c-Jun homodimers can function as a context-specific coactivator

Abstract

Transcription factors can function as DNA-binding-specific activators or as coactivators. c-Jun drives gene expression via binding to AP-1 sequences or as a cofactor for PU.1 in macrophages. c-Jun heterodimers bind AP-1 sequences with higher affinity than homodimers, but how c-Jun works as a coactivator is unknown. Here, we provide in vitro and in vivo evidence that c-Jun homodimers are recruited to the interleukin-1beta (IL-1beta) promoter in the absence of direct DNA binding via protein-protein interactions with DNA-anchored PU.1 and CCAAT/enhancer-binding protein beta (C/EBPbeta). Unexpectedly, the interaction interface with PU.1 and C/EBPbeta involves four of the residues within the basic domain of c-Jun that contact DNA, indicating that the capacities of c-Jun to function as a coactivator or as a DNA-bound transcription factor are mutually exclusive. Our observations indicate that the IL-1beta locus is occupied by PU.1 and C/EBPbeta and poised for expression and that c-Jun enhances transcription by facilitating a rate-limiting step, the assembly of the RNA polymerase II preinitiation complex, with minimal effect on the local chromatin status. We propose that the basic domain of other transcription factors may also be redirected from a DNA interaction mode to a protein-protein interaction mode and that this switch represents a novel mechanism regulating gene expression profiles.

FIG. 1.

c-Jun is required for upregulated expression of the IL-1β gene in activated macrophages. (A) TPA induces IL-1β mRNA in TF-1 progenitor cells (Northern blotting) or RAW macrophages (reverse transcription-PCR), without affecting control mRNAs (GAPDH [glyceraldehyde-3-phosphate dehydrogenase] or S16). The cDNA template is omitted in the negative control. (B) TPA upregulates c-Jun protein, as detected by Western blot analysis of TF-1 and RAW cells, without affecting PU.1 or C/EBPβ. Note that the C/EBPβ isoform detected in hematopoietic cells corresponds to the protein initiated at Met22. (C) A c-Jun antisense RNA disrupts the induction of c-Jun but not JunD proteins in TF-1 cells, as assessed by Western blotting. TF-1 cells expressing the empty vector (MSCV alone) serve as controls. (D) A c-Jun antisense (AS) RNA decreases the induction of IL-1β mRNA by TPA. Northern blots of RNA harvested at the indicated times were quantified by phosphor imaging, and the IL-1β mRNA signal (lower panel) was normalized to that of GAPDH (not shown). MW, molecular weight; α, anti.

FIG. 2.

Synergistic collaboration between PU.1, C/EBPβ, and c-Jun in driving IL-1β promoter activity. (A) TPA induces an increase in IL-1β promoter activity in hematopoietic cells. The IL-1β promoter from −4400 to +11 or −131 to +11 in luciferase reporter constructs was delivered to TF-1 cells. At the indicated times, cells were harvested to determine luciferase activity, shown here as the ratio of enzyme activity observed in TPA-treated cells versus that observed in control cells which were maintained in standard culture medium. In parallel, cells were also electroporated with a control Rous sarcoma virus-luciferase reporter. (B) Dose-dependent activation of the IL-1β131-luciferase reporter by c-Jun in collaboration with C/EBPβ and/or PU.1 in transfected F9 cells. Transcriptional outputs (graph) are expressed as the increase in activation over the output obtained with the reporter vector alone. Western blot analysis of transfected F9 cells (not shown) and COS expressing PU.1, c-Jun, or C/EBPβ individually (lane 1) or together (lane 2) was performed. Note that both C/EBPβ isoforms are detected in transfected cells. (C) The PU.1 and C/EBP DNA sites of IL-1β131 are required for transactivation in F9 cells by PU.1, C/EBPβ, and c-Jun, or for TPA induction in TF-1 cells. Transcriptional outputs are expressed as the increase in induction by TPA for TF-1 cells or the increase in activation over the reporter alone in F9 cells. (D) PU.1 but not c-Jun can bind IL-1β promoter sequences as revealed by EMSA. EMSAs were performed with undirected reticulocyte (ret) lysates (lanes 2 and 6), in vitro translated PU.1 (lane 3), or in vitro translated c-Jun (lanes 4 and 7). ³²P-labeled DNA probes were the human IL-1β 131 (−131 to +11) (lanes 1 to 4) or a TRE-containing an AP-1 binding site (lanes 5 to 7). (E) PU.1, C/EBPβ, and c-Jun occupy the IL-1β promoter in vivo. ChIP assays with the indicated antibodies or control IgGs were performed from RAW cells or from TF-1 cells with or without TPA treatment. IL-1β promoter sequence was amplified by real-time PCR. Data were expressed as the enrichment over control IgGs and a control genomic sequence. Data shown are the average of three different experiments.

