory proteins all share ankyrin repeats, sequester the NF-kappa B complexes in the cytoplasm, and block their binding to kappa B DNA sequences. Initially described as a cytoplasmic protein (28), Ikappa B-alpha has since been detected in the nucleus of transfected Vero cells (31) as well as after serum stimulation (32). The nuclear localization of Ikappa B-alpha is mediated by its second ankyrin repeat, which acts as a nuclear import sequence (33). Once in the nucleus, Ikappa B-alpha can remove NF-kappa B dimers from their kappa B DNA sequences, thus inhibiting NF-kappa B activity (34). When fused to the GAL-4 DNA-binding domain, Ikappa B-alpha displays transactivation abilities (35, 36), a property not possessed by the naturally occurring Ikappa B-alpha protein (32). These results raised the possibility that Ikappa B-alpha can interact with other transcription factors and modulate their activity.

In this report, we demonstrated that Ikappa B-alpha is able to physically interact with the HOXB7 homeodomain-containing protein and to enhance HOXB7 transcriptional activity. We further identified HOXB7 and Ikappa B-alpha domains involved in this interaction. Our results thus demonstrate a novel function of Ikappa B-alpha .

    EXPERIMENTAL PROCEDURES

Cell Cultures-- The MDA-MB231 cell line was obtained from the American Type Tissue Collection (Rockville, MD). The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and antibiotics.

Plasmids-- Coding sequences for the HOXB7 protein and for a naturally occurring protein lacking 2 amino acids in its C-terminal sequence (B7*) (20) were subcloned by PCR2 into the expression vectors pcDNA3 (Invitrogen, San Diego, CA) and pMT2T. The constructs were sequenced to confirm the integrity of the amplified regions. pcDNA3 expression vectors coding for HOXB7 proteins deleted either in the N- or the C-terminal domain were constructed by PCR amplification. The constructs B7-Delta N18, Delta N54, Delta N86, and Delta N129 generate HOXB7 proteins lacking 18, 54, 86, and 129 N-terminal amino acids, respectively. The constructs B7-Delta C12, B7-Delta C34, B7-Delta C80, and B7-Delta C97 encode HOXB7 proteins deleted of 12, 34, 80, and 97 C-terminal amino acids, respectively.

The mammalian PMT2T expression vectors for p50, RelA, and Ikappa B-alpha were previously described (37, 38). The Ikappa B-alpha coding sequence was also subcloned by PCR into the expression vector pcDNA3. The PMT2T expression vectors for Ikappa B-alpha Delta N and Ikappa B-alpha Delta C lacking the first 53 codons and the last 42 codons of Ikappa B-alpha , respectively (39), are schematically illustrated in Fig. 1A. The PMT2T expression vector for Ikappa B-alpha N+C GST codes for a protein where the ankyrin repeats of Ikappa B-alpha have been replaced by the GST peptide (Fig. 1A) as described (39).

Both the pT109 and pTCBS reporter plasmids were provided by Dr. Zappavigna (Laboratory of Gene Expression, Department of Biology and Technology, Instituto Scientifico H. S. Raffaele, Milan, Italy). The pTCBS plasmid contains an 8-fold multimerized form of a homeodomain consensus-binding sequence (CBS) cloned upstream of an HSV-TK promoter and a luciferase reporter gene, whereas the pT109 construct does not contain the CBS sequence and was thus used as a negative control. The kappa B-ICAM-1 reporter plasmid construct has been previously described; it harbors three NF-kappa B-like sites from the ICAM-1 promoter cloned upstream of the herpes simplex virus thymidine kinase minimal promoter and the luciferase gene (40).

For GST interaction experiments, various functional domains of Ikappa B-alpha were subcloned by PCR into the BamHI/EcoRI polylinker of the pGEX-2TK vector (Amersham Pharmacia Biotech) to create GST fusion proteins. These constructs include pGEX Ikappa B-alpha Delta C, pGEX Ikappa B-alpha Delta N, pGEX ankyrins, pGEX NIkappa B, and pGEX CIkappa B and are schematically illustrated in Fig. 1B. The sequence of primer 1 is 5'-TATAGGATCCATGTTCCAGGCGGCC-3'; primer 2, 5'-TATAGGATCCCTCGAGCCGCAGGAGGT-3'; primer 3, 5'-TATAGGATCCAACCTTCAGATGCTGCCAGAG-3'; primer 4, 5'-ATATGAATTCCTCGAGGCGGATCTCCT-3'; primer 5, 5'-ATATGAATTCTTCTAGTGTCAGCTGGCC-3'; and primer 6, 5'-ATATGAATTCTCATAACGTCAGACGCTGGCC-3'.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Schematic illustration of the Ikappa B-alpha expression vectors (A) and the GST-Ikappa B-alpha constructs (B). The ankyrin repeats are illustrated by dark rectangles. The primers designed for PCR amplification are numbered from 1 to 6 and represented by arrows. Primers 4-6 are derived from the complementary strand.

