DNA element downstream of the jB site in the Lcn2 promoter is required for transcriptional activation by IjBf and NF-jB p50 Akira Kohda, Soh Yamazaki and Hideki Sumimoto* Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan
The nuclear protein IjBf activates transcription of a subset of NF-jB-dependent innate immune genes such as Lcn2 encoding the antibacterial protein lipocalin-2. IjBf functions as a coactivator via its interaction with NF-jB p50, which contains a DNA-binding Rel-homology domain but lacks a transcriptional activation domain. However cis-regulatory elements involved in IjBf function have remained unknown. Here, we show that, although IjBf by itself is unable to associate with the Lcn2 promoter, IjBf interacts with the promoter via p50 binding to the NF-jB-binding site (jB site) and the interaction also requires the pyrimidinerich site (CCCCTC) that localizes seven bases downstream of the jB site. The pyrimidine-rich site is also essential for IjBf-mediated activation of the Lcn2 gene. Introduction of both sites into an IjBf-independent gene culminates in IjBf–p50–DNA complex formation and transcriptional activation. Furthermore, spacing between the two sites is crucial for both IjBf– DNA interaction and IjBf-mediated gene activation. Thus, the pyrimidine-rich IjBf-responsive site plays an essential role in productive interaction of IjBf with the p50–DNA complex.
Introduction The NF-jB family of transcription factors includes p50, p52, p65 (also known as RelA), RelB and c-Rel; by forming a homo- or heterodimer, NF-jB regulates expression of a wide spectrum of genes that critically contribute to host defense and inflammation (O’Dea & Hoffmann 2010; Smale 2010; Hayden & Ghosh 2012). In resting cells, NF-jB complexes are sequestered in the cytoplasm via direct association with the classical IjB proteins, such as IjBa, IjBb and IjBe, which contain ankyrin-repeat domain (ARD) composed of six to seven ankyrin repeats. These cytoplasmic IjB proteins are known to inhibit NF-jB function by masking the nuclear localization signal of NF-jB. Upon cell stimulation, the cytoplasmic IjB proteins are rapidly phosphorylated and degraded so that NF-jB translocates to the nucleus and interacts with NF-jB-binding elements (jB sites) Communicated by: Keiichi I. Nakayama *Correspondence: [email protected]
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on promoter/enhancer regions of target genes, leading to transcriptional activation (O’Dea & Hoffmann 2010; Smale 2010; Hayden & Ghosh 2012). In contrast to constitutive expression of the cytoplasmic IjB proteins, another group of ARD-harboring IjB proteins, including Bcl-3 (Bours et al. 1993; Fujita et al. 1993), IjBf (also known as MAIL or INAP; Kitamura et al. 2000; Haruta et al. 2001; Yamazaki et al. 2001) and IjBNS (Fiorini et al. 2002), is absent in resting cells; they are expressed in stimulated cells in a manner dependent on NF-jB activation and accumulated to the nucleus but not to the cytoplasm. These nuclear IjB proteins directly interact via the ARD with NF-jB p50 but not with NF-jB p65 (Bours et al. 1993; Fujita et al. 1993; Yamazaki et al. 2001; Fiorini et al. 2002; Yamamoto et al. 2004; Hirotani et al. 2005; Trinh et al. 2008). As p50 harbors a DNA-binding Rel-homology domain but lacks a transcriptional activation domain, the p50 homodimer requires coactivators to positively regulate transcription. However, nuclear IjB proteins, defective in intrinsic DNA-binding activity,
DOI: 10.1111/gtc.12162 © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
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positively regulates transcription (Fujita et al. 1993; Kitamura et al. 2000; Yamamoto et al. 2004; Touma et al. 2007; Schuster et al. 2012), although they may serve as a negative regulator in a context-dependent manner (Yamazaki et al. 2001; Yamamoto et al. 2004; Hirotani et al. 2005; Kuwata et al. 2006). For instance, IjBf, a key regulator in the immune system (Yamamoto et al. 2004; Okamoto et al. 2010; Okuma et al. 2013), activates transcription of a subset of genes such as Lcn2 (encoding the antibacterial protein lipocalin-2) in cooperation with NF-jB (Cowland et al. 2006; Kayama et al. 2008; Yamazaki et al. 2008). However, it has remained largely unknown about the mechanism whereby nuclear IjB proteins assemble with NF-jB and DNA for subsequent transcriptional activation; in particular, there has been no information on cis-elements involved in nuclear IjB function except jB sites. In this study, we show that interaction of IjBf with the p50–DNA complex requires not only the jB site but also its downstream site in the promoter of Lcn2. This site also plays a crucial role in IjBfmediated activation of the Lcn2 gene. Introduction of the two sites into an IjBf-independent gene culminates in formation of the productive IjBf– p50–DNA complex. Thus, the site downstream of the jB site likely functions as a key IjBf-responsive element.
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Results IjBf binds to the Lcn2 promoter in a manner dependent on p50–DNA interaction
It is known that IjBf-dependent transcriptional activation of Lcn2 requires the jB site GGGAATGTCCC at positions –230/–220 from transcription start site (Cowland et al. 2006; Kayama et al. 2008; Yamazaki et al. 2008), which sequence corresponds well with those of p50 homodimer-binding sites (Ghosh et al. 2012; Siggers et al. 2012). To investigate interaction of the Lcn2 promoter with p50 and IjBf, we purified recombinant IjBf and NF-jB p50 and carried out a DNA-binding assay using protein pull-down followed by PCR amplification of protein-bound DNA (for detail, see ‘Experimental procedures’). When hexahistidine-tagged p50 (His–p50) was incubated with a DNA fragment of the Lcn2 promoter region containing the jB site (–317/–117), the fragment co-pulled down with His– p50 was effectively amplified by PCR (Fig. 1A). The PCR product was not detected when a mutant p50 with the Y57A/E60D substitution, defective in binding to jB sites (Carmody et al. 2007), was used instead of the wild-type protein, or when the jB site sequence was mutated to the sequence AATAATG TTAA (the mutated bases are underlined). These findings confirm that the present assay is suitable for
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Figure 1 Interaction of IjBf with the Lcn2 promoter in a manner dependent on p50 binding to the jB site. His–p50 (wt), His– p50 with deletion of amino acids 358–366 (Δ), or His–p50 (Y57A/E60D) was incubated with (B, C) or without (A) MBP–IjBf in the presence of Lcn2 (–317/–117) or a mutant Lcn2 (–317/–117) carrying the mutated jB site (jBm). After the protein–DNA complex was pulled down with nickel-affinity beads (A) or amylose resins (B, C), the precipitated DNA was amplified by PCR and the product was subjected to agarose gel electrophoresis. The precipitated proteins were subjected to SDS-PAGE, followed by immunoblot with the anti-His antibody (A, C) or by staining with CBB (B). Positions for marker proteins are indicated in kDa. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
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evaluating protein–DNA interactions. By contrast to p50, IjBf by itself failed to interact with the Lcn2 promoter (Fig. 1B, upper panel). In the presence of p50, however, IjBf effectively bound to the Lcn2 promoter. A mutant p50 with truncation of amino acids 358–366, a region required for its interaction with IjBf (Ryzhakov et al. 2013), neither bound to IjBf (Fig. 1B, lower panel) nor supported IjBf binding to DNA (Fig. 1B, upper panel). However, p50 (Y57A/E60D) was able to interact with IjBf but did not induce IjBf–DNA association, and IjBf failed to bind to a mutant Lcn2 promoter with impairment of the jB site (Fig. 1C). Thus, p50 binding to the jB site is required to recruit IjBf to DNA.
