The Nuclear Protein IκBζ Forms a Transcriptionally Active Complex with Nuclear Factor-κB (NF-κB) p50 and the Lcn2 Promoter via the N- and C-terminal Ankyrin Repeat Motifs*

The nuclear protein IκBζ, comprising the N-terminal trans-activation domain and the C-terminal ankyrin repeat (ANK) domain composed of seven ANK motifs, activates transcription of a subset of nuclear factor-κB (NF-κB)-dependent innate immune genes such as Lcn2 encoding the antibacterial protein lipocalin-2. Lcn2 activation requires formation of a complex containing IκBζ and NF-κB p50, a transcription factor that harbors the DNA-binding Rel homology region but lacks a trans-activation domain, on the promoter with the canonical NF-κB-binding site (κB site) and its downstream cytosine-rich element. Here we show that IκBζ productively interacts with p50 via Asp-451 in the N terminus of ANK1, a residue that is evolutionarily conserved among IκBζ and the related nuclear IκB proteins Bcl-3 and IκBNS. Threonine substitution for Asp-451 abrogates direct association with the κB-site-binding protein p50, complex formation with the Lcn2 promoter DNA, and activation of Lcn2 transcription. The basic residues Lys-717 and Lys-719 in the C-terminal region of ANK7 contribute to IκBζ binding to the Lcn2 promoter, probably via interaction with the cytosine-rich element required for Lcn2 activation; glutamate substitution for both lysines results in a loss of transcriptionally active complex formation without affecting direct contact of IκBζ with p50. Both termini of the ANK domain in Bcl-3 and IκBNS function in a manner similar to that of IκBζ to interact with promoter DNA, indicating a common mechanism in which the nuclear IκBs form a regulatory complex with NF-κB and promoter DNA via the invariant aspartate in ANK1 and the conserved basic residues in ANK7.

Nuclear factor-B (NF-B) 2 plays central roles in host defense and inflammation as a homo-or heterodimer of NF-B/Rel family proteins by controlling the expression of genes for pro-inflammatory cytokines, chemokines, and antibacterial proteins (1)(2)(3)(4). The mammalian NF-B family is com-posed of five structurally related polypeptides: p50, p52, p105 (the precursor of p50), p100 (the precursor of p52), p65 (also known as RelA), RelB, and c-Rel. They share the Rel homology region, which mediates dimerization, nuclear translocation, binding to specific DNA sequences known as NF-B-binding elements (B sites), and association with one of the IB family proteins (1)(2)(3)(4). Among the members of the family, p65, RelB, and c-Rel have an ability to activate transcription by themselves via the C-terminal trans-activation domain, which is absent in the smaller p50 and p52 proteins. In resting cells, NF-B dimers are retained in the cytoplasm by associating with a member of the prototypical/cytoplasmic IB proteins including IB␣, IB␤, and IB⑀ (1)(2)(3)(4). Cell activation with appropriate stimuli such as bacterial LPS leads to phosphorylation-induced degradation of cytoplasmic IBs and resultant liberation of NF-B dimers (1,5,6). The released NF-B dimers subsequently translocate to the nucleus and thus induce the expression of primary response genes via binding to B sites on their promoter/enhancer regions (1)(2)(3)(4).
The primarily induced gene products include the atypical/ nuclear IB proteins Bcl-3 (7,8), IB (also known as MAIL or INAP) (9 -11), and IB NS (12), which are not expressed in resting cells. In contrast to the cytoplasmic IBs, which preferentially associate with NF-B dimers that possess at least one p65 or c-Rel subunit such as the p50-p65 heterodimer (13), the nuclear IBs prefer the p50 (or p52) homodimer as a partner to directly regulate the activation of secondary response genes for appropriate host defense responses (14 -19). Thus, a part of the secondary response genes is controlled by NF-B-dependent induction of nuclear IBs and their subsequent association with NF-B. For instance, the secondarily induced genes Lcn2 (encoding the antibacterial protein lipocalin-2) and Ptx3 (encoding the antibacterial protein pentraxin 3) are activated by the nuclear IB protein IB (20 -24), which serves as a key regulator in the immune system (16,18,25,26). On the other hand, Bcl-3 participates in transcriptional control of the chemokine-encoding genes Ccl2 for monocyte chemoattractant protein-1 (MCP-1) and Cxcl10 for interferon-␥-induced protein 10 (IP-10) (27), and IB NS regulates transcription of Il6 (encoding the pro-inflammatory cytokine interleukin-6) and Il12b (encoding interleukin-12 subunit p40) (28,29).
The IB proteins are characterized by the presence of the ankyrin repeat domain (ARD). The ARD of the cytoplasmic or nuclear IBs contains six or seven ankyrin repeat (ANK) motifs, respectively. The ANK is an evolutionarily conserved protein motif of about 33 amino acid residues that forms an L-shaped structure comprising a ␤ hairpin and two antiparallel ␣ helices. Consecutive ANK motifs generally stack together to serve as an underlying architecture of a modular specific protein-interacting interface (30 -32). The ARD of IBs mediates the association with NF-B dimers via direct binding to the NF-B Rel homology region (23,33,34). Although the ARD of nuclear IBs displays a high sequence similarity to that of cytoplasmic IBs except for the additional seventh motif ANK7 (10,12,35,36), the preference for NF-B dimer species differs between the nuclear and cytoplasmic IBs, as described above (7-9, 12, 16, 37). Furthermore, there exists a difference in interaction with B DNA. Although IB␣ promotes the dissociation of a p65containing dimer from the promoter DNA (38), the nuclear IBs are generally assumed to interact indirectly with the B site via binding to NF-B p50 or p52 (7,8,23,33,34). In addition, we have recently shown that IB interaction with the Lcn2 promoter also requires a region downstream of the B site (23). However, the molecular mechanism underlying assembly of the nuclear IB-containing regulatory complex has not been well elucidated.