In Vitro Translation-- In vitro transcription and translation were performed using the Wheat Germ TNT kit provided by Promega (Madison, WI) with 1 µg of various DNA templates and [35S]methionine, according to the protocol provided by the manufacturer.

In Vitro Protein-Protein Interactions-- GST fusion proteins were produced in the Escherichia coli BL21 bacterial strain. Bacteria were grown in 500 ml of Luria broth to an A600 nm of 0.6, induced with 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside for 3 h and harvested. Bacterial pellets were washed once with phosphate-buffered saline, resuspended in 10 ml of NENT buffer (250 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8, Nonidet P-40 1.5%) and sonicated three times for 15 s at 4 °C. Insoluble materials were removed by centrifugation. GST fusion proteins were purified after incubation of 1 ml of the supernatant with 10 µl of glutathione-Sepharose beads for 1 h at 4 °C (Amersham Pharmacia Biotech). The beads were then washed twice with 1 ml of NENTM buffer (NENT + 0.5% milk) and once with 1 ml of TWB buffer (20 mM Hepes, pH 7.9, 60 mM NaCl, 1 mM dithiothreitol, 6 mM MgCl2, 8.2% glycerin, 0.1 mM EDTA). In each case, the expected fusion proteins were visualized on a 12% polyacrylamide gel stained by Coomassie Blue. Protein-protein interactions were performed by incubating an aliquot of the GST-Ikappa Balpha fusion protein bound to the glutathione-Sepharose beads with 10 µl of in vitro translated protein in 200 µl of TWB buffer for 1 h at 4 °C. Beads were then washed six times with 1 ml of NENTM buffer, resuspended into migrating buffer, and loaded on an SDS-polyacrylamide gel before autoradiography.

Transient Transfections and Luciferase Assays-- Transfections of MDA-MB231 cells were performed as described (41), using 1 µg of reporter plasmid (either pTCBS or pT109) and various amounts of vectors expressing HOXB7, RelA, p50, and/or Ikappa B-alpha . Total amounts of transfected DNA were kept constant throughout by adding appropriate amounts of either pcDNA3 or pMT2T empty vectors. Cells were harvested 48 h after transfection, and luciferase activities were measured with the Luciferase Reporter Gene Assay kit (Boehringer Mannheim), as recommended by the manufacturer. The luciferase activities were normalized to the protein concentration of the extracts.

    RESULTS

p50, RelA, and Ikappa B-alpha Enhance Transactivation by the HOXB7 Protein-- To investigate whether the HOXB7 protein can interact with transcription factors from other families, we transiently transfected MDA-MB231 cells with a HOXB7 expression vector and a variety of constructs coding for different members of the NF-kappa B/Ikappa B families. Both the pTCBS and pT109 constructs were used as reporter plasmids: the pTCBS plasmid contains a luciferase reporter gene driven by a multimerized HOX CBS that is recognized by most HOX proteins, whereas the pT109 vector does not harbor any HOX-binding sequence and was used as a negative control (42). A 3.6-fold induction over basal luciferase activity was measured when the HOXB7 expression vector was transfected with pTCBS (Fig. 2, column 3), as described previously (20, 41). This effect was mediated by the binding of the HOXB7 protein to the CBS sequence, because no significant effect was observed with the pT109 reporter plasmid (Fig. 2, column 4).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2.   NF-kappa B and Ikappa B-alpha increase transactivation by HOXB7 through a HOX consensus DNA-binding sequence. MDA-MB231 cells were transfected with 1 µg of HOXB7, p50, RelA, and/or Ikappa B-alpha expression vectors together with 1 µg of reporter plasmid, as indicated in the figure. The pT109 does not contain any HOX-binding sequence and was used as a negative control. The figure shows the relative luciferase activity over the basal activity observed with 1 µg of the pTCBS or pT109 reporter plasmid alone. Each value represents the mean (± S.D.) of at least three independent experiments after normalization to the protein concentration of the extracts.

When p50 and RelA expression vectors were transfected with the pTCBS or pT109 reporter plasmids, weak inductions of luciferase activity were observed (Fig. 2, columns 5 and 6). Moreover, a very weak increase in luciferase activity was observed when the plasmid encoding Ikappa B-alpha was co-transfected with either the pTCBS or pT109 reporter constructs (Fig. 2, columns 7 and 8), indicating that, as expected, Ikappa B-alpha did not transactivate through these promoters in MDA-MB231 cells. When the HOXB7 expression construct was co-transfected with p50, RelA, and pTCBS, a 4.9-fold induction over basal luciferase activity was observed (Fig. 2, column 9), indicating that NF-kappa B members enhanced HOXB7 transcriptional activity. To determine whether Ikappa B-alpha could inhibit the transactivation observed with HOXB7 and p50-RelA, we co-transfected an Ikappa B-alpha expression vector with the plasmids generating the HOXB7, p50, and RelA proteins as well as with the pTCBS construct. Surprisingly, a further increase in luciferase activity (7.2-fold induction over basal luciferase activity) was measured (Fig. 2, columns 11). Moreover, the luciferase activity was even more elevated (13.7-fold induction over basal luciferase activity) when we co-transfected only the HOXB7 and Ikappa B-alpha expression vectors with the pTCBS reporter plasmid (Fig. 2, column 13).