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IjBf activates Lcn2 transcription in a manner dependent on p50–DNA interaction
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To study gene activation by IjBf and p50, we prepared mouse embryonic fibroblasts (MEFs) doubly deficient in p50 and IjBf (p50-/IjBf-deficient MEFs) and tested the activity of a luciferase reporter regulated by the Lcn2 promoter. As expected, the Lcn2 activity was increased synergistically by p50 and IjBf (Fig. 2A), supporting the idea that the present system recapitulates the IjBf dependence shown previously for the in situ gene (Cowland et al. 2006; Kayama et al. 2008; Yamazaki et al. 2008). The activation was abrogated by the mutation in the jB site, indicative of its crucial role. The mutation of p50, defective in either binding to the jB site (Y57A/E60D) or interacting with IjBf (deletion of amino acids 358–366), also led to a loss of transcriptional activation of Lcn2. Thus, IjBf binds to DNA and activates Lcn2 by interacting with the p50–DNA complex. The E-selectin (a.k.a. ELAM-1)-encoding gene Sele undergoes NF-jB-dependent activation and contains the jB site GAGAATTTCC in the promoter region (–94/–85; Becker-Andre et al. 1992; Whitley et al. 1994), a 10-base sequence that agrees well with the consensus sequences for binding to the p50–p65 heterodimer (Ghosh et al. 2012; Siggers et al. 2012). As expected, luciferase activity of the Sele upstream region (–1023/+105) was strongly increased by co-expression of p50 and p65. However, p50 was unable to activate Sele in combination with IjBf (Fig. 2B). Consistent with the inability, IjBf did not form a complex with the Sele promoter (Fig. 2C), suggesting that Sele is an IjBf-independent gene. 622
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Figure 2 Transcriptional activation of Lcn2 and Sele by NF-jB and IjBf. (A, B) p50-/IjBf-deficient MEFs were transfected with the following plasmids: the luciferase reporter plasmid pGL3-Basic containing the upstream region of Lcn2 (–1031/+54) (A) or Sele (–1023/+105) (B); the internal control plasmid pRL–TK; and pcDNA3 for expression of FLAG– IjBf, HA–p50 (wt), HA–p50 with deletion of amino acids 358–366 (Δ), HA–p50 (Y57A/E60D) or HA–p65. Luciferase activities were determined as described in ‘Experimental procedures.’ Each graph represents the mean SD obtained from three independent transfections. Cell lysates were analyzed by immunoblot with the anti-FLAG, anti-HA or anti-b-actin antibody. (C) MBP–IjBf was incubated with His–p50 in the presence of Lcn2 (–1031/+54) or Sele (–1023/+105). The protein–DNA complex was analyzed as in Fig. 1; MBP–IjBf or His–p50 was detected with CBB or immunoblot, respectively.
Region downstream of the jB site is required for both IjBf binding to DNA and IjBf-mediated activation of Lcn2
To determine cis-elements for IjBf interaction other than the jB site, we prepared Sele-based reporters, in which sequences around the jB site from the Lcn2 promoter were swapped into the Sele gene (SL1 to SL4; Fig. 3A). As shown in Fig. 3B, IjBf interacted
© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
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well with SL2 to SL4 but not with SL1, suggesting the requirement for the CCCCTC sequence that locates downstream of the jB site in the Lcn2 promoter. Co-expression of p50 and IjBf induced activation of these promoters except SL1 (Fig. 3C). Furthermore, replacement of the pyrimidine-rich site abrogated IjBf binding to the Lcn2 promoter (Fig. 3D) without affecting interaction of p50 with the jB site (Fig. 3E). The replacement also resulted in a defective activation of Lcn2 by p50 and IjBf
(Fig. 3F). These findings indicate that the pyrimidine-rich site is required for both IjBf binding to DNA and IjBf-mediated activation of Lcn2. Presence of both jB and its downstream sites is sufficient for IjBf binding to DNA and IjBfmediated activation of Lcn2
To test whether the presence of both jB and pyrimidine-rich sites are sufficient for synergy
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Figure 3 Role for the jB site-containing region of Lcn2 in IjBf binding to DNA and IjBf-mediated transcriptional activation. (A) Nucleotide sequences of the jB site-containing region of Lcn2, Sele and their mutants used here. The jB sites in Lcn2 and Sele are underlined; a putative STAT-binding site in Sele is indicated by dotted line. (B, D, E) His–p50 was incubated with (B, D) or without (E) MBP–IjBf in the presence of the following DNA fragment: Lcn2 (–500/+50), Sele (–445/+105) or the indicated mutant. The protein–DNA complex was analyzed as in Fig. 1. (C, F) p50-/IjBf-deficient MEFs were transfected with the following plasmids: pGL3-Basic containing Lcn2 (–500/+50), Sele (–445/+105) or their indicated mutant; pRL–TK; and pcDNA3FLAG–IjBf and pcDNA3-HA–p50. Luciferase activities were determined as in Fig. 2. Each graph represents the mean SD obtained from three independent transfections. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
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between p50 and IjBf, we prepared luciferase reporters in which the Lcn2 jB site, the pyrimidinerich site or both were introduced into the Sele promoter (Fig. 4A). Simultaneous introduction of the two sites culminated in IjBf–p50–DNA complex formation (Fig. 4B and C) and cooperative transcriptional activation (Fig. 4D). Swapping the Lcn2 jB site for the Sele jB site by itself rendered the Sele promoter in a state accessible for p50 (Fig. 4B) but not for IjBf (Fig. 4C). Co-expression of p50 and IjBf induced transcription from the Lcn2 jB sitecontaining Sele promoter (SL6) but to a much lower extent than that from the promoter with both the Lcn2 jB and pyrimidine-rich sites (SL5; Fig. 4E). However, the downstream IjBf-sensitive site by itself was not sufficient to mediate the recruitment of IjBf to DNA (Fig. 4C) or IjBf-mediated gene activation (Fig. 4E). Thus, the presence of both jB and pyrimidine-rich sites of Lcn2 is sufficient for IjBf–p50– DNA complex formation and synergistic transcriptional activation. Spacing between jB and its downstream pyrimidine-rich sites is crucial for both IjBf binding to DNA and IjBf-mediated activation of Lcn2
