A hyper-dynamic equilibrium between promoter-bound and nucleoplasmic dimers controls NF-κB-dependent gene activity
2006; Springer Nature; Volume: 25; Issue: 4 Linguagem: Inglês
10.1038/sj.emboj.7600977
ISSN1460-2075
AutoresDaniela Bosisio, Ivan Marazzi, A Agresti, Noriaki Shimizu, Marco E. Bianchi, Gioacchino Natoli,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoArticle9 February 2006free access A hyper-dynamic equilibrium between promoter-bound and nucleoplasmic dimers controls NF-κB-dependent gene activity Daniela Bosisio Daniela Bosisio Institute for Research in Biomedicine, Bellinzona, SwitzerlandPresent address: General Pathology and Immunology, University of Brescia, Viale Europa 11, Brescia, Italy Search for more papers by this author Ivan Marazzi Ivan Marazzi Institute for Research in Biomedicine, Bellinzona, Switzerland Search for more papers by this author Alessandra Agresti Alessandra Agresti San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Noriaki Shimizu Noriaki Shimizu Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan Search for more papers by this author Marco E Bianchi Corresponding Author Marco E Bianchi San Raffaele University, Milan, Italy Search for more papers by this author Gioacchino Natoli Corresponding Author Gioacchino Natoli European Institute of Oncology, Milan, Italy Search for more papers by this author Daniela Bosisio Daniela Bosisio Institute for Research in Biomedicine, Bellinzona, SwitzerlandPresent address: General Pathology and Immunology, University of Brescia, Viale Europa 11, Brescia, Italy Search for more papers by this author Ivan Marazzi Ivan Marazzi Institute for Research in Biomedicine, Bellinzona, Switzerland Search for more papers by this author Alessandra Agresti Alessandra Agresti San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Noriaki Shimizu Noriaki Shimizu Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan Search for more papers by this author Marco E Bianchi Corresponding Author Marco E Bianchi San Raffaele University, Milan, Italy Search for more papers by this author Gioacchino Natoli Corresponding Author Gioacchino Natoli European Institute of Oncology, Milan, Italy Search for more papers by this author Author Information Daniela Bosisio1,‡, Ivan Marazzi1,‡, Alessandra Agresti2,‡, Noriaki Shimizu3, Marco E Bianchi 4 and Gioacchino Natoli 5 1Institute for Research in Biomedicine, Bellinzona, Switzerland 2San Raffaele Scientific Institute, Milan, Italy 3Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan 4San Raffaele University, Milan, Italy 5European Institute of Oncology, Milan, Italy ‡These authors contributed equally to this work *Corresponding authors: San Raffaele University, Via Olgettina 58, 20132, Milan, Italy. Tel.: +39 02 26434 763; Fax: +39 02 26434 861; E-mail: [email protected] of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, 20141, Milan, Italy. Tel.: +39 02 5748 9953; Fax: +39 02 5748 9851; E-mail: [email protected] The EMBO Journal (2006)25:798-810https://doi.org/10.1038/sj.emboj.7600977 Present address: General Pathology and Immunology, University of Brescia, Viale Europa 11, Brescia, Italy PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Because of its very high affinity for DNA, NF-κB is believed to make long-lasting contacts with cognate sites and to be essential for the nucleation of very stable enhanceosomes. However, the kinetic properties of NF-κB interaction with cognate sites in vivo are unknown. Here, we show that in living cells NF-κB is immobilized onto high-affinity binding sites only transiently, and that complete NF-κB turnover on active chromatin occurs in less than 30 s. Therefore, promoter-bound NF-κB is in dynamic equilibrium with nucleoplasmic dimers; promoter occupancy and transcriptional activity oscillate synchronously with nucleoplasmic NF-κB and independently of promoter occupancy by other sequence-specific transcription factors. These data indicate that changes in the nuclear concentration of NF-κB directly impact on promoter function and that promoters sample nucleoplasmic levels of NF-κB over a timescale of seconds, thus rapidly re-tuning their activity. We propose a revision of the enhanceosome concept in this