To further characterize the transcriptional properties of the HOXB7 protein, additional transient expression experiments were performed using the kappa B-ICAM-1 reporter plasmid harboring three kappa B-like binding sites upstream of a CAT gene. As expected, transfection of the p50 and RelA expression vectors induced CAT activity (Fig. 3, column 2), and this effect was inhibited by simultaneous expression of Ikappa B-alpha (Fig. 3, column 3). Transfection of increasing amounts of HOXB7 expression vector did not lead to any significant induction of CAT activity (Fig. 3, columns 4-6). Moreover, when we co-transfected HOXB7 with both p50 and RelA expression vectors, the CAT activity was close to that measured in the absence of HOXB7 (Fig. 3, columns 2 and 8) and was attenuated by the inhibitor Ikappa B-alpha (Fig. 3, columns 10-12). No significant induction of CAT activity was measured when HOXB7 and Ikappa B-alpha expressing vectors were co-transfected (Fig. 3, columns 14-16). These results suggest that the HOXB7 protein does not significantly modulate the transcriptional abilities of NF-kappa B members.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 3.   HOXB7 does not modify NF-kappa B transcriptional activity. MDA-MB231 cells were transfected with 1 µg of p50, RelA, and/or Ikappa B-alpha expression vectors together with various amounts of HOXB7 expression vector (0.5, 1, or 2 µg) and 1 µg of the kappa B-ICAM-1 reporter plasmid, as indicated in the figure. The figure shows the relative CAT activity over the basal activity observed with 1 µg of the kappa B-ICAM-1 reporter plasmid alone. Each value represents the mean (± S.D) of at least three independent experiments after normalization to the protein concentration of the extracts.

Ikappa B-alpha Physically Interacts in Vitro with the N-terminal Domain of HOXB7-- To determine whether Ikappa B-alpha physically interacted with the HOXB7 protein, purified GST-Ikappa B-alpha fusion protein bound to glutathione-Sepharose beads was incubated with in vitro translated HOXB7. After precipitation of the beads, a positive signal was detected (Fig. 4, lane 2), whereas HOXB7 did not interact with the GST protein (lane 3), thus demonstrating the existence of an in vitro interaction between HOXB7 and Ikappa B-alpha . To map the HOXB7 domain involved in this process, we designed several constructs generating HOXB7 gene products progressively deleted in their N-terminal domain and designated as B7-Delta N18, Delta N54, Delta N86, and Delta N129. All these HOXB7 proteins shared an intact homeodomain, whereas only the Delta N129 peptide was deleted of the pentapeptide. These products were then in vitro translated and incubated with the GST-Ikappa B-alpha fusion protein as described above. None of these proteins were able to significantly interact with Ikappa B-alpha (Fig. 4, lanes 5, 8, 11, and 14).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 4.   In vitro protein-protein interaction between HOXB7 and Ikappa B-alpha requires the HOXB7 N-terminal domain. The HOXB7 expression vectors are schematically represented. B7-Delta N18, Delta N54, Delta N86, and B7-Delta N129 products are deleted in their N-terminal domain. The homeodomain is illustrated by a large shaded rectangle, whereas the pentapeptide is represented by a small shaded box upstream of the homeodomain, and the acidic C-terminal tail is shown as a cross-hatched box. The expected molecular mass of the resulting proteins is mentioned on the right. 35S-Labeled in vitro translated wild-type and deleted HOXB7 proteins were incubated with a GST-Ikappa B-alpha fusion protein attached to glutathione-Sepharose beads (lanes 2, 5, 8, 11, and 14), precipitated and run on an SDS-polyacrylamide gel. Beads carrying the GST protein alone were used as negative controls (lanes 3, 6, 9, 12, and 15). In vitro translated proteins (10% of the amounts used in the precipitation experiments) were run on lanes 1, 4, 7, 10, and 13.