In the present study, we show that IB, containing the N-terminal trans-activation domain and the C-terminal ARD, forms a transcriptionally active complex with the Lcn2 promoter via both Asp-451-mediated association with the B-sitebinding protein p50/p52 and Lys-717/Lys-719-dependent interaction with the downstream extra B site on the Lcn2 promoter. Asp-451, an invariant residue among the nuclear IBs, is present in the N terminus of ANK1; Lys-717 and Lys-719 exist in the region C-terminal to the second ␣ helix of ANK7, and the corresponding sites are also occupied by basic residues in Bcl-3 and IB NS . Both termini of the ARD in these proteins serve in a manner similar to that of IB, indicative of a common mechanism by which nuclear IBs form a p50/p52-containing regulatory complex on target gene promoters.

Results
The Invariant Aspartate in ANK1 of IB and Other Nuclear IBs Is Crucial for Interaction with NF-B-It is known that the ARD of the nuclear IB proteins IB, IB NS , and Bcl-3 ( Fig.  1A) is responsible for binding to NF-B p50 (33,34). To determine the N-terminal boundary of the IB region required for interaction with p50, we expressed and purified a series of N-terminally truncated IB as GST-fused proteins and tested their ability to bind to p50. As shown in Fig. 1B, IB-(449 -728) fully interacted with p50, as did IB-(414 -728). On the other hand, IB-(453-728) failed to bind to p50 (Fig. 1B), suggesting a role for amino acid residues 449 -452. Among the four residues, Asp-451 in ANK1 is the only one that is completely conserved during evolution (Fig. 1A). Intriguingly, the aspartate also exists in the other nuclear IB proteins IB NS and Bcl-3 (Asp-60 and Asp-127, respectively) but is replaced by threonine or serine in cytoplasmic IB members (Fig. 1A). Consistent with the conservation, the replacement of Asp-451 by threonine impaired IB binding to p50 in a GST pulldown assay (Fig. 1C). The D451T substitution in IB also resulted in a loss of its co-immunoprecipitation with p50 when FLAG-tagged full-length IB and HA-p50 were expressed in HEK293T cells (Fig. 1D). Thus, the invariant residue Asp-451 in ANK1 plays a crucial role in IB interaction with p50.
We next investigated the role of the corresponding aspartate residue in other nuclear IB proteins (Asp-60 in IB NS and Asp-127 in Bcl-3). Threonine substitution for Asp-60 in IB NS led to an impaired interaction with p50 both in a GST pulldown assay using purified proteins (Fig. 1E) and in a co-immunoprecipitation assay using proteins expressed in HEK293T cells (Fig.  1F). Similarly, compared with wild-type Bcl-3, a mutant protein with threonine substitution for Asp-127 interacted with p50 much less efficiently both in vitro (Fig. 1E) and in vivo (Fig. 1G). These findings highlight a conservative role for the invariant aspartate of ANK1 in direct interaction of nuclear IBs with p50.
Nuclear IB proteins are also known to form a complex with NF-B p52, a protein homologous to p50 (7,12,37). As expected from the homology, in vitro complex formation with p52 was impaired by threonine substitution for the invariant aspartate in ANK1 of nuclear IBs: Asp-451 in IB, Asp-60 in IB NS , and Asp-127 in Bcl-3 ( Fig. 2A). The critical role for the aspartates was confirmed by co-precipitation of p52 with IB ( Fig. 2B), IB NS (Fig. 2C), and Bcl-3 ( Fig. 2D) when ectopically expressed in HEK293T cells. Thus, p52 likely interacts with nuclear IB proteins in a manner similar to the way p50 does.
Asp-451 of IB Is Involved in Transcriptional Activation-As we have shown previously (23), expression of IB along with p50 results in transcriptional activation of the promoter of the lipocalin-2-encoding gene Lcn2 in p50-/IB-deficient mouse embryonic fibroblasts (MEFs) (Nfkb1 Ϫ/Ϫ ;Nfkbiz Ϫ/Ϫ MEFs) (Fig. 3A). To examine the role for Asp-451 of IB in p50-dependent Lcn2 activation, we used p50-/IB-deficient MEFs in which a luciferase reporter is regulated by the Lcn2 promoter (23). As shown in Fig. 3A, IB (D451T), defective in associating with p50, activated the Lcn2 promoter much less effectively than the wild-type protein even in the presence of p50. We next tested the role for Asp-451 of ANK1 in IBmediated activation of the endogenous Lcn2 gene. As shown in Fig. 3B, exogenous expression of wild-type IB in IB-deficient bone marrow-derived macrophages (BMMs) resulted in time-dependent activation of the endogenous Lcn2 gene in response to LPS. On the other hand, IB (D451T), a mutant protein defective in direct interaction with NF-B p50 (Fig. 1), failed to activate the endogenous Lcn2 gene, although IB (D451T) was expressed at a level similar to that of the wild-type protein (Fig. 3B). Thus, the interaction between IB and p50 appears to be involved in transcriptional activation of Lcn2.
We also investigated the function of Bcl-3 Asp-127, a residue that corresponds to Asp-451 in IB and is crucial for binding to p50 and p52 (Figs. 1 and 2), in gene activation. As shown in Fig. 3C, Bcl-3 (D127T) activated a reporter of the Ccl2 promoter in LPS-stimulated RAW264.7 cells that ectopically expressed p52 but to a significantly lesser extent than the wildtype protein. Thus, Bcl-3 binding to p52 plays a role in Ccl2 activation. On the other hand, IB did not activate Lcn2 in combination with p52 under conditions where p50 fully supported IB (Fig. 3D). This raises the possibility that p52 is incapable of directly interacting with the Lcn2 promoter DNA, although p52 binds to IB as p50 does (Fig. 2B). To address this question, we analyzed the formation of the protein-promoter complex using a DNA-binding assay in which DNA bound to a tagged protein is pulled down with tag affinity beads and amplified by PCR (for details, see "Experimental Procedures"). As shown in Fig. 3E, the Lcn2 promoter efficiently interacted with wild-type p50 but not with p50 (Y57A/E60D), a mutant protein defective in binding to the B site (23), or p65. In contrast to p50, wild-type p52 failed to directly bind to the Lcn2 promoter (Fig. 3E). Thus, IB appears to activate Lcn2 by specifically interacting with p50.
