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Short Communication |
B-mediated stimulation of the human interleukin-6 promoter
1 Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3US, UK
2 Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA
Correspondence
Richard Caswell
CaswellR{at}cf.ac.uk
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ABSTRACT |
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B (NF-B). As the interleukin-6 (IL-6) promoter can be activated by both NF-B and IE2p86, it was examined whether there is competition between these two factors. Here, it is reported that both wild-type and mutant IE2p86 can block activation of the IL-6 promoter in response to interleukin-1
. By using an artificial activator in which the activation domain of NF-B is directed to the promoter by the GAL4 DNA-binding domain, it is shown that the mutant form of IE2p86 can inhibit NF-B-mediated activation at a step subsequent to promoter recruitment. These data therefore suggest a novel mechanism for inhibition of NF-B by IE2p86. Supplementary figures showing binding of IE2p86 and CAD291 to basal transcription factors in vitro and the sequence of the human IL-6 promoter are available with the online version of this paper.
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MAIN TEXT |
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; Song & Stinski, 2002). More recently, it has emerged that IE2p86 can inhibit the expression of some cellular genes, particularly those activated by nuclear factor B (NF-B) (Taylor & Bresnahan, 2005, 2006a, b), which limits expression of pro-inflammatory genes following infection. The interleukin (IL)-6 gene promoter can be activated by NF-B in response to inflammatory stimuli such as IL-1. Interestingly, IE2p86 activates the IL-6 promoter, although deletion analysis suggests that there may be competition or interference between the activation mechanisms used by cellular transcription factors and IE2p86 (Gealy et al., 2005). To determine whether IE2p86 interferes with cellular control of the IL-6 promoter and to separate putative activation and inhibition functions of IE2p86, we utilized a mutant that lacks the ability to activate the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) (Yeung et al., 1993).
IE2p86 contains two regions that act as independent activation domains (ADs) when fused to the GAL4 DNA-binding domain (DBD). One of these, referred to here as AD2, lies at the C terminus of IE2p86 (aa 544–579; Pizzorno et al., 1991). Previously, a series of mutants of IE2p86 (strain Towne) were described in which acidic residues in AD2 were replaced by valines (Yeung et al., 1993). These include CAD291, in which all seven acidic residues were substituted (Fig. 1a). Initially, we compared the ability of this mutant and wild-type IE2p86 (strain Towne) to activate the IL-6 promoter. An expression vector for wild-type IE2p86 (Towne) was constructed by cloning full-length IE2p86 cDNA from plasmid pRG360 (a gift from Richard Greaves, formerly of the Department of Virology, Faculty of Medicine, Imperial College London, UK) into pcDNA3 (Invitrogen), to yield pcDNA-IE2[Towne]. To generate a cDNA expression vector for CAD291, a region encoding aa 89–579 was excised from pcDNA-IE2[Towne] by digestion with EcoNI and XhoI, and replaced with an EcoNI–SalI fragment from plasmid pCAD291 (Yeung et al., 1993), to yield pcDNA-IE2[mt291]. A similar strategy was used to generate expression vectors for mutants CAD342 and CAD841. As expected (Gealy et al., 2005), wild-type IE2p86 activated the core IL-6 promoter (Fig. 1b); however, as observed with the HIV-1 LTR (Yeung et al., 1993), mutant CAD291 was completely defective for transactivation. This is consistent with reports that IE2p86 can activate both the IL-6 promoter and the HIV-1 LTR via core elements (Gealy et al., 2005; Walker et al., 1992), implying similar transcriptional mechanisms. Interestingly, mutants CAD342 and CAD841, which have intermediate numbers of acidic residues in AD2, retained partial activity, as they had against HIV-1 LTR (Yeung et al., 1993). In these experiments, expression levels of wild-type IE2p86 and mutants CAD342 and CAD841 were essentially identical. Expression of CAD291 was slightly lower, at about half that of the other proteins (data not shown); however, this lower expression alone was not sufficient to account for the complete loss of activation.