To determine whether other domains were involved in the interaction, additional constructs including a naturally occurring HOXB7 mutant that lacks two amino acids at the C-terminal tail (B7*) (20) and HOXB7 products deleted in their C-terminal domain (B7-Delta C12, B7-Delta C34, B7-Delta C80, and B7-Delta C97) were in vitro translated (Fig. 5, lanes 1, 4, 7, 10, 13, and 16) and incubated with the GST-Ikappa B-alpha fusion protein bound to glutathione-Sepharose beads. All these proteins were still able to interact with Ikappa B-alpha despite the absence of a complete homeodomain sequence for B7-Delta C80 and of the pentapeptide region for B7-Delta C97 (Fig. 5, lanes 5, 8, 11, 14, and 17). These results indicate that a HOXB7/Ikappa B-alpha physical interaction can occur independently of the homeodomain sequence and depends exclusively on an intact HOXB7 N-terminal sequence.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 5.   In vitro protein-protein interaction between HOXB7 and Ikappa B-alpha does not require the HOXB7 C-terminal domain. The HOXB7 expression vector generating proteins deleted in their C-terminal domain are schematically represented (B7-Delta C12, B7-Delta C34, B7-Delta C80, and B7-Delta C97). The B7* protein is a naturally occurring mutant deleted of two C-terminal amino acids. The homeodomain is illustrated by a large shaded rectangle, whereas the pentapeptide is represented by a small shaded box upstream of the homeodomain, and the acidic C-terminal tail is shown as a cross-hatched box. The expected molecular mass of the resulting proteins are mentioned on the right. 35S-Labeled in vitro translated wild-type and deleted HOXB7 proteins were incubated with a GST-Ikappa B-alpha fusion protein attached to glutathione-Sepharose beads (lanes 2, 5, 8, 11, 14, and 17), precipitated, and run on an SDS-polyacrylamide gel. Beads carrying the GST protein alone were used as negative controls (lanes 3, 6, 9, 12, 15, and 18). In vitro translated proteins (10% of the amounts used in the precipitation experiments) were run on lanes 1, 4, 7, 10, 13, and 16.

The N-terminal Domain of the HOXB7 Protein Is Required for the Interaction with Ikappa B-alpha in Vivo-- We previously demonstrated that both the N-terminal domain and the acidic C-terminal tail of the HOXB7 protein mediated its transcriptional properties.1 Because the N-terminal domain of HOXB7 was required for the interaction with Ikappa B-alpha in vitro, we transfected MDA-MB231 cells with the B7Delta N129 expression vector and the pTCBS or pT109 reporter plasmid. The B7Delta N129 product, alone or co-expressed with Ikappa B-alpha , did not induce any luciferase activity (Fig. 6). Moreover, the B7-Delta C12 protein, which lacks the acidic C-terminal domain but still interacts with Ikappa B-alpha in vitro (Fig. 5), did not behave as a transcriptional activator (Fig. 6). Interestingly, when both the B7-Delta C12 and Ikappa B-alpha expression vectors were transfected simultaneously with the pTCBS reporter plasmid, an induction of the luciferase activity similar to that measured with both HOXB7 wild-type and Ikappa B-alpha proteins was observed (Fig. 6). These results suggest that the inhibitor Ikappa B-alpha potentiates HOXB7 transactivating activities through a physical interaction with the HOXB7 N-terminal domain.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6.   The HOXB7 N-terminal domain is required for transactivation by HOXB7/Ikappa B-alpha . MDA-MB231 cells were transfected with 1 µg of expression vectors coding for wild-type or deleted HOXB7 and 1 µg of Ikappa B-alpha expression vector together with 1 µg of reporter plasmid. The figure shows the relative luciferase activity over the basal activity observed with the pTCBS or the pT109 reporter plasmid alone. Each value represents the mean (± S.D.) of at least three independent experiments after normalization as described above.

The Ankyrin Repeats and the C-terminal Domain of Ikappa B-alpha Are Required for in Vitro and in Vivo Interaction with HOXB7-- To map the Ikappa B-alpha domain(s) involved in the physical interaction with HOXB7, we inserted the ankyrin domain of Ikappa B-alpha in the pGex-2TK vector (Fig. 7A, lane 2) and produced the corresponding fusion protein. Incubation of this protein with in vitro translated HOXB7 demonstrated a physical interaction between the two proteins (Fig. 7A, lane 2), whereas HOXB7 could not interact with the GST protein purified from E. coli (Fig. 7A, lane 3). Because the signal was weaker than that observed with the full-length GST-Ikappa B-alpha fusion protein (Fig. 7A, lane 1), it is likely that other functional domains were also involved in this process. An Ikappa B-alpha peptide deleted of the C-terminal domain was fused to GST (Fig. 7B) and purified. As illustrated in Fig. 7B (lane 4), this fusion protein bound to glutathione-Sepharose beads interacted with HOXB7, although this interaction was weaker than that obtained with the full-length Ikappa B-alpha product (Fig. 7B, lane 1). Moreover, a GST-Ikappa B-alpha -C-terminal domain fusion protein was still able to interact with HOXB7 (Fig. 7B, lane 5). To confirm that both the ankyrin and C-terminal domains of Ikappa B-alpha were responsible for the interaction, a fusion protein that does not contain the Ikappa B-alpha -N-terminal domain was produced and incubated with HOXB7. As illustrated in Fig. 7C (lane 6), a signal similar to that obtained with the wild-type Ikappa B-alpha protein was observed, suggesting that the N-terminal domain of Ikappa B-alpha is not involved in the interaction with HOXB7. Indeed, incubation of the GST-Ikappa B-alpha -N-terminal domain fusion protein with HOXB7 did not generate any significant signal (Fig. 7C, lane 7). These results indicate that both the Ikappa B-alpha ankyrin and C-terminal domains are required for physical in vitro interaction with HOXB7.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 7.   In vitro protein-protein interaction between HOXB7 and Ikappa B-alpha requires Ikappa B-alpha ankyrin repeats and the C-terminal domain. The GST-Ikappa B-alpha constructs are schematically illustrated. 35S-Labeled in vitro translated wild-type HOXB7 protein was incubated with full-length (lane 1) or deleted (lanes 2-7) GST-Ikappa B-alpha fusion proteins attached to glutathione-Sepharose beads, precipitated, and run on an SDS-polyacrylamide gel. Beads carrying the GST protein alone were used as negative controls (lane 3).