Lys-717 and Lys-719 in ANK7 of IB Participate in Lcn2 Activation-We next studied the role for the C-terminal region of the IB ARD, which comprises seven ANK repeats (Fig. 4A).
For this purpose, we expressed a series of C-terminally truncated IB proteins in Nfkb1 Ϫ/Ϫ ;Nfkbiz Ϫ/Ϫ MEFs to test their ability to activate the Lcn2 promoter. As shown in Fig. 4B, IB-(1-721) was as active as full-length IB of 728 amino acids. By contrast, Lcn2 was activated much more weakly by IB-(1-718) and only marginally by IB-(1-716) (Fig. 4B), indicating a crucial role for the C-terminal region of IB ANK7 (amino acids 717-721) in Lcn2 activation. On the other hand, this region was dispensable for direct contact of IB with p50, as estimated by the GST pulldown assay (Fig. 4C). The dispensability is in contrast with the involvement of IB ANK1 in direct interaction with p50 ( Fig. 1) as well as in Lcn2 activation (Fig. 3). The IB C-terminal region crucial for Lcn2 activation contains the basic residues Lys-717 and Lys-719, both of which are evolutionarily well conserved (Fig. 4A). The Lcn2 promoter was activated by a mutant IB carrying substitution of the acidic residue glutamate for Lys-717, but to a lesser extent than the wild-type protein (Fig. 4D), and double glutamate substitution for Lys-717 and Lys-719 led to an almost complete loss of Lcn2 activation (Fig. 4D) without affecting the ability to directly interact with p50 ( Fig. 4E). Furthermore, LPS-induced activation of the endogenous Lcn2 gene was not observed in BMMs expressing a mutant IB with the K717E/K719E substitution (Fig. 3B). In contrast, simultaneous replacement of Lys-717 and Lys-719 by the other basic residue arginine hardly affected Lcn2 activation (Fig. 4F). These observations imply a possible role for the positive charge at amino acid positions 717 and 719 in IB ANK7.
Lys-717 and Lys-719 in ANK7 of IB Participate in Complex Formation with the Lcn2 Promoter and Not via p50 -It should be noted that the basic residues lysine and arginine are both capable of not only forming a salt bridge with a phosphate group of DNA but also of directly interacting with a base of DNA, especially guanine (39,40). Furthermore, we have shown recently that a cytosine-rich region downstream of the B site is involved in formation of the active three-species complex IB-p50-DNA (23), suggesting that the extra-B site makes a direct contact with p50, IB, or both. It seems thus possible that the arginine-replaceable residues Lys-717 and Lys-719 in ANK7 participate in IB interaction with the Lcn2 promoter DNA. To test this possibility, we analyzed the in vitro formation of the IB-p50-DNA complex using the method used in Fig. 3E.
Under conditions where the Lcn2 promoter fragment was fully co-precipitated with wild-type IB, the co-precipitation was not caused by IB (D451T), defective in direct binding to p50 (Fig. 5A). The D451T substitution also disrupted IB interaction with the endogenous promoter of Lcn2, as shown by ChIP analysis using cells that stably expressed the wild-type or mutant protein (Fig. 5B). Further analysis with the anti-RNA polymerase II antibody revealed that the impairment of IB-p50 association resulted in loss of the in vivo formation of a transcriptionally active complex on the Lcn2 promoter (Fig.  5B). The requirement of IB-p50 association for interaction with the promoter is in good agreement with the following observation. Both p50 protein and the p50-binding site on the promoter (the B site) were required for IB interaction with DNA ( Fig. 5A). Thus, Asp-451 indirectly participates in complex formation with the Lcn2 promoter via direct binding to p50. On the other hand, Lys-717 and Lys-719 in IB may directly interact with target DNA because the K717E/K719E substitution abolished not only the recruitment to the Lcn2 endogenous promoter for subsequent active complex formation ( Fig. 5B) but also the in vitro interaction with the target DNA without affecting binding to p50 (Fig. 5A). As indicated from the finding that arginine can replace Lys-717 and Lys-719 in Lcn2 activation (Fig. 4F), the replacement did not affect active complex formation with the Lcn2 promoter (Fig. 5D). These findings indicate that IB interacts with the Lcn2 promoter via the arginine-replaceable residues Lys-717 and Lys-719 in a manner independent of direct binding to p50. Furthermore, proline substitution for the evolutionarily well conserved residue Gly-718, which locates between Lys-717 and Lys-719 in IB ANK7 (Fig. 4A), resulted in a loss of both interaction with the Lcn2 promoter ( Fig. 5A) and Lcn2 activation (Fig. 5C). The observation suggests that correct orientation of Lys-717 and Lys-719 toward the target DNA may be required for association of IB to the Lcn2 promoter. On the other hand, neither S720A nor I721S substitution affected Lcn2 activation (Fig. 5C).
Acetylation of IB Does Not Seem to Be Involved in Lcn2 Activation-The significance of Lys-717 and Lys-719 of IB in Lcn2 activation may suggest the involvement of posttransla-tional modification of these lysine residues, such as acetylation.