dynamic framework. Introduction After degradation of their cytoplasmic inhibitors (IκBs), the homo- and hetero-dimers composed of the five NF-κB/Rel proteins (p65/RelA, c-Rel, RelB, p50 and p52) (Thanos and Maniatis, 1995a; Verma et al, 1995; Ghosh et al, 1998) translocate to the nucleus and bind thousands of 9–10 nt κB sites dispersed in the genome (Martone et al, 2003; Natoli et al, 2005) in order to activate transcription of 200–300 genes implicated in inflammation, immune response and antiapoptosis. Mechanistic understanding of NF-κB-regulated transcription requires the definition of the kinetic properties of NF-κB interaction with this large number of sites. In vitro results indicate that in comparison with most transcription factors (TFs) (whose affinity for cognate sites is generally close to 10−9 M), NF-κB dimers have a very high affinity for κB sites (from 10−13 to 10−10 M) (Urban and Baeuerle, 1990; Chen-Park et al, 2002), and generate very stable complexes with a half-life of 45 min (Zabel and Baeuerle, 1990). NF-κB–DNA complexes are further stabilized by cooperative protein–protein interactions with other sequence-specific TFs and architectural proteins, giving rise to multimolecular protein–DNA complexes called enhanceosomes. In vitro the interferon beta (IFN-β) enhanceosome (Maniatis et al, 1998) remains completely stable for over 10 h (Yie et al, 1999), but the existence of stable transcription complexes containing NF-κB in vivo remains to be proven. Here, we carried out an in vivo kinetic and quantitative analysis of NF-κB activation, recruitment to and persistence on target genes, and transcriptional induction. We found that NF-κB association with target sites contained within transcriptionally active genes results in its transient immobilization, that is, a measurable reduction in mobility as compared to the nucleoplasmic NF-κB molecules. Such transient immobilization generates a window of opportunity during which encounter with partner TFs and recruitment of the transcriptional machinery must occur for transcription to start. This window of opportunity is, however, very short: complete turnover of NF-κB on activated chromatin occurs in less than 30 s. The fast dynamics of NF-κB binding and unbinding generates a dynamic equilibrium between promoter-bound and nucleoplasmic NF-κB dimers: cyclic oscillations in the nucleoplasmic pool of NF-κB induce parallel oscillations in promoter occupancy and transcriptional activity, which are dissociated from the occupancy of the same promoter by other TFs. Taken together, these data indicate that NF-κB and the TFs interacting with adjacent binding sites do not generate stable enhanceosomes, but rather undergo short and individual interactions with their respective binding sites. Thus, the enhanceosome can be considered a well-defined set of molecular species, but a hyperdynamic one: each molecular species is represented inside the enhanceosome by individual molecules that belong to it for a very short time. Results Mobility of nucleoplasmic NF-κB We first used fluorescence recovery after photobleaching (FRAP) to analyze the average mobility of nucleoplasmic GFP-tagged NF-κB dimers. The GFP tag did not affect the ability of p65 to bind endogenous genes and to activate their transcription, indicating that this fusion protein is fully functional (Supplementary Figure S1). Because the kinetic properties of a given protein are similar among several different cell types (Phair et al, 2004b), we chose HeLa cells for ease of transfection. In tumor necrosis factor (TNF)-stimulated HeLa cells, p65-GFP showed very rapid recovery kinetics, even faster than that of GR (Figure 1A and B), which is known to turn over rapidly on its binding sites (McNally et al, 2000). The high nuclear mobility of p65 cannot be accounted for by overexpression, as p65-GFP represents only a fraction of endogenous p65 (on average, about 50%; data not shown); moreover, cells expressing low and high levels of fluorescence, and therefore with lower and higher levels of endogenous plus transfected p65, had similar FRAP recovery kinetics (data not shown). Figure 1.Evaluation of p65/RelA nuclear mobility in living cells. All curves were