We finally tested the effect of the position of the pyrimidine-rich sequence (CCCCTC) on IjBf binding to DNA and IjBf-mediated transcriptional activation; for this purpose, we prepared variant probes that differed in the spacing of the IjBf-responsive pyrimidine-rich site in relation to the jB site (SL8 and SL9). As shown in Fig. 4F, increasing and decreasing of the space each abrogated IjBf binding to DNA. Consistent with this, cells carrying these variant promoters failed to exhibit a luciferase activity upon co-expression of p50 and IjBf (Fig. 4G), indicating that localizing the IjBf-responsive sequence seven bases downstream of the jB site is crucial for synergy between p50 and IjBf in transcriptional activation.
Discussion In the present study, we show that IjBf interacts with the Lcn2 promoter not only via p50 binding to the jB site but also via the pyrimidine-rich site (CCCCTC) that localizes seven bases downstream of the jB site in the Lcn2 promoter, and that the latter site is also required for IjBf-mediated activation of the Lcn2 gene. Introduction of both the jB and pyrimidine-rich sites into the IjBf-independent gene Sele culminates in IjBf–p50–DNA 624
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complex formation and transcriptional activation, in which spacing between the two sites plays a crucial role. Thus, the pyrimidine-rich IjBfresponsive site in the Lcn2 promoter participates in productive interaction of IjBf with the p50–DNA complex. In spite of the lack of the Lcn2 pyrimidine-rich site, SL6 (a Sele-based promoter with the Lcn2 jB site) is slightly activated by p50 and IjBf, although the activation is much lower than that of SL5 harboring the two sites (Fig. 4E). However, p50 and IjBf induce no reporter activity of SL1, another promoter that contains the Lcn2 jB site but not the pyrimidine-rich sequence (Fig. 3C). Although the reason for this discrepancy is presently unknown, it might be possible that a region upstream of the jB site in the Sele promoter affects p50-/IjBf-mediated activation of SL6; the region contains a consensus sequence for STAT family transcription factors (Decker et al. 2012), which is disrupted in SL1 (Fig. 3A). In contrast to the crucial role of the extra-jB site in IjBf-mediated Lcn2 activation, Bcl-3, another member of the nuclear IjB family, is considered to function as a coactivator in a manner dependent on a base within jB sites (Leung et al. 2004; Wang et al. 2012). Although Bcl-3 is incapable of directly binding to DNA, Bcl-3 interacts with NF-jB p50 and p52 and thus functions via a jB site in promoters/enhancers of target genes (Fujita et al. 1993; Kitamura et al. 2000). Indeed the p52 homodimer in complex with Bcl-3 efficiently activates transcription from jB sites with the central base pair G/C but not from A/T-centric jB sites (Leung et al. 2004; Wang et al. 2012). It is possible that IjBf may also function in a manner dependent on an intra-jB site; the possibility should be tested in future studies. The present study also shows that localization of the IjBf-responsive element seven bases downstream of the jB site is important for both IjBf binding to DNA and IjBf-mediated gene activation (Fig. 4). The spacing may enable the p50–IjBf complex to make direct contacts with the IjBf-responsive element, although it remains to be determined whether IjBf or p50 (or both) directly interacts with the pyrimidine-rich site. A cis-element-dependent cooperation also occurs between the interferon regulatory factor IRF4 and the Ets family protein PU.1 (Brass et al. 1999; Escalante et al. 2002). IRF4 by itself interacts only weakly with IRF sites (with high affinity for IRF1 and IRF2), but it strongly binds together with
© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
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Figure 4 Role for the Lcn2 pyrimidine-rich site in IjBf binding to DNA and IjBf-mediated transcriptional activation. (A) Nucleotide sequences of the jB site-containing region of Lcn2, Sele and mutants used here. The jB sites in Lcn2 and Sele are underlined. (B, C, F) His–p50 was incubated with (C, F) or without (B) MBP–IjBf in the presence of Lcn2 (–500/+50), Sele (–445/+105) or the indicated mutant. The protein–DNA complex was analyzed as in Fig. 1. (D, E, G) p50-/IjBf-deficient MEFs were transfected with the following plasmids: pGL3-Basic containing Lcn2 (–500/+50), Sele (–445/+105) or the indicated mutant; pRL–TK; and pcDNA3-FLAG–IjBf and pcDNA3-HA–p50. Luciferase activities were determined as in Fig. 2. Each graph represents the mean SD obtained from three independent transfections.
PU.1 to composite DNA elements containing overlapping Ets and IRF sites in promoters/enhancers of target genes, leading to cooperative transcriptional activation. IRF4 also cooperates with the AP-1 transcription factors BATF and JUN via binding to AP-1–IRF composite elements in a similar manner (Glasmacher et al. 2012; Murphy et al. 2013). It is tempting to postulate that the pyrimidine-rich IjBf-responsive element in the Lcn2 promoter
(CCCCTC) or a related sequence functions also in other IjBf-dependent genes. A preliminary search of ours suggests that the sequences do not seem to exist several bases downstream of jB sites in promoter regions of genes encoding IL-6 and G-CSF, which are regulated by IjBf (Yamamoto et al. 2004). The question whether other IjBf-dependent genes are regulated via a DNA cis-element out of jB sites should be addressed in the future studies.