To test this possibility, we treated cells with the potent histone acetyltransferase inhibitor anacardic acid (41,42) and nicotinamide, an agent that inhibits the NAD ϩ -dependent class III (sirtuin) family of histone deacetylases (HDACs) (43,44). Lcn2 activation by IB remained unaffected by these inhibitors (Fig.  6, A and B). Under conditions where histone acetylation was significantly enhanced by the presence of trichostatin A (TSA), an inhibitor of class I/II HDAC (43,44), lysine residues in IB were not acetylated (Fig. 6C). Furthermore, a mobility shift of IB on an SDS-PAGE gel, which was expected to occur by posttranslational modification, was not induced by TSA (Fig.   FIGURE 3. Asp-451 of IB and Asp-127 of Bcl-3 participate in transcriptional activation. A and D, the role of IB Asp-451 in Lcn2 activation. p50-/IBdeficient MEFs were transfected with the following plasmids: the luciferase reporter plasmid pGL3-Basic containing the upstream region of Lcn2 (Ϫ1031/ϩ54), the internal control plasmid pRL-TK, pcDNA3 for expression of FLAG-IB (WT) or FLAG-IB (D451T), and HA-p50 (A) or HA-p52 (D). Luciferase activities were determined as described under "Experimental Procedures." Each graph represents the mean Ϯ S.D. obtained from three independent transfections. Cell lysates were analyzed by immunoblot with anti-FLAG, anti-HA, or anti-␤-tubulin antibody. MW, molecular weight. B, IB-mediated activation of the endogenous Lcn2 gene. IB-deficient BMMs were retrovirally transduced for expression of WT IB or a mutant protein with the D451T or K717E/K719E substitution. Proteins in the cell lysate were analyzed by immunoblot with the anti-IB or anti-␤-tubulin antibody (left panel). The transduced BMMs were stimulated for the indicated time with LPS, and the relative amounts of mRNA transcribed from the endogenous Lcn2 gene were estimated by quantitative real-time RT-PCR as described under "Experimental Procedures" (right panel). Each graph represents the mean Ϯ S.D. in triplicate determinations. C, the role of Bcl-3 Asp-127 in Ccl2 activation. RAW264.7 cells were transfected with the following plasmids: the luciferase reporter plasmid pGL3-Basic containing the upstream region of Ccl2 (Ϫ2777/ϩ76), the internal control plasmid pRL-TK, and pcDNA3 for expression of FLAG-Bcl-3 (WT) or FLAG-Bcl-3 (D127T) and HA-p52. Luciferase activities were determined as described under "Experimental Procedures." Each graph represents the mean Ϯ S.D. obtained from three independent transfections. Cell lysates were analyzed by immunoblot with anti-FLAG, anti-HA, or anti-␤-actin antibody. E, association of p50 with the Lcn2 gene promoter. His-tagged p50 (WT), p52 (WT), p65 (WT), or p50 (Y57A/E60D) was incubated with the Lcn2 gene promoter (Ϫ317/Ϫ117). After the protein-DNA complex was pulled down with COSMOGELா His-Accept, the co-precipitated DNA was amplified by PCR, and the product was analyzed by agarose gel electrophoresis. The precipitated proteins were subjected to immunoblot analysis with anti-His antibody. Positions for marker proteins are indicated in kilodaltons. 6C). These observations suggest that acetylation of Lys-717 or Lys-719 of IB does not participate in Lcn2 activation, which also appears to be supported by the finding that neither gene activation nor promoter association are prevented by replacement of the lysines with arginine, a residue that does not undergo acetylation (Figs. 4F and 5D).
Basic Residues in ANK7 of IB NS and Bcl-3 Are Involved in Association with Target DNA-Lys-717 and Lys-719 of IB are predicted to follow the second ␣ helix in ANK7, and basic residues also exist at the corresponding sites in ANK7 of IB NS and Bcl-3 (Fig. 7A). As shown in Fig. 7B, association of IB NS with the promoter of the IB NS -regulated gene Il6 was prevented by simultaneous glutamate substitution for Lys-316, Arg-317, and Arg-319 in ANK7, although the substitution did not affect IB NS binding to p50. Similarly, glutamate substitution for Arg-354 and Lys-356 in ANK7 of Bcl-3 resulted in a loss of complex formation with the promoter of the Bcl-3-dependent gene Cxcl10 without affecting Bcl-3 interaction with p50 (Fig. 7C), and Bcl-3 (R354E/K356E) also failed to associate with the endogenous promoter of Cxcl10, as indicated by ChIP analysis (Fig. 7D). Thus, in IB NS and Bcl-3, basic residues that follow the second ␣ helix in ANK7 appear to be involved in recognition of their target gene promoters. Furthermore, the R354E/ K356E substitution in Bcl-3 resulted in loss of the p52-dependent Ccl2 activation in LPS-stimulated RAW264.7 cells (Fig.  7E), confirming the significance of the basic residues in ANK7. The DNA-binding assays also revealed that the invariant aspartate residues in ANK1 of IB NS (Asp-60) (Fig. 7B) and Bcl-3 (Asp-127) (Fig. 7, C and D) participate in association with their target gene promoters via direct binding to p50, similar to the corresponding residue of IB (Asp-451) (Fig. 5A).