averaged from 15 cells each; bars represent standard error. (A) FRAP analysis of GFP-wt p65 and of a GFP fusion with a mutant p65 that does not bind κB sites. GFP-p65 is cytoplasmic in >95% of HeLa cells before TNF treatment (data not shown). FRAP analysis was started 20 min after TNF stimulation. The difference between the wt and mutant p65-GFP curves is statistically highly significant (P<0.0001). (B) FRAP analysis of GFP-GR (in cells treated with dexamethasone for 20′) and of a nuclear GFP (NLS-GFP) (Bonaldi et al, 2003). (C) Quantitative analysis of FRAP in cells coexpressing p50 and GFP-wt p65 or GFP-mut p65. The difference between the two curves is significant (P A; 26 E>D) (Saccani et al, 2004; G Ghosh, personal communication). Mutant p65-GFP showed a FRAP recovery curve substantially faster than those of wild-type (wt) p65-GFP and of GFP-GR (Figure 1A and B) but much slower than that of GFP alone (Figure 1B), indicating that mutant p65 still interacts with chromatin. We also performed similar FRAP experiments in HeLa cells cotransfected with p65-GFP and p50, and therefore expressing a p50/p65-GFP heterodimer. The results were similar to those obtained with the p65 homodimer: the wt p50/p65-GFP heterodimer has a fast recovery curve, and the heterodimer containing the mutated p65-GFP has an even faster one (Figure 1C). These results are consistent with the idea that the measured kinetics of wt p65 is dominated by the binding to high-affinity sites, which may exceed 10–14 000 in mammalian genomes (Martone et al, 2003; discussed in Natoli et al, 2005). Rapid p65/RelA exchange on cognate binding sites in vivo To further prove the specific binding of GFP-tagged NF-κB species to high-affinity κB sites, we constructed an array of 384 canonical κB sites (Figure 2A) and stably transfected it into HeLa cells. In many cells belonging to several different clones, a bright spot of about 1.5 μm in diameter was evident after transfection with p50/p65-GFP; similar spots were never observed in control cells. A representative nucleus containing one such cluster, and three other clusters from different clones, are shown in Figure 2B. Clearly, a high concentration of κB sites produces a high local concentration of fluorescent p65-GFP molecules. Figure 2.NF-κB binding to high-affinity κB sites in living cells. (A) Schematic representation of the 384-κB construct, containing 128 repetitions of a basic unit of three tandem κB sites. (B) Visualization of an array of 384 κB sites in living cells. Cells bearing the 384-κB construct were cotransfected with p50 and GFP-p65, stimulated with TNF for 20 min, and viewed with a confocal microscope. Four representative nuclei (from different clones) are shown. (C) Selected FRAP images showing bleaching and recovery of one κB site array and a different area in the nucleus (encircled). (D) FRAP curves for p50/p65-GFP averaged from 15 nondescript areas in the nucleus ('nucleus') and eight 384 κB site arrays ('cluster'). Bars represent standard error. (E) FLIP analysis of NF-κB exchange on the κB site array. A representative nucleus is shown before and after the design of the bleach area (red). The image series shows the fluorescence loss from the array over time. FLIP curves (generated as described in Supplementary Figure S2) are averaged from seven 384 κB site arrays and 15 nondescript areas in the nucleus. Bars represent standard error. The difference between the two curves is statistically nonsignificant. Download figure Download PowerPoint We next measured FRAP recovery curves. Recovery on the arrays of κB sites was slower than on surrounding chromatin (Figure 2C and D), suggesting that NF-κB is immobilized onto high-affinity sites for longer than onto average genomic sequences, which contain κB sites with a wide range of affinities. Nonetheless, recovery was fast in absolute terms even on arrayed κB sites: In all, 50% recovery occurred in less than 1 s and 80% recovery was observed in about 5 s. Fluorescence on the 384 κB sites did not recover completely within 15 s, whereas fluorescence in other areas of the nucleus did. There are two most likely explanations for this behavior: a high probability of recapture of bleached NF-κB