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Experimental procedures Plasmid construction The mouse cDNA encoding full-length IjBf (amino acids 1–728), IjBf-(414–728), p50-(1–366) and p65-(1–549) was prepared as previously described (Yamazaki et al. 2001, 2005). The cDNA for p50-(1–357) was prepared by PCR using the p50-(1–366) cDNA as a template. Mutations leading to the Y57A/E60D substitution were introduced by PCR-mediated site-directed mutagenesis. The cDNAs were ligated to the following expression vector: pMALc2 (New England BioLabs) for expression as protein fused to maltosebinding protein (MBP) in Escherichia coli; pRSFDuet-1 (Novagen; Yuzawa et al. 2011) for expression as hexahistidine (His)-tagged protein in E. coli; and pcDNA3 (Invitrogen) for expression as FLAG- or HA-tagged protein in mammalian cells. Upstream fragments of the lipocalin-2-encoding gene (Lcn2) and the E-selectin-encoding gene (Sele) were amplified by PCR using mouse genomic DNA and subcloned into pGL3-Basic vector (Promega) for binding and luciferase reporter analysis. All of the constructs were sequenced for confirmation of their identity.
Analysis of protein–DNA interaction His-tagged p50 proteins and MBP-tagged IjBf were purified as previously described (Yuzawa et al. 2011; Kamakura et al. 2013). For pull-down of His-tagged protein, His–p50-(1–366), His–p50-ΔC (1–357) or His–p50-(1–366/Y57A/E60D) at 30 pmol was incubated in 500 lL of buffer A (150 mM NaCl, 5% glycerol, 1 mM DTT, 0.5% Triton X-100 and 25 mM TrisHCl, pH 8.0) containing salmon sperm DNA (0.1 mg/mL), followed by further incubation with the indicated DNA fragment (0.1 pmol) and COSMOGELâ His-Accept (nacalai tesque). After washing with buffer A containing 25 mM imidazole, the protein–DNA complex was eluted from the resin with buffer A containing 1 M imidazole. For MBP pull-down, MBP–IjBf(414–728) was used because this region is required and sufficient for IjBf binding to p50 and DNA (Trinh et al. 2008). A Histagged protein (30 pmol except Fig. 1B, where 900 pmol of His-tagged proteins was used) and/or MBP–IjBf-(414–728) (150 pmol) were incubated for 20 min at 4 °C in 500 lL of buffer B (137 mM NaCl, 2.7 mM KCl, 0.5% Triton X-100, 1 mM DTT, 8.1 mM Na2HPO4 and 1.5 mM KH2PO4, pH 7.4) containing salmon sperm DNA (0.1 mg/mL). 0.1 pmol of the indicated DNA fragment of Lcn2 or Sele promoter, flanked by 79-bp and 263-bp regions linked to either side of the multicloning site of the pGL3-Basic vector, was subsequently added, followed by further incubation for 40 min at 4 °C in the presence of Amylose Resin (New England Biolab). After washing with buffer B, the protein–DNA complex was eluted from the resin with buffer B containing 20 mM maltose. The eluted protein was applied to SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining or immunoblot analysis, the latter of which was carried out using the anti-His5 antibody (Qiagen); the blots were developed using ECL-Prime (GE Healthcare Biosciences)
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for visualization of the antibodies. The eluted DNA was analyzed by PCR using the following primer pairs: 50 -TACA GGGTTATGGGAGTGGAC-30 and 50 -TCTGTTGAAAT ACTTGGCAAGAT-30 for detection of the Lcn2 promoter region, and 50 -TACGGATCCATGGAAGACGCCAAAAAC ATA-30 and 50 -CATATCGTTTCATAGCTTCTGC-30 for detection of the 263-bp region.
Luciferase reporter assay MEFs doubly deficient in NF-jB p50 and IjBf were prepared from fetuses obtained by mating of Nfkb1/;Nfkbiz+/ mice; p50-deficient (Nfkb1/) mice were purchased from The Jackson Laboratory, and IjBf-deficient (Nfkbiz/) mice were generated as previously described (Yamamoto et al. 2004). All animals were housed and maintained in a specific pathogen-free animal facility at Kyushu University. All experiments were carried out in accordance with the guidelines for Proper Conduct of Animal Experiments (Science Council of Japan). The experimental protocol was approved by Kyushu University (Permit Number: A22-005). p50-/IjBf-deficient (Nfkb1/;Nfkbiz/) MEFs were immortalized by transfection with pEF321-T encoding the SV40 large T antigen (Kim et al. 1990). p50-/IjBf-deficient MEFs were transfected with the luciferase reporter plasmid pGL3-Basic containing the indicated promoter, the internal control plasmid pRL–TK (Promega) and the indicated expression plasmids, using X-tremeGENE HP DNA Transfection Reagent (Roche Applied Science). The luciferase activity of transfected cells was determined by the Dual-Luciferaseâ Reporter assay system (Promega), as previously described (Yamazaki et al. 2008). For estimation of protein levels, cell lysates were analyzed by immunoblot with the anti-FLAG (M2; Sigma-Aldrich), anti-HA (3F10; Roche Applied Science) or anti-b-actin (sc-47778; Santa Cruz Biotechnology) antibody.
Acknowledgements We thank Prof. Sumio Sugano (University of Tokyo) for kindly providing pEF321-T; Yohko Kage (Kyushu University) and Namiko Kubo (Kyushu University) for technical assistance. This work was supported in part by MEXT (the Ministry of Education, Culture, Sports, Science and Technology) and by JSPS (Japan Society for the Promotion of Science).