Activation of Lcn2 Involves Promoter Association with IB via an Extra-B Site in a Sequence-specific Manner-Activation of the mouse Lcn2 gene requires promoter association with the IB-p50 complex via both the B site (5Ј-GGGAAT-GTCCC-3Ј at positions Ϫ230 to Ϫ220 relative to the transcription start site) and its downstream region of 5Ј-CCCCTC-3Ј at positions Ϫ212 to Ϫ207 (23) (see Fig. 8A). To elucidate base specificity in the downstream sequence, we constructed a series of mutant Lcn2 promoters and tested their ability. Substitution of either guanine or adenine for cytosine at position Ϫ212 led to a loss of both interaction with IB and IB-mediated Lcn2 activation (Fig. 8B). On the other hand, they were only marginally impaired by thymine replacement at the corresponding position (Fig. 8B). Adenine but not thymine or guanine partially replaced cytosine at position Ϫ211 (Fig. 8C). At positions Ϫ210 (Fig. 8D) and Ϫ209 (Fig. 8E), cytosine was strictly required for both IB binding to the Lcn2 promoter and IB-mediated Lcn2 activation. By contrast, any of four bases fully functioned at positions Ϫ208 (Fig. 8F) and Ϫ207 (Fig. 8G). In the sequence CCCCTC at positions Ϫ212 to Ϫ207 of the mouse Lcn2 promoter, the preference for cytosine at positions Ϫ212 to Ϫ209 but not at position Ϫ208 or Ϫ207 (Fig. 8, B-G) appears to be consistent with the conservation of the first four cytosines but not the last two bases in the corresponding region from other mammals (Fig. 8A). These mutational analyses indicate that the (Ϫ317/Ϫ117). After the protein-DNA complex was pulled down with glutathione-Sepharose-4B beads, the co-precipitated DNA was amplified by PCR, and the product was analyzed by agarose gel electrophoresis. The precipitated proteins were subjected to SDS-PAGE, followed by staining with CBB or immunoblot with anti-His antibody. MW, molecular weight. B, formaldehyde-fixed chromatin was prepared from RAW264.7 cells stably expressing FLAG-IB (WT), FLAG-IB (K717E/K719E), or FLAG-IB (D451T) (top panel) and subjected to ChIP assay using anti-FLAG (M2) mouse monoclonal antibody or anti-RNA polymerase II antibody (bottom panel). Precipitated DNA was analyzed by PCR using primers corresponding to the Lcn2 locus. The results are representative of experiments from at least three independent experiments. IP, immunoprecipitation. C, the role of Gly-718, Ser-720, and Ile-721 in Lcn2 activation. p50-/IB-deficient MEFs were transfected with the following plasmids: the luciferase reporter plasmid pGL3-Basic containing the upstream region of Lcn2 (Ϫ1031/ϩ54), the internal control plasmid pRL-TK, and pcDNA3 for expression of FLAG-IB with the indicated amino acid substitution and HA-p50. Luciferase activities were determined as described under "Experimental Procedures." Each graph represents the mean Ϯ S.D. obtained from three independent transfections. Cell lysates were analyzed by immunoblot with anti-FLAG, anti-HA, or anti-␤-tubulin antibody. Positions for marker proteins are indicated in kilodaltons.
IB-responsive element (positions Ϫ212 to Ϫ209) requires the sequence 5Ј-YCCC-3Ј (Y is pyrimidine). Taken together with the present findings, IB activates the endogenous Lcn2 gene both via Asp-451-dependent direct interaction with p50 and Lys-717/Lys-719-involved association with the sequence 5Ј-YCCC-3Ј downstream of the B site.

Discussion
The nuclear IB proteins IB, Bcl-3, and IB NS are thought to regulate NF-B-dependent transcription by directly interacting with a p50 or p52 homodimer that binds to the B site on target genes. However, the mechanism for formation of the regulatory complex has not been fully elucidated. In the present study, we show that IB, comprising the N-terminal transactivation domain and the C-terminal ARD composed of seven ANK motifs, forms a transcriptionally active complex on its target gene Lcn2 both via Asp-451-mediated binding to p50 and via Lys-717/Lys-719-dependent interaction with the extra-B site of the Lcn2 promoter. Asp-451 is present in the N-terminal region of ANK1, whereas the basic residues Lys-717 and Lys-719 exist in the C-terminal region of ANK7. We also demonstrate similar roles for both termini of the ARD in Bcl-3 and IB NS , proposing a model for a common mechanism by which nuclear IBs form a p50/p52-containing complex on target gene promoters.
Asp-451 in IB ANK1 is strictly conserved during evolution (Fig. 1). Replacement of Asp-451 by threonine abrogates both association with a homodimer of the NF-B subunit p50 (Fig. 1) and activation of Lcn2 via formation of the IB-p50-DNA complex on the promoter (Figs. 3 and 5). The aspartate residue is also conserved among the nuclear IBs, including Bcl-3 and IB NS ; however, it is replaced by threonine or serine in cytoplasmic IBs such as IB␣ and IB␤ (Fig. 1), which associate with a p50/p52 homodimer much less efficiently (13). The conservation among the nuclear IBs is consistent with the present finding that the corresponding aspartate residues (Asp-127 in Bcl-3 and Asp-60 in IB NS ) are also crucial for interaction with a p50 or p52 homodimer ( Figs. 1 and 2). It should be noted that Asp-451 in IB is one of the very few residues that are completely conserved among nuclear IBs but replaced in cytoplasmic IBs. On the other hand, the presence of aspartate at this position by itself does not seem to be sufficient because a mutant IB␣ carrying aspartate substitution for Thr-71 at the corresponding position of ANK1 as well as the wild-type protein fails to bind to a p50 homodimer (data not shown).
Although little is known about the tertiary structure of nuclear IB⅐NF-B complexes, crystal structures of the cytoplasmic IB proteins IB␣ and IB␤ complexed with a p65-p50 heterodimer and a p65 homodimer, respectively, have been solved (45)(46)(47). If IB interacts with NF-B subunits in a manner similar to the cytoplasmic IBs, then it seems likely that Asp-451 in IB is positioned toward the C-terminally localized nuclear localization signal (NLS) in p50, a region that is required not only for nuclear localization of p50 but also for direct interaction of p50 with IB (23,48). Because of the dual role of the p50 NLS-containing region, the requirement of direct p50-IB interaction for gene activation has not been established, although an NLS-truncated p50 protein did not activate IB-dependent transcription, and thus IB was assumed to interact with the promoter DNA via association with p50, which directly binds to the B site (23, 48). The conclusion appears to be strongly supported by the present findings that the D451T substitution in IB abrogates interaction with p50 ( Fig. 1), complex formation on both endogenous and exogenous Lcn2 promoter (Fig. 5), and activation of Lcn2 transcription (Fig. 3).