molecules dissociating from the tightly clustered κB sites, or the presence of an immobile fraction of NF-κB molecules. To distinguish between these two possibilities, we used a modified fluorescence loss in photobleaching (FLIP) protocol. More than 90% of the nucleus (excluding the array or a control area) was repeatedly bleached (red area in Figure 2E), and fluorescence loss from the array (or the control area) into the bleached area was measured (Figure 2E). Fluorescent molecules on the array remain fluorescent if they do not move, whereas those leaving the array are bleached as soon as they enter the area targeted by the laser. In these conditions, even a minute amount of fluorescent molecules remaining stably bound to the cluster should stand out clearly. Differently from FRAP, which is affected by both the association and dissociation rates, our modified FLIP is intended to measure the dissociation rate of fluorescent molecules from the array. Some loss of fluorescence is due to photobleaching inherent in repeated imaging of the nucleus: we corrected for this effect as shown in Supplementary Figure S2. Even after correction, analysis of the curve and the errors (Supplementary Figure S3) indicated that less than 1% of NF-κB molecules remain on the cluster after 20 s. These results indicate that the maximal residence time of NF-κB on high-affinity sites in vivo is less than 20 s; this residence time is not much different from that on average chromatin, suggesting once again that NF-κB mobility in average chromatin is dominated by interactions with high-affinity sites. NF-κB turnover on transcriptionally functional promoters The array of κB sites is artificial and, most important, not transcriptionally active. We then set out to measure the kinetics of NF-κB exchange at arrays of transcriptionally active NF-κB-dependent genes. We constructed a basic unit consisting of the human immunodeficiency virus (HIV) 5′ long terminal repeat (LTR), which contains two canonical κB sites, cloned upstream of a reporter gene encoding a cyan fluorescent protein (CFP) directed to peroxysomes via a C-terminal SKL tripeptide (Figure 3A). This NF-κB-responsive gene unit was cloned into the pSFV-dhfr vector. This plasmid contains both a mammalian replication initiation origin and a matrix attachment region from the Chinese hamster dhfr gene: when integrated into the genome, it initiates events similar to gene amplification in cancer cells, and it generates tandem repeats of up to 10 000 copies (Shimizu et al, 2001). We obtained arrays of several hundred plasmid copies in immortalized fibroblasts lacking both the p50 and p65 NF-κB subunits. Clone 2.24 was estimated by real-time polymerase chain reaction (PCR) to bear a repeat made of almost 2000 plasmid units (Supplementary Figure S4). After transfection of p65 and p50, TNF stimulation enhanced transcription of the CFP-SKL genes, as indicated by the increased CFP mRNA levels (Figure 3B), thus demonstrating that the array is transcriptionally active and NF-κB-regulated. Under the same conditions, HA-tagged p65 was efficiently recruited on the array, as shown by an anti-HA chromatin immunoprecipitation (ChIP) (Figure 3C). The HIV-CFP-SKL array was readily visualized upon TNF stimulation of cells reconstituted with untagged p50 and p65 tagged with monomeric red fluorescent protein (mRFP) (Figure 3D, left), although it was much less bright than the 384-κB site array (indicating a comparatively low density of κB sites). The morphology of the clusters varied from diffused (Figure 3D, top line) to beaded structures (bottom line), and was similar to the morphology of actively transcribed chromosome fibers and GR-responsive MMTV clusters (Muller et al, 2004). Figure 3.An array of transcriptionally functional NF-κB-regulated gene units. (A) Schematic representation of the NF-κB-regulated gene units amplified in clones 2.24 and 4.14. (B) NF-κB-dependent induction of CFP-SKL and luciferase mRNA. Clones 2.24 and 4.14 were transfected with p65 and left untreated (lanes '1') or stimulated with TNF-α for 1 h (lanes '2'). (C) Anti-HA-p65 ChIP on the HIV-LTR-CFP-SKL array. Clone 2.24 was transfected with HA-p65+p50 and stimulated with TNF-α for 