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.1999 Nov;19(11):7589-99.
doi: 10.1128/MCB.19.11.7589.

CHOP enhancement of gene transcription by interactions with Jun/Fos AP-1 complex proteins

Affiliations

CHOP enhancement of gene transcription by interactions with Jun/Fos AP-1 complex proteins

M Ubeda et al. Mol Cell Biol.1999 Nov.

Abstract

The transcription factor CHOP (C/EBP homologous protein 10) is a bZIP protein induced by a variety of stimuli that evoke cellular stress responses and has been shown to arrest cell growth and to promote programmed cell death. CHOP cannot form homodimers but forms stable heterodimers with the C/EBP family of activating transcription factors. Although initially characterized as a dominant negative inhibitor of C/EBPs in the activation of gene transcription, CHOP-C/EBP can activate certain target genes. Here we show that CHOP interacts with members of the immediate-early response, growth-promoting AP-1 transcription factor family, JunD, c-Jun, and c-Fos, to activate promoter elements in the somatostatin, JunD, and collagenase genes. The leucine zipper dimerization domain is required for interactions with AP-1 proteins and transactivation of transcription. Analyses by electrophoretic mobility shift assays and by an in vivo mammalian two-hybrid system for protein-protein interactions indicate that CHOP interacts with AP-1 proteins inside cells and suggest that it is recruited to the AP-1 complex by a tethering mechanism rather than by direct binding of DNA. Thus, CHOP not only is a negative or a positive regulator of C/EBP target genes but also, when tethered to AP-1 factors, can activate AP-1 target genes. These findings establish the existence of a new mechanism by which CHOP regulates gene expression when cells are exposed to cellular stress.