FIG. 3.

Detection of PU.1, C/EBPβ, and c-Jun from macrophages on IL-1β promoter templates by DNA pull-down assay. (A) Schematic representation of the DNA pull-down assay. (B) Diagram of the immobilized templates used in DNA pull-down assays. Mutations of DNA binding sites for PU.1 and/or C/EBPβ are indicated by X. The black box represents the AP-1/TRE binding site. (C) NE from TPA-treated RAW cells were incubated with immobilized DNA templates. After the beads were washed, bound proteins were eluted in SDS-PAGE sample buffer for Western blotting. MW, molecular weight; α, anti.

FIG. 4.

c-Jun is recruited to the IL-1β promoter via DNA-bound PU.1 and C/EBPβ as shown by DNA pull-down assays. (A) Western blot analysis of NE derived from COS cells overexpressing individually C/EBPβ, PU.1, or c-Jun. (B) PU.1 or C/EBPβ binding to the IL-1β promoter requires the integrity of their binding sites at −45 or −90, respectively. DNA pull-down assays and detection of bound proteins are as described in the legend of Fig. 3C. (C) PU.1 and/or C/EBPβ recruit c-Jun to IL-1β promoter templates. PU.1, C/EBPβ, or c-Jun COS NE were incubated with the IL-1β promoter templates as described in the legend of Fig. 3C, either individually or in combination. Bound proteins (upper blots) and unbound c-Jun (bottom blot) were revealed by Western blotting. (D) The experiment is the same as in panel C but with the use of recombinant c-Jun purified from E. coli with the IL-1β template (lanes 1 and 2) or the TRE template in the absence (lane 3) or the presence (lane 4) of a 300-fold molar excess of free double-stranded TRE competitor. (E) c-Jun recruitment to the IL-1β promoter template depends on PU.1 concentration (left) and the integrity of the PU.1 and C/EBP binding sites (right). DNA binding was performed as in panel C with wt (lanes 1 to 3 and 7) or mutant (lanes 4 to 6) DNA templates, a sixfold lower PU.1 concentration (lane 1) or a 300-fold molar excess of free double-stranded oligonucleotide containing the TRE binding site (lane 7). Cβ, C/EBPβ; P, PU.1; J, c-Jun; MW, molecular weight; α, anti.

FIG. 5.