15′ or 30′ as indicated. (D) Clones 2.24 and 4.14 were transfected with mRFP-p65 and GFP-rpbI; cells were stimulated with TNF for 30′ and analyzed in vitro. Details of representative nuclei are shown, in which the arrays display a different morphology. Download figure Download PowerPoint In response to TNF stimulation, the HIV-CFP-SKL array recruited RNA-Pol II (GFP-RpbI) with a pattern that extensively overlapped the one generated by mRFP-p65 (Figure 3D, left), indicating that NF-κB recruitment coincides with productive transcription (or at least with the assembly of a preinitiation complex). FRAP on the gene array showed 80% recovery in less than 3 s, and apparently complete recovery in about 30 s (Figure 4A). Recovery was anyway slower than that measured in the nucleoplasm, suggesting a transient immobilization of p65 on the array. The modified FLIP protocol described in the preceding section showed that the rate of NF-κB loss from the array was detectably smaller than the rate of loss from the control area (Figure 4B), demonstrating (in agreement with FRAP data) that NF-κB molecules spend a longer time on the cluster containing the HIV-1 LTR than in nondescript areas of the nucleus. However, all or nearly all NF-κB fluorescence was lost both from the array and the control area, suggesting that NF-κB molecules are not stably engaged with their sites. The experimental error does not allow us to formally and conclusively rule out the existence of an immobile fraction; however, similar to what we found for the array of κB sites (Supplementary Figure S3), if an immobile fraction exists it must be smaller than 2% (data not shown). Figure 4.Dynamics of NF-κB exchange on trascriptionally active chromatin. FRAP (A) and FLIP (B) curves on clone 2.24 (HIV-LTR) and 4.14 (synthetic gene). Curves are averaged from 10 clusters per type, and 20 nondescript areas in the nuclei (the two types of clones have been merged as there is no statistical difference between them). Bars represent standard error. The differences between the curves representing the two types of clusters are statistically nonsignificant, whereas the difference between any of the clusters and the rest of the nucleus is highly significant (P<0.001). In (B), the image series show the fluorescence loss over time at the cluster from one cell each of clones 2.24 and 4.14. FLIP curves are averaged from 10 cells each; bars represent standard error. The differences between the curves representing the two types of clusters are statistically nonsignificant, whereas the difference between any of the clusters and the rest of the nucleus is highly significant (P<0.002). (C) FRAP and (D) FLIP for S536A p65-GFP on the HIV-LTR cluster (clone 2.24). Curves are averaged from eight clusters and 10 nondescript areas in the nuclei; bars represent standard error. Download figure Download PowerPoint We also generated a second cell line (4.14) with an array of about 900 copies of a gene unit containing a synthetic NF-κB-regulated promoter (with three κB sites upstream of a minimal SV-40 promoter) driving the expression of a luciferase reporter (Figure 3A, B and D, right). Remarkably, results were indistinguishable from those obtained with the HIV-LTR (Figure 4A and B). Therefore, the kinetics of NF-κB exchange on transcriptionally active chromatin was similar in two completely unrelated gene arrays. These results indicate that NF-κB interactions with target genes last a few seconds; as a consequence, the NF-κB-dependent events leading to transcriptional activation must take place on a similar timescale. Active removal of NF-κB from chromatin Such a fast exchange can be explained by at least two types of mechanisms. First, as NF-κB interaction with DNA is extremely salt-sensitive (Phelps et al, 2000), the exchange might occur spontaneously at the relatively high salt concentration in the cell (170 mM). The second possibility is that rapid exchange is active and catalyzed at least in part by nucleosome remodeling and proteasomal degradation, as demonstrated for GR recycling (Fletcher et al, 2002; Nagaich et al, 2004; Stavreva et al, 2004; Agresti et al, 2005). To directly address this point, we employed a p65 mutant (S536A) showing