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), the wild-type AD2 fusion activated the E1B promoter in a Gal4 site-dependent manner in Vero cells; however, AD2 from CAD291 lacked any such activity (Fig. 1c
). Moreover, the wild-type AD2–GAL4 fusion also activated the core IL-6 promoter in Vero cells, whereas the mutant AD2 lacked any activity (Fig. 1d). This indicates that, when directed to the promoter, the wild-type AD2 is sufficient for transcriptional activation in Vero cells, but that the mutations present in CAD291 abolish this function. Interestingly, the wild-type AD2 fusion was unable to activate the IL-6 promoter in U373-MG cells, showing that other regions of IE2p86 are also required for activation in these cells (data not shown).
As the loss of transactivation activity by full-length CAD291 could not be accounted for by lower expression alone, we examined other properties of this protein. Previous reports suggest that the extent of SUMO (small ubiquitin-like modifier) modification of IE2p86 is important for activation of some promoters (Barrasa et al., 2003; Hofmann et al., 2000). To examine SUMO modification of CAD291, we transfected U373-MG cells with either pcDNA-IE2[Towne] or pcDNA-IE2[mt291]; 40 h post-transfection, cells were lysed in RIPA buffer containing 4 mM N-ethylmaleimide, then immunoprecipitated overnight at 4 °C with 1 µg anti-IE antibody (MAB810; Chemicon) and 50 µl protein A/G PLUS–agarose beads (Santa Cruz Biotechnology). After washing, samples were analysed by SDS-PAGE and Western blotting for IE2p86, as described previously (Gealy et al., 2005). In both samples, a similar proportion of total IE2p86 was present in a higher-molecular-mass form corresponding to SUMO-conjugated IE2p86 (the identity of the SUMO-conjugated form was confirmed by reprobing using a SUMO-1-specific antibody; data not shown). Thus, the lack of transcriptional activity of CAD291 cannot be attributed to a defect in SUMO modification; moreover, CAD291 retains sufficient structural integrity to be recognized and modified by the SUMOylation machinery. To examine subcellular localization, we cloned full-length coding regions for wild-type IE2p86 or mutants CAD291 or CAD841 into plasmid pEGFP-C2 (Clontech) for expression of fusions to enhanced green fluorescent protein (eGFP). After transfection into human fibroblasts, cells were fixed, stained with 4'-6-diamidino-2-phenylindole (DAPI) and prepared for microscopy as described previously (Gealy et al., 2005). All fusion proteins were expressed in the nucleus in a pattern typical of wild-type IE2p86 (Fig. 2b). Again, this indicates that AD2 mutants retain the ability to be recognized and targeted to the appropriate cellular compartment. Moreover, in vitro protein–protein interaction assays indicated that CAD291 binds GST fusions to TATA-binding protein (TBP), TBP-associated factor (TAF) 4 and TFIIB with affinity similar to that of wild-type IE2p86 (see Supplementary Fig. S1, available in JGV Online). Taken together, these results suggest that the inability of CAD291 to activate transcription is probably due to a specific defect in AD2, rather than to a loss of expression or ‘global’ functions of IE2p86.