In addition to direct association with the B-site-binding protein p50, IB activates Lcn2 transcription by interacting with the extra-B site of the Lcn2 promoter in which the basic residues Lys-717 and Lys-719 in the C-terminal region of IB ANK7 likely play a major role (Figs. 4 and 5). The association does not appear to be mediated via direct binding to p50 because the K717E/K719E substitution in IB leads to a loss of both promoter association and Lcn2 activation without affect- ing direct contact of IB with p50 (Figs. 4 and 5). In addition, the function of the lysine residues does not seem to require their modification, such as acetylation (Fig. 6), which is supported by the finding that arginine, a residue insusceptible to acetylation, fully serves in place of them (Figs. 4 and 5).
The extra-B site required for IB-dependent Lcn2 activation localizes seven bases downstream of the B site in the Lcn2 promoter (23) and strictly requires the sequence 5Ј-YCCC-3Ј as an IB-responsive element, as shown in this study (Fig. 8). In both promoter association and Lcn2 activation, Lys-717 and Lys-719 in IB are fully replaced by arginine residues (Figs. 4 and 5). It is known that lysine and arginine, but not glutamate, are often involved in direct interaction with DNA, which can be mediated not only via nonspecific interaction with the phos- FIGURE 7. Basic residues in ANK7 of IB NS and Bcl-3 participate in association with their target gene promoter. A, amino acid sequence alignment of ANK7 in the mouse nuclear IB proteins IB, Bcl-3, and IB NS . The two antiparallel ␣ helices in ANK7 of Bcl-3 were determined in crystal structures of Bcl-3 (36), whereas those of IB and IB NS were predicted from their primary sequences by using the program PSIPRED (45,46). Residues boxed in gray are the basic residues that follow the second ␣ helix of ANK7. B, complex formation of IB NS with p50 and the Il6 promoter. GST-fused WT IB NS or a mutant IB NS with the K316E/R317E/R319E or D60T substitution was incubated with or without His-p50 in the presence of WT Il6 (Ϫ167/Ϫ38) or a mutated B site (Bm)-carrying Il6 (Ϫ167/Ϫ38). After the protein-DNA complex was pulled down with glutathione-Sepharose-4B beads, the co-precipitated DNA was amplified by PCR, and the product was analyzed by agarose gel electrophoresis. The precipitated proteins were subjected to SDS-PAGE, followed by staining with CBB. MW, molecular weight. C, complex formation of Bcl-3 with p50 and the Cxcl10 promoter. MBP-fused WT Bcl-3-(122-362) or a mutant protein with the R354E/K356E or D127T substitution was incubated with or without His-p50 in the presence of Cxcl10 (Ϫ162/Ϫ58) or a mutated B site (Bm)-carrying Cxcl10 (Ϫ162/Ϫ58). After the protein-DNA complex was pulled down with amylose resins, the co-precipitated DNA was analyzed as in B. The precipitated proteins were subjected to SDS-PAGE, followed by staining with CBB or immunoblot with the anti-His antibody. D, formaldehyde-fixed chromatin was prepared from RAW264.7 cells stably expressing FLAG-Bcl-3 (WT), FLAG-Bcl-3 (R354E/K356E), or FLAG-Bcl-3 (D127T) (top panel) and subjected to ChIP assay using anti-FLAG (M2) mouse monoclonal antibody (bottom panel). Precipitated DNA was analyzed by PCR using primers corresponding to the Cxcl10 locus. The results are representative of experiments from at least three independent experiments. IP, immunoprecipitation. E, the role of Arg-354 and Lys-356 of Bcl-3 ANK7 in Ccl2 activation. RAW264.7 cells were transfected with the following plasmids: the luciferase reporter plasmid pGL3-Basic containing the upstream region of Ccl2 (Ϫ2777/ϩ76), the internal control plasmid pRL-TK, and pcDNA3 for expression of FLAG-Bcl-3 (WT) or FLAG-Bcl-3 (R354E/K356E) and HA-p52. Luciferase activities were determined as described under "Experimental Procedures." Each graph represents the mean Ϯ S.D. obtained from three independent transfections. Cell lysates were analyzed by immunoblot with anti-FLAG, anti-HA, or anti-␤-actin antibody. Positions for marker proteins are indicated in kilodaltons.
phate moiety of DNA but also via recognition of a DNA base, especially a guanine (39,40). In this context, it should be noted that the IB-responsive element (5Ј-YCCC-3Ј) of the Lcn2 promoter is abundant in the Lys/Arg-recognizing base guanine in the antisense strand. The presence of multiple guanines in the element may be in agreement with the present conclusion that Lys-717 and Lys-719 each contribute to element recognition because single glutamate substitution for either lysine residue results in only a partial loss of the activity of IB (Fig. 4). Correct orientation of the side chains of Lys-717 and Lys-719 toward target DNA also seems to be important for element recognition, as indicated by the finding that replacement of the flexible residue Gly-718, which is the intervening amino acid between the lysines and is strictly conserved during evolution, by the inflexible residue proline abrogates both binding to the Lcn2 promoter and transcription of Lcn2 (Fig. 5).
