Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300

Abstract

We report that interferon gamma (IFN-gamma) inhibits transcription of the macrophage scavenger receptor gene by antagonizing the Ras-dependent activities of AP-1 and cooperating ets domain transcription factors, apparently as a result of competition between AP-1/ets factors and activated STAT1 for limiting amounts of CBP and p300. Consistent with this model, STAT1 alpha interacts directly with CBP in cells, and microinjection of anti-CBP and anti-p300 antibodies blocks transcriptional responses to IFN-gamma. Cells lacking STAT1 fail to inhibit AP-1/ets activity, and overexpression of CBP both potentiates IFN-gamma-dependent transcription and relieves AP-1/ets repression. Thus, CBP and p300 integrate both positive and negative effects of IFN-gamma on gene expression by serving as essential coactivators of STAT1 alpha, modulating gene-specific responses to simultaneous activation of two or more signal transduction pathways.

Figure 1

IFN-γ antagonizes M-CSF-dependent expression of scavenger receptor hGH transgenes. (A) SR–hGH fusion genes used to generate transgenic mice are illustrated, indicating the approximate positions of binding sites for AP-1, ets2, and PU.1 proteins. SRmAE–hGH contains point mutations in the AP-1/ets binding motifs that abolish AP-1 and ets2 interaction. (B) Regulation of the SR6.5-hGH, SR-EP–hGH, and SRmAE–hGH transgenes in bone marrow progenitor cells and progenitor cells treated for 3 days with M-CSF (20 ng/ml). (C) IFN-γ inhibition of M-CSF-dependent expression of SR6.5-hGH and SRmEP–hGH transgenes in bone-marrow-derived macrophages. (D) Inhibition of TPA-dependent expression of SR6.5–luciferase and SR-EP–luciferase fusion genes in THP-1 cells. The SR6.5 and SR-EP regulatory elements illustrated in A were fused to a luciferase reporter gene and transfected into THP-1 cells. Cells were treated with TPA and/or IFN-γ, and luciferase activity was quantitated 12 hr later. (E) IFN-γ inhibits TPA and Val-12 Ras-dependent activation of an AP-1/ets-dependent promoter. A luciferase reporter gene containing three copies of the composite AP-1/ets element from the SR-A promoter fused to the minimal (−36 to +33) rat prolactin promoter was transfected into HeLa cells. To stimulate AP-1 and ets activity, cells were either cotransfected with a vector directing the expression of a constitutively active form of Ras (Val-12 Ras) or treated with TPA.

Figure 2

IFN-γ inhibits the transcriptional activities of AP-1 and cooperating ets factors. (A) IFN-γ inhibits the activities of the c-jun transactivation domain. The N-terminal transactivation domain of c-jun was fused in frame to the GAL4 DNA binding domain and used to direct expression of a Gal4-dependent promoter. Transactivation domain function was stimulated by cotransfection of a Val-12 Ras expression plasmid. (B) Effects of IFN-γ on c-Jun kinase and ets2 kinase activities. HeLa cells were rendered quiescent by culture in serum-free media for 16 hr and then treated with IFN-γ, TPA, and/or serum as indicated. Cell lysates were then assayed for Jun N-terminal kinase and ets2 kinase activities (24). (C) Overexpression of CBP relieves IFN-γ antagonism of AP-1/ets activities. The 3× AP-1/ets-promoter was cotransfected into THP-1 cells with 1 μg of a CBP expression plasmid or an equivalent amount of empty expression plasmid and treated with IFN-γ and/or TPA. (D) Overexpression of CBP potentiates transcriptional responses to IFN-γ. A luciferase reporter gene containing eight GASs linked to a minimal prolactin promoter was cotransfected into HeLa cells with increasing amounts of a vector directing expression of CBP (24). Error bars for A, C, and D are standard deviations.

Figure 3

CBP and p300 are required for IFN-γ-dependent transcription. (A) Microinjection of a CBP-specific antiserum inhibits IFN-γ-dependent transcription. Rat-1 fibroblasts were injected with a lacZ reporter gene under the transcriptional control of a minimal promoter containing eight copies of a consensus GAS. Cells were co-injected with control rabbit IgG (1, 2, 4, 5) or rabbit anti-CBP IgG (3, 6), treated with vehicle (1, 4) or IFN-γ (2, 3, 5, 6), and analyzed 12 hr later. Expression of the lacZ reporter gene was determined by 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal) staining (–6). Injected cells were visualized by nuclear staining for IgG using a rhodamine anti-rabbit IgG (1, 2, 3) and by the presence of X-Gal staining, which partially quenches the rhodamine signal. Photomicrographs of representative injected cells are shown. (B) Co-injection of anti-CBP and anti-p300 IgG strongly inhibits IFN-γ-dependent transcription. Cells were injected with the GAS–lacZ reporter gene and anti-CBP IgG, anti-CBP preincubated with the CBP peptide antigen, anti-p300 IgG, or a combination of anti-CBP IgG and anti-p300 IgG as shown. The bar graph indicates the percentage of injected cells that exhibited detectable X-Gal staining. (C) Injection of anti-p300 IgG has no effect on basal levels of expression of a SP1-dependent (SV40)–lacZ reporter gene. For B and C, error bars represent the mean ± SEM of a minimum of three experiments in which at least 200 cells were injected.

Figure 4

Interactions of STAT1α with CBP (A) STAT1α interacts with CBP in cells. 293 cells were transfected with a vector directing expression of an epitope (FLAG)-tagged form of CBP and then treated with IFN-γ or vehicle for 15 min, and whole-cell lysates were prepared. Lysates were immunoprecipitated with an anti-STAT1 antiserum (lanes 2 and 3) or preimmune serum (lane 4). Immunoprecipitates or unprecipitated lysate (lane 1) were resolved by SDS/PAGE, transferred to nitrocellulose membranes, and subjected to Western blotting using an anti-CBP antiserum. (B) IFN-γ-dependent interactions of STAT1α with the N terminus of CBP. The indicated regions of CBP were linked in frame to the DNA binding domain of GAL4 and transfected into 293 cells. Cells were subsequently treated with IFN-γ or vehicle for 15 min, and whole-cell lysates were prepared. Lysates were immunoprecipitated with an anti-STAT1 antiserum and subjected to Western blotting using an anti-GAL4 DNA binding domain antibody. (C) Direct interactions of STAT1α with the N terminus of CBP. ³⁵S-labeled STAT1α or STAT1ΔC truncated at amino acid 705 were produced by translation in vitro and incubated with GST–CBP1–451, GST–CBP-bromo domain, or GST alone. After washing and SDS/PAGE, specifically associated proteins were detected by autoradiography.

Figure 5

AP-1/ets inhibition requires STAT1. The luciferase reporter gene containing three copies of the composite AP-1/ets element described in Fig. 1E was transfected into U3A cells that lack STAT1 or parental 2fTGH cells that contain functional STAT1. Cells were treated with TPA and/or IFN-γ, as indicated, before assay for relative luciferase activity. Error bars are standard deviations