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Yamazaki, S., Muta, T., Matsuo, S. & Takeshige, K. (2005) Stimulus-specific induction of a novel nuclear factor-jB regulator, IjB-f, via Toll/interleukin-1 receptor is mediated by mRNA stabilization. J. Biol. Chem. 280, 1678–1687. Yamazaki, S., Muta, T. & Takeshige, K. (2001) A novel IjB protein, IjB-f, induced by proinflammatory stimuli, negatively regulates nuclear factor-jB in the nuclei. J. Biol. Chem. 276, 27657–27662. Yuzawa, S., Kamakura, S., Iwakiri, Y., Hayase, J. & Sumimoto, H. (2011) Structural basis for interaction between the conserved cell polarity proteins Inscuteable and Leu-Gly-Asn repeat-enriched protein (LGN). Proc. Natl Acad. Sci. USA 108, 19210–19215. Received: 16 May 2014 Accepted: 18 May 2014
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The nuclear protein IjBf activates transcription of a subset of NF-jB-dependent innate immune genes such as Lcn2 encoding the antibacterial protein lipocalin-2. IjBf functions as a coactivator via its interaction with NF-jB p50, which contains a DNA-binding Rel-homology domain but lacks a transcriptional activation domain. However cis-regulatory elements involved in IjBf function have remained unknown. Here, we show that, although IjBf by itself is unable to associate with the Lcn2 promoter, IjBf interacts with the promoter via p50 binding to the NF-jB-binding site (jB site) and the interaction also requires the pyrimidinerich site (CCCCTC) that localizes seven bases downstream of the jB site. The pyrimidine-rich site is also essential for IjBf-mediated activation of the Lcn2 gene. Introduction of both sites into an IjBf-independent gene culminates in IjBf–p50–DNA complex formation and transcriptional activation. Furthermore, spacing between the two sites is crucial for both IjBf– DNA interaction and IjBf-mediated gene activation. Thus, the pyrimidine-rich IjBf-responsive site plays an essential role in productive interaction of IjBf with the p50–DNA complex.
Introduction The NF-jB family of transcription factors includes p50, p52, p65 (also known as RelA), RelB and c-Rel; by forming a homo- or heterodimer, NF-jB regulates expression of a wide spectrum of genes that critically contribute to host defense and inflammation (O’Dea & Hoffmann 2010; Smale 2010; Hayden & Ghosh 2012). In resting cells, NF-jB complexes are sequestered in the cytoplasm via direct association with the classical IjB proteins, such as IjBa, IjBb and IjBe, which contain ankyrin-repeat domain (ARD) composed of six to seven ankyrin repeats. These cytoplasmic IjB proteins are known to inhibit NF-jB function by masking the nuclear localization signal of NF-jB. Upon cell stimulation, the cytoplasmic IjB proteins are rapidly phosphorylated and degraded so that NF-jB translocates to the nucleus and interacts with NF-jB-binding elements (jB sites) Communicated by: Keiichi I. Nakayama *Correspondence: [email protected]
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on promoter/enhancer regions of target genes, leading to transcriptional activation (O’Dea & Hoffmann 2010; Smale 2010; Hayden & Ghosh 2012). In contrast to constitutive expression of the cytoplasmic IjB proteins, another group of ARD-harboring IjB proteins, including Bcl-3 (Bours et al. 1993; Fujita et al. 1993), IjBf (also known as MAIL or INAP; Kitamura et al. 2000; Haruta et al. 2001; Yamazaki et al. 2001) and IjBNS (Fiorini et al. 2002), is absent in resting cells; they are expressed in stimulated cells in a manner dependent on NF-jB activation and accumulated to the nucleus but not to the cytoplasm. These nuclear IjB proteins directly interact via the ARD with NF-jB p50 but not with NF-jB p65 (Bours et al. 1993; Fujita et al. 1993; Yamazaki et al. 2001; Fiorini et al. 2002; Yamamoto et al. 2004; Hirotani et al. 2005; Trinh et al. 2008). As p50 harbors a DNA-binding Rel-homology domain but lacks a transcriptional activation domain, the p50 homodimer requires coactivators to positively regulate transcription. However, nuclear IjB proteins, defective in intrinsic DNA-binding activity,
DOI: 10.1111/gtc.12162 © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
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positively regulates transcription (Fujita et al. 1993; Kitamura et al. 2000; Yamamoto et al. 2004; Touma et al. 2007; Schuster et al. 2012), although they may serve as a negative regulator in a context-dependent manner (Yamazaki et al. 2001; Yamamoto et al. 2004; Hirotani et al. 2005; Kuwata et al. 2006). For instance, IjBf, a key regulator in the immune system (Yamamoto et al. 2004; Okamoto et al. 2010; Okuma et al. 2013), activates transcription of a subset of genes such as Lcn2 (encoding the antibacterial protein lipocalin-2) in cooperation with NF-jB (Cowland et al. 2006; Kayama et al. 2008; Yamazaki et al. 2008). However, it has remained largely unknown about the mechanism whereby nuclear IjB proteins assemble with NF-jB and DNA for subsequent transcriptional activation; in particular, there has been no information on cis-elements involved in nuclear IjB function except jB sites. In this study, we show that interaction of IjBf with the p50–DNA complex requires not only the jB site but also its downstream site in the promoter of Lcn2. This site also plays a crucial role in IjBfmediated activation of the Lcn2 gene. Introduction of the two sites into an IjBf-independent gene culminates in formation of the productive IjBf– p50–DNA complex. Thus, the site downstream of the jB site likely functions as a key IjBf-responsive element.