To confirm these results in vivo, we performed transient expression experiments using both pT109 and pTCBS constructs as reporter plasmids and a variety of vectors generating distinct Ikappa B-alpha peptides (Fig. 1A). Full-length Ikappa B-alpha , Ikappa B-alpha Delta C, Ikappa B-alpha Delta N, and NIkappa BGSTCIkappa B proteins did not significantly induce luciferase activity when co-transfected with any of the reporter plasmids (Fig. 8, rows 5-12). However, cotransfection of the plasmids generating the Ikappa B-alpha Delta N and HOXB7 peptides led to a 13-fold induction of luciferase activity (Fig. 8, row 17) that was dependent on the binding to the CBS sequence (row 18) and comparable with that measured with the full-length Ikappa B-alpha expression vector (row 19). Cotransfection of the expression plasmids for Ikappa B-alpha Delta C and HOXB7 generated only a 4.3-fold induction over the basal activity (Fig. 8, row 15). To confirm that the ankyrin domain was also required, we cotransfected the pTCBS or the pT109 reporter plasmids with the HOXB7 plasmid and the NIkappa BGSTCIkappa B construct generating an Ikappa B-alpha -related protein in which the ankyrin domain had been replaced by the GST sequence. As illustrated in Fig. 8 (row 13), we observed only a 6.4-fold induction of the luciferase activity. Our results clearly demonstrate that the ankyrin repeats and the C-terminal domain of Ikappa B-alpha are required for in vitro and in vivo interaction with the HOXB7 homeodomain-containing protein.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 8.   The Ikappa B-alpha ankyrin and C-terminal domains are required for transactivation by HOXB7/Ikappa B-alpha . MDA-MB231 cells were transfected with expression vectors coding for HOXB7 (1 µg) and for wild-type or deleted Ikappa B-alpha (1 µg) as well as with the reporter plasmid (1 µg), as indicated in the figure. The figure shows the relative luciferase activity over the basal activity observed with 1 µg of the pTCBS or the pT109 reporter plasmid alone. Each value represents the mean (± S.D) of at least three independent experiments after normalization as described above.


    DISCUSSION

This report has demonstrated a physical interaction between the HOXB7 homeodomain-containing protein and Ikappa B-alpha , resulting in an enhanced transactivation by this HOX gene product. Moreover, we identified the HOXB7 and Ikappa B-alpha functional domains mediating this interaction. These results provide new insights into the transcription properties of the homeodomain-containing proteins and reveal a novel function of the inhibitor Ikappa B-alpha .

All the homeodomain-containing proteins encoded by the 39 HOX genes share a highly conserved 60-amino acid DNA-binding domain, the homeodomain, and bind to very similar sequences in vitro (8-10). Their in vivo specificity may thus involve protein-protein interactions with other transcription factors. In this context, the homeodomain proteins derived from the extradenticle/Pbx genes act as co-factors for HOX gene products that contain a pentapeptide sequence (11), whereas the AbdB-like HOX proteins, which do not harbor a pentapeptide, interact with Meis1 (13). We have provided here evidence that the NF-kappa B proteins, including the p50-p65 heterodimer, can enhance the transcription potential of the HOXB7 protein in transient expression experiments. This effect is presumably mediated by physical interactions between the p50-p65 complex and HOXB7. Preliminary in vitro experiments have indeed confirmed this hypothesis (data not shown). It is tempting to speculate the existence of a p50-p65-HOXB7 complex that could bind the CBS consensus sequence and transactivate through the HOXB7 and p65 activation domains. This complex, however, did not display a similar effect on a kappa B-binding site under our experimental conditions.

We have shown that the inhibitor Ikappa B-alpha can enhance the HOXB7 transactivating effect. This is the first demonstration that Ikappa B-alpha interacts with proteins from other families of transcription factors. Previous reports had demonstrated that Ikappa B-alpha can translocate to the nucleus, using its second ankyrin repeat as a nuclear import sequence (33) and subsequently remove the NF-kappa B complex from its binding site (34). Moreover, Ikappa B-beta can repress the 9-cis-retinoic acid-induced transcriptional activity of retinoid X receptor in lipopolysaccharide-treated cells (43). Thus, in both cases, Ikappa B proteins localized in the nucleus negatively regulate the transcriptional activity of their interacting partners. Surprisingly, our study demonstrates that Ikappa B-alpha can also positively regulate the transcriptional properties of a homeodomain-containing protein. A similar phenomenon was described previously for Bcl3, another member of the Ikappa B protein family. Indeed, Bcl3 can transactivate through kappa B sites when physically associated with p52 and p50 (37, 44), and it has been demonstrated that the N- and C-terminal domains of Bcl3 are transcriptional activation domains (37). Moreover, Bcl3, but not Ikappa B-beta , can also act as a coactivator of the retinoid X receptor (45). These results and the present report strongly suggest that distinct Ikappa B proteins can modulate, positively as well as negatively, the transcriptional properties of their interacting partners, including transcription factors that do not belong to the NF-kappa B family.