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), we asked whether IE2p86 could, conversely, interfere with activation of the IL-6 promoter in response to a physiological stimulus such as IL-1. Cells were transfected with a construct containing nt –234 to +15 of the IL-6 promoter, which includes the NF-B site and is responsive to IL-1 (Fig. 3a). We examined the response to IL-1 in the presence or absence of expression vectors for wild-type IE2p86 or mutant CAD291. As expected (Shimizu et al., 1990; Zhang et al., 1990), IL-1 alone resulted in approximately fourfold stimulation, whereas wild-type IE2p86 activated the promoter by approximately sevenfold (Fig. 3b). However, in the presence of IE2p86, IL-1 was unable to stimulate expression further, suggesting competition or exclusion between the two mechanisms of activation. Moreover, although CAD291 did not activate the promoter, the mutant blocked stimulation by IL-1. Thus, both wild-type IE2p86 and the activation-defective mutant CAD291 interfere with normal cellular control of the IL-6 promoter in response to a physiological stimulus; however, this experiment does not reveal the point at which interference occurs. To investigate this further, we used a reporter containing Gal4 sites upstream of the core IL-6 promoter (Fig. 3a). As reported previously (Vanden Berghe et al., 1998), expression was activated strongly by a synthetic activator in which the GAL4 DBD is fused to aa 286–551 of NF-B p65 (Fig. 3c). Titration of CAD291 resulted in attenuation of activation by GAL4–p65, although CAD291 itself had no effect on expression in the absence of the activator protein. Moreover, although the reporter construct was activated strongly by a GAL4–VP16 fusion, CAD291 had no inhibitory effect on activation by this factor (Fig. 3d). This indicates that CAD291 is not a non-specific attenuator of transcriptional activation, but that its action is (at the IL-6 promoter at least) specific for activation by the AD of NF-B.
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B-mediated activation by preventing binding of this factor to its cognate DNA sites (Taylor & Bresnahan, 2006a). However, this mechanism cannot explain the phenomenon observed here, as the AD of NF-B p65 was targeted to the promoter by the GAL4 DBD. Indeed, the inability of CAD291 to inhibit activation by GAL4–VP16 indicates that CAD291 does not prevent the GAL4 DBD from binding the promoter. Therefore inhibition by CAD291 must occur at a stage subsequent to activator recruitment, presumably by interference with the molecular interactions by which the NF-B p65 AD stimulates transcription. It is known that wild-type IE2p86 can interact with multiple components of the basal transcription machinery in vitro (Caswell et al., 1993; Hagemeier et al., 1992; Jupp et al., 1993; Lukac et al., 1997; Sommer et al., 1994) and with TFIID in vivo (Lukac et al., 1997), although the functional consequences of this interaction on DNA binding and other activities of TFIID are largely unknown. Similarly, NF-B interacts with several basal transcription factors, including TFIIB and subunits of TFIID (Buss et al., 2004; Chen & Greene, 2004; Yamit-Hezi & Dikstein, 1998). It is possible that IE2p86 disrupts such interactions or that IE2p86 ‘reprograms’ TFIID in such a way as to make it unresponsive to NF-B. As mutant CAD291 appears to retain the ability to interact with these factors, such mechanisms might also explain the effects seen here. Alternatively, as IE2p86 can interact with co-activators of NF-B, such as P/CAF and p300/CBP (Bryant et al., 2000; Schwartz et al., 1996), it is possible that IE2p86 competes with NF-B for binding to these cofactors. Moreover, although the GAL–p65 fusion used here contains only aa 286–551 of p65 and thus lacks regions that are acetylated by P/CAF and p300/CBP to regulate DNA binding, it retains aa 310, which is a target for p300/CBP acetylation in the regulation of the transactivation function of NF-B p65 (Chen & Greene, 2004).
Clearly, many questions remain as to the mechanism by which IE2p86 both activates transcription and inhibits NF-B. The effect of mutations in AD2 suggests that this region of IE2p86 makes as-yet-unknown contacts with the transcriptional machinery that are vital for transcriptional activation and are abolished by the mutations present in CAD291. Analysis of such putative interactions, and of transcription-factor recruitment to the IL-6 and other promoters, lies beyond the scope of this report. Nevertheless, our data indicate that mutant CAD291 can be used to separate the functions of transcriptional activation and attenuation of NF-B-dependent gene expression, which are observed with wild-type IE2p86, and that, at the IL-6 promoter, the inhibition of NF-B-dependent expression occurs at a stage subsequent to the recruitment of the activator to the promoter. This suggests a novel mechanism for the attenuation of NF-B-dependent gene expression in the HCMV-infected cell.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 February 2007;
accepted 22 May 2007.
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Deceased. 