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Results IjBf binds to the Lcn2 promoter in a manner dependent on p50–DNA interaction
It is known that IjBf-dependent transcriptional activation of Lcn2 requires the jB site GGGAATGTCCC at positions –230/–220 from transcription start site (Cowland et al. 2006; Kayama et al. 2008; Yamazaki et al. 2008), which sequence corresponds well with those of p50 homodimer-binding sites (Ghosh et al. 2012; Siggers et al. 2012). To investigate interaction of the Lcn2 promoter with p50 and IjBf, we purified recombinant IjBf and NF-jB p50 and carried out a DNA-binding assay using protein pull-down followed by PCR amplification of protein-bound DNA (for detail, see ‘Experimental procedures’). When hexahistidine-tagged p50 (His–p50) was incubated with a DNA fragment of the Lcn2 promoter region containing the jB site (–317/–117), the fragment co-pulled down with His– p50 was effectively amplified by PCR (Fig. 1A). The PCR product was not detected when a mutant p50 with the Y57A/E60D substitution, defective in binding to jB sites (Carmody et al. 2007), was used instead of the wild-type protein, or when the jB site sequence was mutated to the sequence AATAATG TTAA (the mutated bases are underlined). These findings confirm that the present assay is suitable for
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Figure 1 Interaction of IjBf with the Lcn2 promoter in a manner dependent on p50 binding to the jB site. His–p50 (wt), His– p50 with deletion of amino acids 358–366 (Δ), or His–p50 (Y57A/E60D) was incubated with (B, C) or without (A) MBP–IjBf in the presence of Lcn2 (–317/–117) or a mutant Lcn2 (–317/–117) carrying the mutated jB site (jBm). After the protein–DNA complex was pulled down with nickel-affinity beads (A) or amylose resins (B, C), the precipitated DNA was amplified by PCR and the product was subjected to agarose gel electrophoresis. The precipitated proteins were subjected to SDS-PAGE, followed by immunoblot with the anti-His antibody (A, C) or by staining with CBB (B). Positions for marker proteins are indicated in kDa. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
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evaluating protein–DNA interactions. By contrast to p50, IjBf by itself failed to interact with the Lcn2 promoter (Fig. 1B, upper panel). In the presence of p50, however, IjBf effectively bound to the Lcn2 promoter. A mutant p50 with truncation of amino acids 358–366, a region required for its interaction with IjBf (Ryzhakov et al. 2013), neither bound to IjBf (Fig. 1B, lower panel) nor supported IjBf binding to DNA (Fig. 1B, upper panel). However, p50 (Y57A/E60D) was able to interact with IjBf but did not induce IjBf–DNA association, and IjBf failed to bind to a mutant Lcn2 promoter with impairment of the jB site (Fig. 1C). Thus, p50 binding to the jB site is required to recruit IjBf to DNA.
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IjBf activates Lcn2 transcription in a manner dependent on p50–DNA interaction
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To study gene activation by IjBf and p50, we prepared mouse embryonic fibroblasts (MEFs) doubly deficient in p50 and IjBf (p50-/IjBf-deficient MEFs) and tested the activity of a luciferase reporter regulated by the Lcn2 promoter. As expected, the Lcn2 activity was increased synergistically by p50 and IjBf (Fig. 2A), supporting the idea that the present system recapitulates the IjBf dependence shown previously for the in situ gene (Cowland et al. 2006; Kayama et al. 2008; Yamazaki et al. 2008). The activation was abrogated by the mutation in the jB site, indicative of its crucial role. The mutation of p50, defective in either binding to the jB site (Y57A/E60D) or interacting with IjBf (deletion of amino acids 358–366), also led to a loss of transcriptional activation of Lcn2. Thus, IjBf binds to DNA and activates Lcn2 by interacting with the p50–DNA complex. The E-selectin (a.k.a. ELAM-1)-encoding gene Sele undergoes NF-jB-dependent activation and contains the jB site GAGAATTTCC in the promoter region (–94/–85; Becker-Andre et al. 1992; Whitley et al. 1994), a 10-base sequence that agrees well with the consensus sequences for binding to the p50–p65 heterodimer (Ghosh et al. 2012; Siggers et al. 2012). As expected, luciferase activity of the Sele upstream region (–1023/+105) was strongly increased by co-expression of p50 and p65. However, p50 was unable to activate Sele in combination with IjBf (Fig. 2B). Consistent with the inability, IjBf did not form a complex with the Sele promoter (Fig. 2C), suggesting that Sele is an IjBf-independent gene. 622
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Figure 2 Transcriptional activation of Lcn2 and Sele by NF-jB and IjBf. (A, B) p50-/IjBf-deficient MEFs were transfected with the following plasmids: the luciferase reporter plasmid pGL3-Basic containing the upstream region of Lcn2 (–1031/+54) (A) or Sele (–1023/+105) (B); the internal control plasmid pRL–TK; and pcDNA3 for expression of FLAG– IjBf, HA–p50 (wt), HA–p50 with deletion of amino acids 358–366 (Δ), HA–p50 (Y57A/E60D) or HA–p65. Luciferase activities were determined as described in ‘Experimental procedures.’ Each graph represents the mean SD obtained from three independent transfections. Cell lysates were analyzed by immunoblot with the anti-FLAG, anti-HA or anti-b-actin antibody. (C) MBP–IjBf was incubated with His–p50 in the presence of Lcn2 (–1031/+54) or Sele (–1023/+105). The protein–DNA complex was analyzed as in Fig. 1; MBP–IjBf or His–p50 was detected with CBB or immunoblot, respectively.