Interestingly, we demonstrated that both the Ikappa B-alpha ankyrin and C-terminal domains mediate interaction with HOXB7. The same domains are also required for the regulation of c-Rel by Ikappa B-alpha in the nucleus (46). Taken together, these results suggest a critical role for the ankyrin repeats and C-terminal domains in the function of Ikappa B-alpha in the nucleus.

Several models, which are not mutually exclusive, can explain how Ikappa B-alpha enhances HOXB7 transcriptional activity (Fig. 9). The first model does not imply a direct interaction between HOXB7 and Ikappa B-alpha but rather an indirect mechanism mediated by NF-kappa B. Indeed, we can postulate that NF-kappa B activates the expression of HOX genes encoding repressors. Therefore, NF-kappa B inhibition by Ikappa B-alpha would lead to a decreased expression of these HOX genes and to increased luciferase activity in transient expression experiments (Fig. 9A). This first model is the only one that does not require Ikappa B-alpha nuclear localization. The second model is based on a report demonstrating that ankyrin repeats stabilize the DNA binding of other transcription factors (47). The enhanced HOXB7 transcriptional activity would then be mediated by a stronger HOXB7 DNA binding affinity for its target sequence in the presence of Ikappa B-alpha (Fig. 9B). This hypothesis is supported by the observation that the physical interaction between HOXB7 and Ikappa B-alpha requires the ankyrin repeats. In the third model, a HOXB7-Ikappa B-alpha complex is bound to the CBS sequence through the HOXB7 homeodomain and transactivates through Ikappa B-alpha . This hypothesis is supported by previous studies demonstrating that Ikappa B-alpha can transactivate when fused to a GAL4 DNA-binding domain (35, 36). Moreover, Bcl3, another member of the Ikappa B-alpha family, also harbors transactivating domains (37). The fourth model implies a HOXB7-RelA-Ikappa B-alpha complex activating the luciferase gene through both the HOXB7 and RelA transactivation domains. The last two hypotheses are supported by the induction of luciferase activity observed with Ikappa B-alpha and B7Delta C12 expression vectors, whereas the B7Delta C12 protein is not able to transactivate by itself but can physically interact with Ikappa B-alpha . However, the last model cannot account for the fact that the transcriptional activity was higher after transfection of the HOXB7 and Ikappa B-alpha expression vectors than in the presence of the same vectors plus RelA (Fig. 2). Further experiments are required to determine which of these models is correct. Unfortunately, the available HOXB7 antibodies did not allow us to study more precisely the HOXB7 multimeric complexes in transfected or unmodified cells.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 9.   Models for HOXB7 transcriptional activation by NF-kappa B/Ikappa B-alpha proteins. A, the NF-kappa B heterodimer transactivates the expression of a HOX gene that codes for a transcription repressor. Upon transfection of Ikappa B-alpha , the inhibitor sequesters NF-kappa B in the cytoplasm, thus preventing the expression of the HOX target gene. The luciferase gene is consequently activated. This first model does not imply a direct interaction between HOXB7 and Ikappa B-alpha . B, Ikappa B-alpha stabilized the HOXB7 binding to the CBS sequence, thus allowing the induced expression of the luciferase gene. C, upon transfection of Ikappa B-alpha , a HOXB7-Ikappa B-alpha complex is formed on the CBS sequence and activates the expression of the luciferase gene through HOXB7 and Ikappa B-alpha transactivation domains. D, upon transfection of Ikappa B-alpha , a HOXB7-p65-Ikappa B-alpha complex is formed on the CBS sequence and activates the expression of the luciferase gene through HOXB7 and p65 transactivation domains.

The functional link between NF-kappa B-Ikappa B-alpha and homeodomain proteins was unexpected because of the distinct physiological processes they control. However, a first link between these two families during the outgrowth of the vertebrate limb has recently been described (48, 49). Indeed, NF-kappa B gene expression has been detected during limb morphogenesis and the alteration of NF-kappa B activity causes an arrest of the outgrowth (48, 49). Moreover, Ikappa B-alpha is the human homologue of cactus, a protein that plays a crucial role in the dorsoventral patterning of the Drosophila embryo (21). Because HOX genes are clearly required to establish the anteroposterior axis of the developing embryo, it is tempting to speculate that the interaction between Ikappa B-alpha and HOX proteins might determine the antero-posterior and dorsoventral polarities of the embryo.

    ACKNOWLEDGEMENTS

We thank Dr. P. T. van der Saag for providing the kappa B-ICAM-1 reporter plasmid and are grateful to Dr. Zappavigna for the pTCBS and pT109 plasmids.