severely impaired signal-induced proteasomal degradation (Lawrence et al, 2005). FRAP recovery curves of S536A-p65-GFP clearly showed a large fraction of residual fluorescence on the clusters of HIV LTR even after 45 s; a similar result was obtained by FLIP (Figure 4C and D). These results demonstrate that NF-κB can reside for longer times on active genes, if its turnover is not catalyzed. A dynamic equilibrium between promoter-bound and nucleoplasmic NF-κB A prediction of the kinetic microscopy data is that NF-κB bound to target promoters should be in dynamic equilibrium with nucleoplasmic NF-κB molecules: if promoters are freely accessible, any change in nuclear abundance of NF-κB should cause an immediate and comparable change in target gene occupancy. To test this prediction, we used a quantitative ChIP assay to measure NF-κB recruitment to endogenous promoters in a cellular system in which NF-κB nuclear levels oscillate over time because of the rapid sequence of activation events (degradation of the IκBs and nuclear import) and negative feedback mechanisms (IκBα resynthesis) (Hoffmann et al, 2002). In Raw264.7 murine macrophages, TNF-α and LPS induced different patterns of NF-κB oscillations (Figure 5A and B) that were detected by electrophoretic mobility shift assays (EMSA) carried out with a canonical κB site as a probe (5′-GGGACTTTCC-3′). TNF-α induced a rapid and short pulse of NF-κB activity, followed by two additional pulses (peaking at about 120′ and 195′; Figure 5A, upper panel and data not shown). LPS-induced pulses were wider than those induced by TNF (Figure 5B, upper panel). Downregulation of NF-κB activity after the first pulse was less complete than with TNF-α; the second pulse of LPS-induced NF-κB activity was comparable in amplitude to the first one and was associated with a detectable simultaneous reduction in IκBα steady-state levels (Figure 5B). Figure 5.Pulses of NF-κB activity and recruitment of NF-κB to target genes. (A, B) Upper panels: EMSAs showing NF-κB activity in TNF-α- and LPS-stimulated Raw264.7 cells, respectively. A canonical κB site was used as a probe. Kinetics of IκBα degradation and resynthesis are shown. Lower panels: ChIP assays were carried out with an anti-p65 (red) or an anti-c-Rel (light blue) antibody, and recruitment to the IκBα or MIP-2 genes was measured by Q-PCR. Conventional ChIPs migrated on EtBr-stained gels are shown in small insets. Data are from a duplicate experiment and are representative of three independent experiments with similar results. (C) Re-ChIPs were carried out using the indicated antibodies on unstimulated and LPS-stimulated (15′) Raw264.7 cell extracts. The chromatin immunoprecipitated in the first ChIP was divided into five aliquots and re-precipitated as indicated. Download figure Download PowerPoint NF-κB activity induced by both stimuli comprised the typical p50/p65 dimer and the p65/c-Rel heterodimer, which in many cell types is as abundant as p50/p65 (Hansen et al, 1994) and is best detected using the κB site from the human urokinase plasminogen activator (uPA) gene (5′-GGGAAAGTAC-3′) (Supplementary Figure S5). The contribution of other complexes (e.g. p50/c-Rel, p52/c-Rel) to NF-κB activity was quantitatively small. In response to TNF-α stimulation, p65/RelA and c-Rel occupancy of the IκBα and MIP-2 gene promoters (Figure 5A) closely resembled the profile of the bulk NF-κB activity as detected by EMSA: a high-amplitude cycle of NF-κB activity was followed by a second, low-amplitude cycle. In response to LPS stimulation, again both Rel proteins were detected onto DNA by ChIP in a manner that closely paralleled the cycles of NF-κB activity detected by EMSA (Figure 5B). Notably, the profiles of NF-κB activity induced by TNF-α and LPS were different; this indicates that the coincidence between total NF-κB activity and promoter occupancy is a causal relationship, and not a chance occurrence. Other promoters behaved identically to MIP-2 and IκBα (Supplementary Figure S6). The identity of the NF-κB dimers recruited to chromatin in response to stimulation was further investigated by Re-ChIP experiments, in which the chromatin pulled-down in the first round of immunoprecipitation