Region downstream of the jB site is required for both IjBf binding to DNA and IjBf-mediated activation of Lcn2
To determine cis-elements for IjBf interaction other than the jB site, we prepared Sele-based reporters, in which sequences around the jB site from the Lcn2 promoter were swapped into the Sele gene (SL1 to SL4; Fig. 3A). As shown in Fig. 3B, IjBf interacted
© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
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well with SL2 to SL4 but not with SL1, suggesting the requirement for the CCCCTC sequence that locates downstream of the jB site in the Lcn2 promoter. Co-expression of p50 and IjBf induced activation of these promoters except SL1 (Fig. 3C). Furthermore, replacement of the pyrimidine-rich site abrogated IjBf binding to the Lcn2 promoter (Fig. 3D) without affecting interaction of p50 with the jB site (Fig. 3E). The replacement also resulted in a defective activation of Lcn2 by p50 and IjBf
(Fig. 3F). These findings indicate that the pyrimidine-rich site is required for both IjBf binding to DNA and IjBf-mediated activation of Lcn2. Presence of both jB and its downstream sites is sufficient for IjBf binding to DNA and IjBfmediated activation of Lcn2
To test whether the presence of both jB and pyrimidine-rich sites are sufficient for synergy
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Figure 3 Role for the jB site-containing region of Lcn2 in IjBf binding to DNA and IjBf-mediated transcriptional activation. (A) Nucleotide sequences of the jB site-containing region of Lcn2, Sele and their mutants used here. The jB sites in Lcn2 and Sele are underlined; a putative STAT-binding site in Sele is indicated by dotted line. (B, D, E) His–p50 was incubated with (B, D) or without (E) MBP–IjBf in the presence of the following DNA fragment: Lcn2 (–500/+50), Sele (–445/+105) or the indicated mutant. The protein–DNA complex was analyzed as in Fig. 1. (C, F) p50-/IjBf-deficient MEFs were transfected with the following plasmids: pGL3-Basic containing Lcn2 (–500/+50), Sele (–445/+105) or their indicated mutant; pRL–TK; and pcDNA3FLAG–IjBf and pcDNA3-HA–p50. Luciferase activities were determined as in Fig. 2. Each graph represents the mean SD obtained from three independent transfections. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
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between p50 and IjBf, we prepared luciferase reporters in which the Lcn2 jB site, the pyrimidinerich site or both were introduced into the Sele promoter (Fig. 4A). Simultaneous introduction of the two sites culminated in IjBf–p50–DNA complex formation (Fig. 4B and C) and cooperative transcriptional activation (Fig. 4D). Swapping the Lcn2 jB site for the Sele jB site by itself rendered the Sele promoter in a state accessible for p50 (Fig. 4B) but not for IjBf (Fig. 4C). Co-expression of p50 and IjBf induced transcription from the Lcn2 jB sitecontaining Sele promoter (SL6) but to a much lower extent than that from the promoter with both the Lcn2 jB and pyrimidine-rich sites (SL5; Fig. 4E). However, the downstream IjBf-sensitive site by itself was not sufficient to mediate the recruitment of IjBf to DNA (Fig. 4C) or IjBf-mediated gene activation (Fig. 4E). Thus, the presence of both jB and pyrimidine-rich sites of Lcn2 is sufficient for IjBf–p50– DNA complex formation and synergistic transcriptional activation. Spacing between jB and its downstream pyrimidine-rich sites is crucial for both IjBf binding to DNA and IjBf-mediated activation of Lcn2
We finally tested the effect of the position of the pyrimidine-rich sequence (CCCCTC) on IjBf binding to DNA and IjBf-mediated transcriptional activation; for this purpose, we prepared variant probes that differed in the spacing of the IjBf-responsive pyrimidine-rich site in relation to the jB site (SL8 and SL9). As shown in Fig. 4F, increasing and decreasing of the space each abrogated IjBf binding to DNA. Consistent with this, cells carrying these variant promoters failed to exhibit a luciferase activity upon co-expression of p50 and IjBf (Fig. 4G), indicating that localizing the IjBf-responsive sequence seven bases downstream of the jB site is crucial for synergy between p50 and IjBf in transcriptional activation.
Discussion In the present study, we show that IjBf interacts with the Lcn2 promoter not only via p50 binding to the jB site but also via the pyrimidine-rich site (CCCCTC) that localizes seven bases downstream of the jB site in the Lcn2 promoter, and that the latter site is also required for IjBf-mediated activation of the Lcn2 gene. Introduction of both the jB and pyrimidine-rich sites into the IjBf-independent gene Sele culminates in IjBf–p50–DNA 624
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complex formation and transcriptional activation, in which spacing between the two sites plays a crucial role. Thus, the pyrimidine-rich IjBfresponsive site in the Lcn2 promoter participates in productive interaction of IjBf with the p50–DNA complex. In spite of the lack of the Lcn2 pyrimidine-rich site, SL6 (a Sele-based promoter with the Lcn2 jB site) is slightly activated by p50 and IjBf, although the activation is much lower than that of SL5 harboring the two sites (Fig. 4E). However, p50 and IjBf induce no reporter activity of SL1, another promoter that contains the Lcn2 jB site but not the pyrimidine-rich sequence (Fig. 3C). Although the reason for this discrepancy is presently unknown, it might be possible that a region upstream of the jB site in the Sele promoter affects p50-/IjBf-mediated activation of SL6; the region contains a consensus sequence for STAT family transcription factors (Decker et al. 2012), which is disrupted in SL1 (Fig. 3A). In contrast to the crucial role of the extra-jB site in IjBf-mediated Lcn2 activation, Bcl-3, another member of the nuclear IjB family, is considered to function as a coactivator in a manner dependent on a base within jB sites (Leung et al. 2004; Wang et al. 2012). Although Bcl-3 is incapable of directly binding to DNA, Bcl-3 interacts with NF-jB p50 and p52 and thus functions via a jB site in promoters/enhancers of target genes (Fujita et al. 1993; Kitamura et al. 2000). Indeed the p52 homodimer in complex with Bcl-3 efficiently activates transcription from jB sites with the central base pair G/C but not from A/T-centric jB sites (Leung et al. 2004; Wang et al. 2012). It is possible that IjBf may also function in a manner dependent on an intra-jB site; the possibility should be tested in future studies. The present study also shows that localization of the IjBf-responsive element seven bases downstream of the jB site is important for both IjBf binding to DNA and IjBf-mediated gene activation (Fig. 4). The spacing may enable the p50–IjBf complex to make direct contacts with the IjBf-responsive element, although it remains to be determined whether IjBf or p50 (or both) directly interacts with the pyrimidine-rich site. A cis-element-dependent cooperation also occurs between the interferon regulatory factor IRF4 and the Ets family protein PU.1 (Brass et al. 1999; Escalante et al. 2002). IRF4 by itself interacts only weakly with IRF sites (with high affinity for IRF1 and IRF2), but it strongly binds together with
© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
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Figure 4 Role for the Lcn2 pyrimidine-rich site in IjBf binding to DNA and IjBf-mediated transcriptional activation. (A) Nucleotide sequences of the jB site-containing region of Lcn2, Sele and mutants used here. The jB sites in Lcn2 and Sele are underlined. (B, C, F) His–p50 was incubated with (C, F) or without (B) MBP–IjBf in the presence of Lcn2 (–500/+50), Sele (–445/+105) or the indicated mutant. The protein–DNA complex was analyzed as in Fig. 1. (D, E, G) p50-/IjBf-deficient MEFs were transfected with the following plasmids: pGL3-Basic containing Lcn2 (–500/+50), Sele (–445/+105) or the indicated mutant; pRL–TK; and pcDNA3-FLAG–IjBf and pcDNA3-HA–p50. Luciferase activities were determined as in Fig. 2. Each graph represents the mean SD obtained from three independent transfections.