C/EBPβ enhances PU.1 binding to the IL-1β template at limiting concentrations as shown by DNA pull-down and DNase I footprinting. (A) C/EBPβ enhances PU.1 binding to the IL-1β promoter by DNA pull-down assay (as described in the legend of Fig. 4) when the concentration of PU.1 is limiting, corresponding to fourfold less PU.1-containing COS NE (lanes 1 and 2) in a −45 and −90 site-dependent manner (lanes 3 and 4). (B) Schematic representation of the DNase I footprint assay adapted from the DNA pull-down assay. Incubation of COS NE with immobilized ³²P-labeled IL-1β promoter templates, DNase I treatment, and detection of the ³²P-nicked fragments are described in Materials and Methods. (C) Footprints with C/EBPβ, PU.1, and c-Jun alone or in combination. Untransfected (untransf) COS cells showed no protection (compare lanes 1 and 2), whereas the −90 site was protected by C/EBPβ COS-extracts. Protection of the −45 PU.1 DNA binding site by a low concentration of PU.1 (as in panel A) is detectable only in the presence of C/EBPβ (compare lane 5 with lane 3 or 4). No additional protection is observed when Jun is present in the assay with C/EBPβ and a low concentration of PU.1 (compare lane 6 with lane 5) or a higher concentration of PU.1 that allows nearly maximal protection of the −90 and −45 sites (compare lane 8 with lane 7). Cβ, C/EBPβ; P, PU.1; J, c-Jun; P(low), low concentration of PU.1; α, anti.

FIG. 6.

PU.1, c-Jun, and C/EBPβ interact in vitro and in vivo. (A) Pull-down assays using GST, GST-PU.1, or GST-C/EBPβ and ³⁵S-labeled in vitro translated c-Jun, c-Jun ΔB, JunB, or JunD. (B) Schematic representation of Jun proteins and the percentage homology between their TDs or basic leucine zipper domains, c-Jun ΔB, and c-Jun ΔTD. (C) Specificity of c-Jun in transcription activation. The GAL4 reporter (5×GAL4_UAS-tk109-luciferase) was cotransfected in F9 cells with expression vectors for the GAL4_DBD-PU.1 chimera and wt or mutant c-Jun (ΔB and ΔTD), JunB, or JunD. Transcriptional outputs are expressed as synergy (n-fold) (the ratio of the output obtained with both proteins over the sum of their individual outputs). (D) The experiment is the same as that in panel C but with the IL-1β131-luciferase reporter and expression vector for wt PU.1. (E) Transcription activation by c-Jun is decreased by c-Fos. The experiment is the same as that in panel D with the additional use of expression vectors for C/EBPβ and c-Fos, as indicated. (F) c-Jun phosphorylation sites at positions 63 and 73 are dispensable for transcriptional synergy with PU.1. The IL-1β131-luciferase reporter construct was cotransfected in F9 cells with the PU.1 expression vector alone or with expression vectors for wt and c-JunAA mutant (described in the legend of Fig. 7). Outputs are represented as the increase in activation (n-fold) over the reporter alone. Basic/LZ, basic leucine zipper domain.

FIG. 7.

Essential role of c-Jun homodimeric state and of its DBD in physical interactions with PU.1 and C/EBPβ. (A) Schematic representation of HA-tagged c-Jun mutants (left) harboring the indicated point mutations in the DBD (M13, M14, M14b, and M13-14b; + indicates phosphate contacting amino acid residues and a box indicates DNA contacting amino acid residues), in the leucine zipper domain (M17 and M22-23), or in Ser63/Ser73 phospho-acceptor sites (JunAA) and their consequences on DNA binding, dimerization, or phosphorylation state (amino acid positions are according to references and 59). Outputs from pull-down assays between GST-PU.1 (open bars) or GST-C/EBPβ (filled bars) and ³⁵S-labeled in vitro translated wt or mutant c-JunHA proteins are shown as percentages of bound protein compared to wt c-JunHA considered as 100%.

FIG. 8.