The NF-B family proteins serve as a homo-or heterodimer to bind to a B DNA response element in the promoters of distinct inducible genes, thereby playing their respective roles in gene regulation (1)(2)(3)(4). The transcriptional specificity of NF-B dimers is generally thought to be coded within the B site sequences (27,49). Indeed, in the case of the Lcn2 promoter, its B site effectively interacts with the NF-B p50 homodimer but not with the p52 homodimer (Fig. 3), although both homodimers are capable of binding to IB (Fig. 2). As a result, in contrast to p50, p52 fails to induce IB-dependent activation of the Lcn2 gene (Fig. 3). In addition to the B site sequence itself, the ability of NF-B dimers to function on a particular promoter is also considered to be determined by extra-B sites recognized via co-activator proteins (16,50). However, such a co-activator was not previously identified. This study demonstrates that IB, a co-activator of p50, activates the Lcn2 gene not only via binding to p50 but also by associating with the IB-responsive element (5Ј-YCCC-3Ј) downstream of the B site, providing evidence that an extra-B site and its interacting co-activator do determine the transcriptional specificity of NF-B dimers.
Bcl-3 and IB NS are the putative second and third examples that determine the transcriptional specificity of NF-B dimers via association with extra-B sites. These nuclear IB proteins, capable of binding to a p50 and p52 homodimer ( Figs. 1 and 2), have conserved basic residues that follow the second ␣ helix in the C-terminal region of ANK7; the residues appear to be involved in association of Bcl-3 and IB NS with promoters of their target genes (Fig. 7). The crucial role for Arg-354 and Lys-356 of Bcl-3 ANK7 in promoter binding and gene activation agrees well with a model in which the C-terminal region of Bcl-3 ARD in complex with the p50 homodimer is placed near the promoter DNA (36). Future studies should aim to elucidate a Bcl-3-or IB NS -interacting cis element out of B sites in Bcl-3-or IB NS -dependent gene promoters, respectively.
Combined with the present observation that the aspartate residue in the N terminus of the ARD of Bcl-3 and IB NS as well as that of IB plays a crucial role in direct interaction with a p50 or p52 homodimer (Figs. 1 and 2), we propose a common mechanism for nuclear-IB-mediated regulation of NF-B-dependent genes. The nuclear IB proteins with seven ANK motifs (IB, Bcl-3, and IB NS ) form a p50/p52-containing regulatory complex on promoter DNA via the following two interactions: direct association with the B-site-binding protein p50/p52 via the invariant aspartate residue in the N-terminal . For estimation of Lcn2 activation, p50-/IB-deficient MEFs were transfected with the following plasmids: the luciferase reporter plasmid pGL3-Basic containing the upstream region of wild-type Lcn2 (Ϫ500/ϩ50); a mutant Lcn2 with the indicated base replacement, or the IB-independent gene Sele (Ϫ445/ϩ105); the internal control plasmid pRL-TK; and pcDNA3 for expression of FLAG-IB and HA-p50. Luciferase activities were determined as described under "Experimental Procedures." Each graph represents the mean Ϯ S.D. obtained from three independent transfections. For estimation of protein-DNA complex formation, GST-IB was incubated with His-p50 in the presence of the DNA fragment of wild-type Lcn2 (Ϫ500/ϩ50), a mutant Lcn2 with the indicated base replacement, or Sele (Ϫ445/ϩ105). After the complex was pulled down with glutathione-Sepharose-4B beads, the co-precipitated DNA was amplified by PCR, and the product was analyzed by agarose gel electrophoresis. region of ANK1 and interaction with an extra-B site via recognition by the conserved lysine/arginine residues that locate C-terminally to the second ␣ helix of ANK7.

Experimental Procedures
Cells, Antibodies, and Reagents-MEFs doubly deficient in NF-B p50 and IB (Nfkb1 Ϫ/Ϫ ;Nfkbiz Ϫ/Ϫ MEFs) were prepared as described previously (23). Mouse BMMs were obtained as described previously (22,24). All animals were housed and maintained in a specific pathogen-free animal facility at Kyushu University. All experiments were performed in strict accordance with the guidelines for proper conduct of animal experiments (Science Council of Japan). The experimental protocol was approved by the Animal Care and Use Committee of Kyushu University (permit numbers: A24-042 and A26-102). All efforts were made to minimize the numbers of animals and their suffering. MEFs, BMMs, HEK293T cells, and mouse macrophage-like RAW 264.7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 g/ml) at 37°C under 5% CO 2 .
GST Pulldown Assay-GST-and His-tagged proteins were purified as described previously (52,53) and incubated for 20 min at 4°C in 500 l of buffer A (137 mM NaCl, 2.7 mM KCl, 0.5% Triton X-100, 1 mM DTT, 8.1 mM Na 2 HPO 4 , and 1.5 mM KH 2 PO 4 (pH 7.4)). A slurry of glutathione-Sepharose 4B beads (GE Healthcare) was subsequently added, followed by further incubation for 40 min at 4°C. After washing three times with the buffer above, the proteins were eluted from the beads with 20 mM glutathione in 150 mM NaCl, 2 mM DTT, and 100 mM Tris-HCl (pH 8.8). The eluate was subjected to SDS-PAGE, followed by staining with Coomassie Brilliant Blue (CBB).
Immunoprecipitation Analysis-HEK293T cells were transfected with the indicated expression plasmids using X-treme-GENE HP DNA Transfection Reagent (Roche Applied Science) and cultured for 24 or 48 h. Cells were lysed by sonication at 4°C in 500 l of lysis buffer (137 mM NaCl, 2.7 mM KCl, 1% Nonidet P-40, 8.1 mM Na 2 HPO 4 , and 1.5 mM KH 2 PO 4 (pH 7.4)) supplemented with Complete TM Protease Inhibitor Cocktail (Roche Applied Science). Proteins in the cell lysate were immunoprecipitated using anti-FLAG antibody (M2) and protein G-Sepharose (GE Healthcare). The precipitants were analyzed by immunoblot with the anti-FLAG rabbit polyclonal antibody or the anti-HA (3F10) rat monoclonal antibody. The blots were developed using ECL-Prime (GE Healthcare) for visualization of the antibodies.