    FOOTNOTES

* This work was supported by grants from the National Fund for Scientific Research, Télévie (Belgium) and the Centre Anti Cancéreux (University of Liège, Belgium).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Lab. of Immunoregulation, NIAID, NIH, Bethesda, MD 20892.

Research Associates of the National Fund for Scientific Research (Belgium).

** To whom correspondence should be addressed: Medical Oncology, C.H.U. B35, University of Liege, Sart-Tilman, 4000 Liege, Belgium. Tel.: 32-4-366-24-82; Fax: 32-4-366-45-34; E-mail: vbours{at}ulg.ac.be.

1 A. Chariot, C. van Lint, M. Chapelier, J. Gielen, M.-P. Merville, and V. Bours, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; CBS, consensus-binding sequence; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase.

    REFERENCES
Top
Abstract
Introduction
References
  1. Levine, M., Rubin, G. M., and Tjian, R. (1984) Cell 38, 667-673[Medline] [Order article via Infotrieve]
  2. Levine, M., and Hoey, T. (1988) Cell 55, 537-540[Medline] [Order article via Infotrieve]
  3. Akam, M. (1989) Cell 57, 347-349[Medline] [Order article via Infotrieve]
  4. Favier, B., and Dollé, P. (1997) Mol. Hum. Reprod. 3, 115-131[Abstract]
  5. Gehring, W. J., Affolter, M., and Bürglin, T. (1994) Annu. Rev. Biochem. 63, 487-526[CrossRef][Medline] [Order article via Infotrieve]
  6. Acampora, D., D'Esposito, M., Faiella, A., Pannese, M., Migliacco, E., Morelli, F., Stornaiuolo, A., Nigro, V., Simeone, A., and Boncinelli, E. (1989) Nucleic Acids Res. 17, 10385-10402[Abstract]
  7. Graham, A., Papalopupu, N., and Krumlauf, R. (1989) Cell 57, 367-378[Medline] [Order article via Infotrieve]
  8. Kalionis, B., and O'Farrell, P. H. (1993) Mech. Dev. 43, 57-70[CrossRef][Medline] [Order article via Infotrieve]
  9. Pellerin, I., Schnabel, C., Catron, K. M., and Abate, C. (1994) Mol. Cell. Biol. 14, 4532-4545[Abstract]
  10. Shang, Z., Ebright, Y. M., Iler, N., Shannon Pendergrast, P., Echelard, Y., McMahon, A. P., Ebright, R. H., and Abate, C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 118-122[Abstract]
  11. Chang, C.-P., Shen, W.-F., Rozenfeld, S., Lawrence, H. J., Largman, C., and Clearly, M. (1995) Genes Dev. 9, 663-674[Abstract]
  12. Chang, C.-P., De Vito, I., and Clearly, M. L. (1997) Mol. Cell. Biol. 17, 81-88[Abstract]
  13. Shen, W.-F., Montgomery, J. C., Rozenfeld, S., Moskow, J. J., Jeffrey Lawrence, H., Buchberg, A. M., and Largman, C. (1997) Mol. Cell. Biol. 17, 6448-6458[Abstract]
  14. Simeone, A., Mavilio, F., Acampora, D., Giampaolo, A., Paiella, A., Zappavigna, V., D'Esposito, M., Pannese, M., Russo, G., Boncinelli, E., and Peschle, C. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4914-4918[Abstract]
  15. Shen, W.-f., Largman, C., Lowney, P., Corral, J. C., Detmer, K., Hauser, C. A., Simonitch, T. A., Hack, F. M., and Lawrence, H. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8536-8540[Abstract]
  16. Deguchi, Y., Moroney, J. F., and Kehrl, J. (1991) Blood 78, 445-450[Abstract]
  17. Lill, M. C., Fuller, J. F., Herzig, R., Crooks, G. M., and Gasson, J. C. (1995) Blood 85, 692-697[Abstract/Free Full Text]
  18. Baier, L. J., Hannibal, M. C., Hanley, E. W., and Nabel, G. J. (1991) Blood 78, 1047-1055[Abstract]
  19. Corsetti, M. T., Briata, P., Sanseverino, L., Daga, A., Airoldi, I., Simeone, A., Palmisano, G., Angelini, C., Boncinelli, E., and Corte, G. (1992) Nucleic Acids Res. 20, 4465-4472[Abstract]
  20. Chariot, A., Senterre-Lesenfants, S., Sobel, M. E., and Castronovo, V. (1998) J. Cell. Biochem. 71, 46-54[CrossRef][Medline] [Order article via Infotrieve]
  21. Geisler, R., Bergmann, A., Hiromi, Y., and Nusslein-Volhard, C. (1992) Cell 71, 613-621[Medline] [Order article via Infotrieve]