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Figures

FIG. 1
FIG. 1
CHOP interacts with JunD, when bound to the CRE of the rat somatostatin promoter in nuclear extracts of RIN1027-B2 cells. Data shown are EMSA demonstrating RIN1027-B2 nuclear proteins binding to32P-labeled CRE oligonucleotide probe. (A) The oligonucleotide (CRE31) (46), consisting of the somatostatin promoter containing a CRE (SMS-CRE probe). Binding of the32P-labeled probe is fully competed with a 10-fold excess of unlabeled APRE. NS is a nonspecific oligonucleotide control. Note that addition of wild-type CHOP (WT) to the nuclear extract eliminates binding of the C/EBPs whereas addition of the mutant CHOP (MUT) lacking the leucine zipper dimerization domain (CHOP-LZ−) does not interfere with the binding of the proteins. The unknown slowest-migrating complex (arrow) is eliminated by incubation of the nuclear extract with CHOP. (B) The slower-migrating complex is eliminated by preincubation of the nuclear extract with recombinant CHOP and with an antiserum specific for the detection of JunD.
FIG. 2
FIG. 2
CHOP enhances the transcriptional activation of JunD on the somatostatin promoter CRE, and activation depends on the presence of the leucine zipper dimerization domain of CHOP. (A) JunD activates the transcription of a reporter plasmid containing the somatostatin CRE (CRETK-CAT). The reporter plasmid was cotransfected to JEG-3 cells with expression plasmids for either JunD or CREB, in the presence or absence of 8-bromo cAMP (8Br cAMP). EV (empty vector) is the plasmid vector used for expression of JunD and CREB. The data shown are representative of two experiments with similar results. (B) CHOP augments JunD activation of the somatostatin promoter. The mutant CHOP without the leucine zipper dimerization domain (CHOP-LZ−) does not affect transactivation by JunD. The data are the mean and SEM of three independent experiments.
FIG. 3
FIG. 3
CHOP activates transcription of the JunD promoter as efficiently as JunD does itself. (A) Activation of the −219 promoter of the human JunD gene by a JunD expression plasmid. The autoactivation of the JunD promoter occurs via the phorbol response element −53 to −45 (9). The JunD promoter-CAT reporter and JunD expression plasmids were transfected to NIH 3T3 cells. The experiment was performed twice with similar results. (B) CHOP also activates the JunD promoter and augments the activation by JunD (0.5 μg of expression plasmid), and the activation by CHOP is attenuated by deletion of the leucine zipper dimerization domain (CHOP-LZ−). Data are the mean of four experiments and SEM.
FIG. 4
FIG. 4
CHOP cooperates with c-Jun or c-Fos to activate the human collagenase promoter containing a TRE. CHOP enhances the activation of the human collagenase transcriptional reporter, ColTRE-41TK-CAT, by c-Jun (A) or by c-Fos (B). The enhancement of transcription is attenuated when the leucine zipper of CHOP is deleted (CHOP-LZ−). Transcriptional reporter and transcription factor expression plasmids were transfected to NIH 3T3 cells. At 24 h after transfection, the cells were placed in medium containing only 0.5% serum for an additional 24 h to decrease the expression of endogenous AP1 proteins before harvesting.
FIG. 5
FIG. 5
CHOP directly interacts with JunD, c-Jun, and c-Fos, as shown by coimmunoprecipitation and zipper blot studies. (A) Coimmunoprecipitation studies. CHOP, JunD, and c-Fos were translated or cotranslated in a reticulocyte lysate system in the presence of [35S]methionine (top). The proteins were immunoprecipitated (IP) with antisera (antibody [ab]) to CHOP, JunD, and c-Fos (bottom). The immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography. Note that the antiserum to CHOP coimmunoprecipitates both JunD (lane 8) and c-Fos (lane 10). (B) Zipper blot. The proteins designated at the top were bacterially produced recombinant truncated proteins encompassing their respective bZIP DNA-binding and dimerization domain. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was then incubated with a truncated form of CHOP consisting of the bZIP domain that had been labeled with32P (see Materials and Methods) (38). An autoradiogram of the SDS-PAGE gel shows that CHOP binds to c-Fos and c-Jun, as well as to C/EBPβ (positive control), and not to CREB or bovine serum albumin (BSA) (negative controls).
FIG. 6
FIG. 6
GST pulldown experiments show that CHOP binds c-Jun, c-Fos, and JunD. Either wild-type CHOP (WT) or CHOP-LZ− mutant (MUT) fused to GST was incubated with35S-labeled c-Jun or c-Fos (A and B) or JunD, CREB, or C/EBPβ (C and D), and bound proteins were isolated by capture with a glutathione affinity resin. The proteins were analyzed by SDS-PAGE and autoradiography. Autoradiograms of GST-captured proteins compared to input of proteins (INPUT) are shown in panels A and C. Densitometric quantitation of the film images of panels A and C are shown in panels B and D, respectively. The experiments shown were done twice with similar results.
FIG. 7
FIG. 7
CHOP fails to form new stable complexes with AP-1 factors on a TRE. EMSA with the oligonucleotide containing TRE as a gel shift probe and recombinant or in vitro-translated proteins, c-Jun, c-Fos, JunD, and CHOP or its corresponding deletion mutants were used to test the possibility of direct DNA binding of CHOP–AP-1 complexes. (A) A constant amount of recombinant c-Jun was incubated with increasing amounts of recombinant CHOP, its leucine zipper-minus mutant, and the control GST protein. No additional shifting bands were observed in the presence of CHOP. (B) The sifting pattern produced by c-Jun and c-Fos heterodimers was not changed by the presence of increasing amounts of CHOP. A competitive nonlabeled TRE oligonucleotide, but not an unrelated oligonucleotide (UNR), decreased the formation of c-Jun/c-Fos heterodimers in a dose-dependent manner. (C and D) No new retardation complexes, nor changes in binding affinity, were observed when in vitro-translated JunD and c-Fos (C), or c-Jun and c-Fos (D) full-length proteins were used.
FIG. 8
FIG. 8
Evidence of in vivo physical and functional interactions between CHOP and AP-1 proteins. The mammalian two-hybrid system was used to demonstrate in vivo protein-protein interactions. To determine whether such a protein-protein interaction is also functional, the CHOP transactivation domain was retained instead of being replaced with a heterologous activation domain. (A) Map of the luciferase reporter construct used in these experiments. In this reporter, luciferase expression is driven by three GBS as previously described (49). (B) JunD transactivation of the Gal4 reporter was achieved by fusing the DNA-binding domain of Gal4 (GAL4BD) to JunD to create Gal4-JunD. The maps for the wild-type CHOP (CHOP WT) and the corresponding mutants without the leucine zipper (CHOP LZ−) or the basic region (CHOP ΔBR) are also shown. TAD, transactivation domain. (C) CHOP, but not CHOP LZ− or CHOP ΔBR mutants, enhances the transcriptional activity of Gal4-JunD by severalfold. No direct effect of CHOP was observed on the reporter activity, even when the Gal4 binding domain (G4BD) control was cotransfected along CHOP. These data not only demonstrate that the interaction between CHOP and JunD also occurs in vivo but also indicate that this interaction has a functional relevance.
FIG. 9
FIG. 9
CHOP and ATF6 are capable of gene expression regulation by tethering to a preexisting complex. A proposed model for the interaction between CHOP and the AP-1 complex is shown in parallel to the one previously described for ATF6 and SRF (44, 53). Tethering of bZIP proteins to preexisting DNA-protein complexes is a new mechanism by which bZIP transcription factors regulate gene expression. In this case, both CHOP and ATF6 are downstream effectors of the stress-induced p38 MAPK pathway. The tethering mechanism described may facilitate the regulation of a large number of genes by a limited number of transcription factors. This circumstance may have an important significance when cells are exposed to damaging or stressful conditions.
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