Detection of Acetylated Proteins-FLAG-IB expressed in HEK293T cells was incubated for 6 h with 2 M TSA and immunoprecipitated from the cell lysates as described above, with the exception that 2 M TSA and 20 mM nicotinamide were used during immunoprecipitation to prevent deacetylation. The precipitated proteins were applied to immunoblot analysis with anti-acetylated lysine rabbit polyclonal antibody. Histones were prepared from HEK293T cells by acid extraction as described by Nightingale et al. (54). For detection of acetylated histones, the acid extracts were subsequently analyzed by immunoblot with the antiacetylated lysine rabbit polyclonal antibody.
Luciferase Reporter Assay-Nfkb1 Ϫ/Ϫ ;Nfkbiz Ϫ/Ϫ MEFs or RAW264.7 cells were transfected with the luciferase reporter plasmid pGL3-Basic containing the indicated promoter, the internal control plasmid pRL-TK (Promega), and the indicated plasmids for protein expression using X-tremeGENE HP DNA Transfection Reagent. The luciferase activities were determined by the Dual-Luciferase reporter assay system (Promega). For estimation of protein levels, cell lysates were analyzed by immunoblot with anti-FLAG (M2), anti-HA, anti-␤-tubulin, or anti-␤-actin antibody.
Analysis of Protein-DNA Interaction-Analysis of protein-DNA interaction was performed as described previously (23). GST-IB, GST-IB NS , or MBP-Bcl-3 was incubated with His-p50 for 20 min at 4°C in 500 l of buffer A containing salmon sperm DNA (0.1 mg/ml), or His-p50, His-p52, or His-p65 alone was incubated for 20 min at 4°C in 500 l of buffer B (150 mM NaCl, 5% glycerol, 1 mM DTT, 0.5% Triton X-100, and 25 mM Tris-HCl (pH 8.0)) containing salmon sperm DNA (0.1 mg/ml). A slurry of glutathione-Sepharose 4B, Amylose Resin (New England Biolabs), or COSMOGEL His-Accept (Nacalai Tesque) was added in a GST, MBP, or His pulldown assay, respectively. The indicated DNA fragment (0.1 pmol) was subsequently added to the mixture, followed by further incubation for 40 min at 4°C. After washing with buffer A for a GST or MBP pulldown assay or with buffer B containing 25 mM imidazole for a His pulldown assay, the protein-DNA complex was eluted from glutathione-Sepharose 4B with an elution buffer (20 mM glutathione, 150 mM NaCl, 2 mM DTT, and 100 mM Tris-HCl (pH 8.8)), from amylose resin with buffer A containing 20 mM maltose, or from COSMOGEL His-Accept with buffer B containing 1 M imidazole. The eluted protein was applied to SDS-PAGE, followed by CBB staining or immunoblot analysis with the anti-His 5 monoclonal antibody. The DNA in the eluted complex was analyzed by PCR using the following primer pairs: 5Ј-TACAGGGTTATGGGAGTGGAC-3Ј and 5Ј-TCTGTTGAAATACTTGGCAAGAT-3Ј for detection of the Lcn2 promoter region; 5Ј-CCATGGAAGACGCCAAAA-ACA-3Ј and 5Ј-CATATCGTTTCATAGCTTCTGC-3Ј for the 263-bp region of the pGL3-Basic vector (23); 5Ј-CTTAATAA-GGTTTCCAATCAGCC-3Ј and 5Ј-GTCTCATCTTTATTA-GGAGTCAAC-3Ј for the Il6 promoter region; and 5Ј-TCCA-AGTTCATGGGTCACAA-3Ј and 5Ј-TGATTGGCTGACTT-TGGAGA-3Ј for the Cxcl10 promoter region.
Activation of the Endogenous Lcn2 Gene by IB-Retroviral expression of wild-type IB or a mutant protein with the D451T or K717E/K719E substitution was performed according to the method of He et al. (55). Briefly, HEK293T cells were transfected with the Moloney Murine Leukemia virus-⌿E helper plasmid and pBABE-puro-3ϫFLAG-IB, and the culture supernatant containing the retrovirus was collected. IBdeficient BMMs were infected with the retrovirus and cultured for 2 days. The cells were treated for 4, 8, or 12 h with or without LPS (100 ng/ml). Expression of IB was detected by immunoblot with the anti-IB antibody. The mRNA product of the endogenous Lcn2 gene was estimated by quantitative real-time RT-PCR as described previously (56). Briefly, total RNAs were extracted using TRIsure (BIOLINE) according to the instructions of the manufacturer, and 1 g of the RNA was reverse-transcribed by ReverTra Ace reverse transcriptase (TOYOBO) using an oligo(dT) primer, followed by real-time PCR using SYBR premix Ex Taq TM (Takara Bio) on the Roter-Gene 6200 system (Corbett). The primer pairs used were 5Ј-AAGGAGCTGTCCCCTGA-ACT-3Ј and 5Ј-GGTGGGGACAGAGAAGATGA-3Ј for Lcn2 and 5Ј-AAGCGAAACTGGCGGAAAC-3Ј and 5Ј-TAACCG-ATGTTGGGCATCAG-3Ј for the control gene Rpl32.
Secondary Structure Prediction-The secondary structure of the ANK7 in IB and IB NS was predicted using the serverside program PSIPRED (57,58).
Author Contributions-A. K., S. Y., and H. S. designed the study. A. K. performed the experiments. A. K., S. Y., and H. S. analyzed the data and wrote the paper. All authors analyzed the results and approved the final version of the manuscript.