PU.1 to composite DNA elements containing overlapping Ets and IRF sites in promoters/enhancers of target genes, leading to cooperative transcriptional activation. IRF4 also cooperates with the AP-1 transcription factors BATF and JUN via binding to AP-1–IRF composite elements in a similar manner (Glasmacher et al. 2012; Murphy et al. 2013). It is tempting to postulate that the pyrimidine-rich IjBf-responsive element in the Lcn2 promoter
(CCCCTC) or a related sequence functions also in other IjBf-dependent genes. A preliminary search of ours suggests that the sequences do not seem to exist several bases downstream of jB sites in promoter regions of genes encoding IL-6 and G-CSF, which are regulated by IjBf (Yamamoto et al. 2004). The question whether other IjBf-dependent genes are regulated via a DNA cis-element out of jB sites should be addressed in the future studies.
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Experimental procedures Plasmid construction The mouse cDNA encoding full-length IjBf (amino acids 1–728), IjBf-(414–728), p50-(1–366) and p65-(1–549) was prepared as previously described (Yamazaki et al. 2001, 2005). The cDNA for p50-(1–357) was prepared by PCR using the p50-(1–366) cDNA as a template. Mutations leading to the Y57A/E60D substitution were introduced by PCR-mediated site-directed mutagenesis. The cDNAs were ligated to the following expression vector: pMALc2 (New England BioLabs) for expression as protein fused to maltosebinding protein (MBP) in Escherichia coli; pRSFDuet-1 (Novagen; Yuzawa et al. 2011) for expression as hexahistidine (His)-tagged protein in E. coli; and pcDNA3 (Invitrogen) for expression as FLAG- or HA-tagged protein in mammalian cells. Upstream fragments of the lipocalin-2-encoding gene (Lcn2) and the E-selectin-encoding gene (Sele) were amplified by PCR using mouse genomic DNA and subcloned into pGL3-Basic vector (Promega) for binding and luciferase reporter analysis. All of the constructs were sequenced for confirmation of their identity.
Analysis of protein–DNA interaction His-tagged p50 proteins and MBP-tagged IjBf were purified as previously described (Yuzawa et al. 2011; Kamakura et al. 2013). For pull-down of His-tagged protein, His–p50-(1–366), His–p50-ΔC (1–357) or His–p50-(1–366/Y57A/E60D) at 30 pmol was incubated in 500 lL of buffer A (150 mM NaCl, 5% glycerol, 1 mM DTT, 0.5% Triton X-100 and 25 mM TrisHCl, pH 8.0) containing salmon sperm DNA (0.1 mg/mL), followed by further incubation with the indicated DNA fragment (0.1 pmol) and COSMOGELâ His-Accept (nacalai tesque). After washing with buffer A containing 25 mM imidazole, the protein–DNA complex was eluted from the resin with buffer A containing 1 M imidazole. For MBP pull-down, MBP–IjBf(414–728) was used because this region is required and sufficient for IjBf binding to p50 and DNA (Trinh et al. 2008). A Histagged protein (30 pmol except Fig. 1B, where 900 pmol of His-tagged proteins was used) and/or MBP–IjBf-(414–728) (150 pmol) were incubated for 20 min at 4 °C in 500 lL of buffer B (137 mM NaCl, 2.7 mM KCl, 0.5% Triton X-100, 1 mM DTT, 8.1 mM Na2HPO4 and 1.5 mM KH2PO4, pH 7.4) containing salmon sperm DNA (0.1 mg/mL). 0.1 pmol of the indicated DNA fragment of Lcn2 or Sele promoter, flanked by 79-bp and 263-bp regions linked to either side of the multicloning site of the pGL3-Basic vector, was subsequently added, followed by further incubation for 40 min at 4 °C in the presence of Amylose Resin (New England Biolab). After washing with buffer B, the protein–DNA complex was eluted from the resin with buffer B containing 20 mM maltose. The eluted protein was applied to SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining or immunoblot analysis, the latter of which was carried out using the anti-His5 antibody (Qiagen); the blots were developed using ECL-Prime (GE Healthcare Biosciences)
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for visualization of the antibodies. The eluted DNA was analyzed by PCR using the following primer pairs: 50 -TACA GGGTTATGGGAGTGGAC-30 and 50 -TCTGTTGAAAT ACTTGGCAAGAT-30 for detection of the Lcn2 promoter region, and 50 -TACGGATCCATGGAAGACGCCAAAAAC ATA-30 and 50 -CATATCGTTTCATAGCTTCTGC-30 for detection of the 263-bp region.
Luciferase reporter assay MEFs doubly deficient in NF-jB p50 and IjBf were prepared from fetuses obtained by mating of Nfkb1/;Nfkbiz+/ mice; p50-deficient (Nfkb1/) mice were purchased from The Jackson Laboratory, and IjBf-deficient (Nfkbiz/) mice were generated as previously described (Yamamoto et al. 2004). All animals were housed and maintained in a specific pathogen-free animal facility at Kyushu University. All experiments were carried out in accordance with the guidelines for Proper Conduct of Animal Experiments (Science Council of Japan). The experimental protocol was approved by Kyushu University (Permit Number: A22-005). p50-/IjBf-deficient (Nfkb1/;Nfkbiz/) MEFs were immortalized by transfection with pEF321-T encoding the SV40 large T antigen (Kim et al. 1990). p50-/IjBf-deficient MEFs were transfected with the luciferase reporter plasmid pGL3-Basic containing the indicated promoter, the internal control plasmid pRL–TK (Promega) and the indicated expression plasmids, using X-tremeGENE HP DNA Transfection Reagent (Roche Applied Science). The luciferase activity of transfected cells was determined by the Dual-Luciferaseâ Reporter assay system (Promega), as previously described (Yamazaki et al. 2008). For estimation of protein levels, cell lysates were analyzed by immunoblot with the anti-FLAG (M2; Sigma-Aldrich), anti-HA (3F10; Roche Applied Science) or anti-b-actin (sc-47778; Santa Cruz Biotechnology) antibody.
Acknowledgements We thank Prof. Sumio Sugano (University of Tokyo) for kindly providing pEF321-T; Yohko Kage (Kyushu University) and Namiko Kubo (Kyushu University) for technical assistance. This work was supported in part by MEXT (the Ministry of Education, Culture, Sports, Science and Technology) and by JSPS (Japan Society for the Promotion of Science).
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© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