Essential role of the c-Jun homodimeric state and of its DBD in recruitment to the IL-1β promoter by PU.1 and C/EBPβ and transcriptional synergy. (A) Essential role of c-Jun homodimeric state and of its DBD in functional interactions with PU.1 and C/EBPβ. F9 cells were cotransfected with the IL-1β131-luciferase reporter and expression vectors for PU.1, C/EBPβ, and wt or mutant HA-tagged c-Jun as described in Materials and Methods. Luciferase outputs are represented as the increase in activation over the output obtained from the reporter alone. c-Jun mutants (as described in the legend of Fig. 7) defective in DNA binding (left and middle graphs) or harboring altered dimerization properties (right graph) were compared to the wt protein in functional interaction with PU.1 and C/EBPβ. (B) c-JunHA mutants (as described in the legend of Fig. 7) defective in DNA binding (lanes 2, 7, 8, and 9) or harboring altered dimerization properties (lanes 3 and 5) were compared to the wt protein (lanes 1, 4, and 6) in their capacity to be recruited to the IL-1β promoter by PU.1 and C/EBPβ in DNA pull-down assays as described in the legend of Fig. 4. The asterisk (lane 10) corresponds to nonspecific interactions of c-JunHA to magnetic beads immobilized with the −45 and −90 mutated IL-1β template (as described in the legend of Fig. 3B) that are occasionally observed, depending on the batch of poly(dI-dC). Equal amounts of wt and mutant c-JunHA proteins were used in the assay (input panels, 5% of total).

FIG. 9.

Recruitment of RNA Pol II to the IL-1β promoter in vitro and in vivo. (A) Additive effects of PU.1 (P) and/or C/EBPβ (Cβ) and recombinant c-Jun (J) on RNA Pol II recruitment. The respective COS NE were incubated alone (lanes 6 and 8), in paired combinations (lanes 7, 9, and 10), or all together (lanes 2 to 5 and 11) with wt (lanes 1, 2, and 6 to 11) or mutant (lanes 3 to 5) immobilized IL-1β templates. Untransfected COS NE served as negative controls (lane 1). Binding volumes and reagents were scaled up fivefold to allow for the detection of RNA Pol II. After the binding reaction, DNA-bound (top blot) and free (bottom blot) RNA Pol II was revealed by Western blotting. (B) Occupancy of the IL-1β or Mmp12 promoters by c-Jun, c-Fos, acetylated histone H3 (AcH3), and RNA Pol II in RAW cells prior to (open bars) and after (filled bars) TPA treatment. The IL-1β and the Mmp12 promoters were amplified by quantitative PCR from chromatin extracts immunoprecipitated with antibodies as shown. Data are expressed as enrichment over control IgGs and control genomic sequences as described in Materials and Methods. α, anti.

FIG. 10.

c-Jun can mediate transcriptional stress response in macrophages via two different mechanisms. Differences in the promoter context of stress-responsive genes appear to be a major mechanistic determinant of c-Jun versatility. One determinant, exemplified by the IL-1β promoter (mechanism 1; our study), is the presence of DNA-bound C/EBPβ and PU.1 that specifically tether c-Jun homodimers via protein-protein interactions. C/EBPβ and PU.1 cooperatively drive basal transcription via their respective DNA binding sites, corresponding to basal histone H3 acetylation (indicated as Ac) and the presence of RNA Pol II (indicated by the square box over the transcriptional start site). Upon stress induction, c-Jun homodimers are recruited to this complex, allowing for a better recruitment of RNA Pol II (Activated; bold right arrow and square box) and an increase in histone H3 acetylation. These conditions further activate the IL-1β gene over its transcriptional ground state (Basal; small right arrow). Alternatively, another determinant (mechanism 2) is the presence of an AP-1 site (indicated by the rectangular box), exemplified by the Mmp12 promoter, as shown in Ogawa et al. (57). c-Jun in this context acts as a heterodimer with c-Fos via binding to the AP-1 site and allows stress-dependent activation of a constitutively repressed promoter (57). In this context, derepression requires Ser63/Ser73 phosphorylation of c-Jun (57), which is dispensable in the context of an already active promoter, as shown here for the IL-1β promoter. Finally, we show that Pol II is undetectable on the Mmp12 promoter in the absence of stress (indicated by an absence of square box over the start site) and that the presence of c-Jun and c-Fos correlates with a sharp increase in both Pol II recruitment and histone H3 acetylation.