was re-precipitated with an antibody directed against a different NF-κB protein. Re-ChIPs showed that at a single time point multiple NF-κB dimers were promoter associated (Figure 5C). This indicates that each pulse of NF-κB activity detected by anti-p65 or anti-c-Rel ChIP in fact represents the sum of the signals generated by multiple p65- or c-Rel-containing dimers. These results demonstrate that NF-κB-regulated promoters are occupied in proportion to the NF-κB-binding activity available at any one time, as predicted on the basis of the kinetic microscopy data. Recruitment profile of partner TFs to NF-κB-dependent genes A second prediction of the kinetic microscopy data is that NF-κB should not generate stable enhanceosomes composed of NF-κB and partner TFs bound to target promoters in a cooperative fashion. If stable enhancesomes existed, removal of NF-κB from target genes at the end of the first occupancy cycle detected by ChIP should be accompanied by the simultaneous removal of partner TFs (i.e. by the disassembly of the whole enhanceosome). To address this issue, we analyzed recruitment of multiple TFs to the canonical, rapidly activated, NF-κB-dependent MIP-2 gene. The MIP-2 gene promoter contains an AP-1/CRE site adjacent to the distal κB site (Figure 6A) and a C/EBP site immediately downstream of the proximal κB site. In Raw 264.7 cells, LPS stimulation induced a sustained c-Jun amino-terminal phosphorylation and an increase in c-Jun levels, whereas TNF-α stimulation did not (Figure 6A). LPS induced a sustained occupancy of the MIP-2 promoter by c-Jun (Figure 6B). Remarkably, the c-Jun association profile with MIP-2 was completely different from that of p65/RelA and c-Rel; in particular, c-Jun did not show any cyclic behavior and was detected at the MIP-2 promoter between the two cycles of NF-κB recruitment (Figure 6B). We next analyzed the recruitment to the MIP-2 promoter of additional CRE/AP-1-binding proteins (Jun B and ATF-3) and of C/EBPβ. MIP2 gene occupancy by JunB and ATF-3 (Figure 6D) paralleled the increase in their nuclear concentration induced by LPS stimulation (Figure 6C). Nuclear levels of C/EBPβ did not change within this time frame (Figure 6C), but it was anyway efficiently recruited (Figure 6D). In all cases, these TFs displayed occupancy profiles different from those of NF-κB and could be detected at the promoter irrespective of NF-κB occupancy. Figure 6.Recruitment of partner TFs to the MIP-2 gene promoter is dissociated from NF-κB occupancy. (A) Relative position of the κB, CRE and C/EBP sites in the MIP-2 promoter. Immunoblots of LPS and TNF-stimulated cells were probed with antibodies to phospho-Ser63 c-Jun or total c-Jun. A Coomassie staining of the filter is also shown. (B) Anti-c-Jun ChIPs in LPS and TNF-stimulated Raw264.7 cells. An overlay of p65/RelA (red) and c-Jun (green) recruitment curves in cells stimulated with LPS is shown on the right. (C) Immunoblots of LPS-stimulated Raw264.7 cells were probed with antibodies to JunB, ATF-3 or C/EBPβ. (D) Kinetics of JunB (left), ATF-3 (middle) and C/EBPβ (right) recruitment to the MIP2 promoter in LPS-stimulated Raw264.7 cells were assayed by ChIP followed by real-time PCR. (E) Dynamics of histone H4 acetylation (black) and p65 recruitment profiles (red) at the MIP-2 (left) and IκBα (right) gene promoters in LPS-stimulated Raw264.7 cells. Acetylated and total H3 levels are also shown. Download figure Download PowerPoint Analysis of a slow-induced gene (Rantes/Ccl5) showed that p65 was recruited only during the second cycle of NF-κB nuclear accumulation, whereas c-Jun, JunB and C/EBPβ occupied the promoter already at early time points (Supplementary Figure S7). Therefore, NF-κB and partner TFs do not associate with, and are not released from target promoters as a single and stable complex. Histone acetylation patterns at NF-κB-dependent genes are dynamical but not cyclical NF-κB has been reported to interact with histone acetyltransferases and to mediate their recruitment to chromatin (Perkins et al, 1997; Zhong et al, 1998; Sheppard et al, 1999). Because estrogen receptor cycling on the pS2 gene promoter is associated
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