  22. Siebenlist, U., Franzoso, F., and Brown, K. (1994) Annu. Rev. Cell Biol. 10, 405-455[CrossRef]
  23. Baldwin, A. S. (1996) Annu. Rev. Immunol. 14, 649-681[CrossRef][Medline] [Order article via Infotrieve]
  24. Ghosh, S., May, M. J., and Kopp, E. B. (1998) Annu. Rev. Immunol. 16, 225-260[CrossRef][Medline] [Order article via Infotrieve]
  25. Beg, A. A., Finco, T., Nantermet, P., and Baldwin, A. (1993) Mol. Cell. Biol. 13, 3301-3310[Abstract]
  26. Baeuerle, P. A., and Henkel, T. (1994) Annu. Rev. Immunol. 12, 141-179[CrossRef][Medline] [Order article via Infotrieve]
  27. Baeuerle, P. A., and Baltimore, D. (1988) Science 242, 540-546[Medline] [Order article via Infotrieve]
  28. Haskill, S., Beg, A. A., Tompkins, S. M., Morris, J. S., Yurochko, A. D., Sampson-Johanne, A., Mondal, K., Ralph, P., and Baldwin, A. S., Jr. (1991) Cell 65, 1281-1289[Medline] [Order article via Infotrieve]
  29. Thompson, J. E., Phillips, R. J., Erdjument-Bromage, H., Tempst, P., and Ghosh, S. (1995) Cell 80, 573-582[Medline] [Order article via Infotrieve]
  30. Whiteside, S. T., Epinat, J.-C., Rice, N. R., and Israel, A. (1997) EMBO J. 16, 1413-1426[Abstract/Free Full Text]
  31. Zabel, U., Henkel, T., Silva, M. D. S., and Baeuerle, P. (1993) EMBO J. 12, 201-211[Abstract]
  32. Cressman, D. E., and Taub, R. (1993) Oncogene 8, 2567-2573[Medline] [Order article via Infotrieve]
  33. Sachdev, S., Hoffmann, A., and Hannink, M. (1998) Mol. Cell. Biol. 18, 2524-2534[Abstract/Free Full Text]
  34. Arenzana-Seisdedos, F., Thompson, J., Rodriguez, M. S., Bachelerie, F., Thomas, D., and Hay, R. T. (1995) Mol. Cell. Biol. 15, 2689-2696[Abstract]
  35. Morin, P. J., and Gilmore, T. D. (1992) Nucleic Acids Res. 20, 2452-2458
  36. Morin, P. J., Subramanian, G. S., and Gilmore, T. D. (1993) Nucleic Acids Res. 21, 2157-2163[Abstract]
  37. Bours, V., Franzoso, G., Azarenko, V., Park, S., Kanno, T., Brown, K., and Siebenlist, U. (1993) Cell 72, 729-739[Medline] [Order article via Infotrieve]
  38. Brown, K., Park, S., Kanno, T., Franzoso, G., and Siebenlist, U. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2532-2536[Abstract]
  39. Brown, K., Franzoso, G., Baldi, L., Carlson, L., Mills, L., Lin, Y.-C., Gerstberger, S., and Siebenlist, U. (1997) Mol. Cell. Biol. 17, 3021-3027[Abstract]
  40. van de Stolpe, A., Caldenhoven, E., Stade, B. G., Koenderman, L., Raaijmakers, J. A. M., Johnson, J. P., and van der Saag, P. T. (1994) J. Biol. Chem. 269, 6185-6192[Abstract/Free Full Text]
  41. Chariot, A., Castronovo, V., Le, P., Gillet, C., Sobel, M. E., and Gielen, J. (1996) Biochem. J. 319, 91-97[Medline] [Order article via Infotrieve]
  42. Zappavigna, V., Sartori, D., and Mavilio, F. (1994) Genes Dev. 8, 732-744[Abstract]
  43. Na, S.-Y., Kim, H.-J., Lee, S.-K., Choi, H.-S., Na, D.-S., Lee, M.-O., Chung, M., Moore, D. D., and Lee, J. W. (1998) J. Biol. Chem. 273, 3212-3215[Abstract/Free Full Text]
  44. Fujita, T., Nolan, G. P., Liou, H.-C., Scott, M. L., and Baltimore, D. (1993) Genes Dev. 7, 1354-1363[Abstract]
  45. Na, S.-Y., Choi, H.-S., Kim, J. W., Na, D S., and Lee, J. W. (1998) J. Biol. Chem. 273, 30933-30938[Abstract/Free Full Text]
  46. Luque, I., and Gélinas, C. (1998) Mol. Cell. Biol. 18, 1213-1224[Abstract/Free Full Text]
  47. Batchelor, A. H., Piper, D. E., de la Brousse, F. C., McKnight, S. L., and Wolberger, C. (1998) Science 279, 1037-1041[Abstract/Free Full Text]
  48. Bushdid, P. B., Brantley, D. M., Yull, F. E., Blaeuer, G. L., Hoffman, L. H., Niswander, L., and Kerr, L. D. (1998) Nature 392, 615-618[CrossRef][Medline] [Order article via Infotrieve]
  49. Kanegae, Y., Tavares, A. T., Izpisùa Belmonte, J. C., and Verma, I. M. (1998) Nature 392, 611-614[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.