TRE-dependent transcription activation by JDP2-CHOP10 association

Abstract

The c-Jun dimerization protein 2, JDP2, is a member of the activating protein 1 (AP-1) family of transcription factors. Overexpression of JDP2 has been shown to result in repression of AP-1-dependent transcription and inhibition of cellular transformation. Other studies suggested that JDP2 may function as an oncogene. Here we describe the identification of CHOP10, a member of the CCAAT enhancer binding proteins, as a protein associating with JDP2. In contrast to the inhibition of transcription by JDP2, JDP2-CHOP complex strongly enhances transcription from promoters containing TPA response elements (TRE), but not from those containing cyclic AMP response elements (CRE). The association between JDP2 and CHOP10 involves the leucine zipper motifs of both proteins, whereas, the basic domain of CHOP10 contributes to the association of the JDP2-CHOP10 complex with the DNA. DNA binding of JDP2-CHOP complex is observed both in vitro and in vivo. Finally, overexpression of JDP2 results in increased cell viability following ER stress and counteracts CHOP10 pro-apoptotic activity. JDP2 expression may determine the threshold for cell sensitivity to ER stress. This is the first report describing TRE-dependent activation of transcription by JDP2 and thus may provide an explanation for the as yet unexplored oncogenic properties of JDP2.

Figure 1.

Identification of the JDP2–CHOP association in yeast using the RRS. Yeast diploid transformants expressing the indicated prey and bait proteins were selected and grown on glucose plates lacking both uracil and leucine. Colonies were grown at 24°C for 2 days and subsequently were replica plated onto galactose plates lacking methionine, leucine and uracil, and grown at 36°C. The baits fused to Ras are: the p21 activating kinase regulatory domain (Ras-Pak), the JDP2 leucine zipper domain (Ras-JDP2-LZ) and full-length ATF3. An empty pMyr expression vector or human CHOP10 fused to v-Src myristoylation signal (pMyr-CHOP10) were used as prey proteins.

Figure 2.

Validation of the CHOP and JDP2 interaction using the mammalian two-hybrid system. NIH3T3 cells were co-transfected with expression plasmids encoding either the Gal4 DBD or Gal4 fused to JDP2 (Gal4DBD-JDP2), together with a luciferase reporter gene under the control of five tandem repeats of the Gal4 DNA binding site (UAS), in the presence or absence of plasmids coding for either wild-type CHOP10 or the indicated CHOP10 mutant. CHOP10 mutants either lacked the leucine zipper domain (34) (CHOPΔLZ) or lacked 23 amino acids corresponding to the CHOP10 basic region (CHOPΔBasic). The luciferase activity obtained with the empty expression vector was designated 1, and all other activities were calculated relative to this. The net effect of CHOP10 on Gal4DBD-JDP2 was calculated. The results represent the mean ± SEM of four independent experiments.

Figure 3.

JDP2 interacts with endogenous CHOP10 in Tg-stressed cells. NIH3T3 cells were transfected with pCEFL expression plasmids encoding HA-tagged JDP2 (HA-JDP2), or the empty vector (HA). For the induction of CHOP10 expression, transfected cells were exposed to 400 nM Tg for the indicated time. Nuclear extracts were used for immunoprecipitation with antibodies against the HA-tag. Western blots of 10% of the total input lysates (A) and precipitated proteins (B) were probed with the indicated antibodies. (C) NIH3T3 cells were either not treated or exposed to 400 nM Tg for 16 h. Nuclear extracts were precleared with a nonrelevant (αNR) polyclonal antibody (lanes 3 and 4), and subsequently an antibody against JDP2 was used for immunoprecipitation (lanes 5 and 6). Western blots of 5% of the total input lysates (lanes 1 and 2) and precipitated proteins using the indicated antibody are shown. Antibodies used were: anti-JDP2, mouse anti-CHOP10 and rabbit anti-c-Jun.

Figure 4.

The CHOP–JDP2 interaction strongly potentiates TRE- but not CRE-dependent transcription. (A) NIH3T3 cells were co-transfected with the indicated expression plasmids (1 μg each) together with the indicated luciferase reporter plasmid (1 μg). Twenty-four hours following transfection, cells were harvested and luciferase activity was determined. Expression plasmids used were: pcDNA empty vehicle (vector), pcDNA based Rat JDP2 (JDP2), human CHOP10 (CHOP), human ATF3 (ATF3) and JDP2 fused to the VP16 activation domain (VP16-JDP2). In transfections in which a single expression plasmid was used the total amount of expression plasmid was adjusted with the corresponding pcDNA empty expression vehicle. Reporters used were: CRE-luc, cyclic AMP response element; CycD-luc, −1850 cyclin D1 promoter; −79-Jun-luc, −79 c-Jun promoter; −73-hCol-luc, −73 collagenase promoter. Luciferase activity was determined and the results represent the average ± SEM from three independent experiments. (B) NIH3T3 cells were co-transfected with the –79-Jun luciferase reporter plasmid as described above, except that the amount of JDP2 expression plasmid was kept constant (1 μg) whereas the amount of CHOP10 was varied as indicated. Luciferase activity was determined and the results obtained with the reporter plasmid in the presence of JDP2 only were designated 1.0. The results represent the average ± SEM from three independent experiments. Total cell lysate was subjected to 12.5% SDS–PAGE followed by western blotting. The nitrocellulose membrane was probed with antibodies against CHOP10. Lane 1 represents JDP2-transfected cells in the absence of CHOP10 expression.

Figure 5.

An intramolecular dimer of JDP2–CHOP transactivates selectively the TRE-containing promoter element in a dose-dependent manner. (A) Schematic representation of the different monomer constructs and chimera used. The amino acid sequence of the linker used is indicated by a single letter code. The leucine zipper and the basic region of the corresponding encoded protein are indicated (ZIP and b, respectively) (B) NIH3T3 cells were co-transfected with expression plasmids, using the indicated amounts (μg), encoding the JDP2–CHOP chimera as well as their monomeric components, together with a luciferase reporter plasmid. Luciferase reporter plasmids used were: −79-Jun-luc, CRE-luc (as described in Figure 4) and CHOP-luc. The CHOP10 luciferase reporter corresponds to the CHOP-C/EBP-specific binding site (34). Twenty-four hours following transfection, cells were harvested and luciferase activity was determined. The luciferase activity obtained with the empty expression vector was designated 1.0 and all other luciferase activities were calculated relative to this. The results represent the average ± SEM of three independent experiments. (C). Electromobility shift assay with Jun-TRE DNA probe. Lysate used included; un-programmed reticulocyte lysate control (retic) or reticulocyte lysate programmed with either JDP2 or JDP2-CHOP chimera expression plasmids as indicated. Binding buffer contained 25 mM NaCl (lanes 1–3), 70 mM NaCl (lanes 4–6) or 150 mM NaCl (lanes 7–9). Protein–DNA complexes were separated on 6% nondenaturing gels, dried and exposed to autoradiography. (D) Western blot analysis of nuclear extract derived from NIH3T3 stably transfected with either CHOP10 (NIH-Chop, lanes 1 and 3) or JDP2–CHOP (JDP2–Chop lanes 2 and 4) probed with anti-JDP2 (left panel) and anti-CHOP10 (right panel) (E) NIH3T3-CHOP or NIH3T3-JDP2-CHOP stable cell lines were transfected with the human Jun-luciferase reporter in the presence or absence of JDP2 expression plasmid, respectively. 24 h following transfection, cells were formaldehyde fixed, lysed and DNA was fragmented by sonication. Cell lysate was either incubated in high salt to reverse cross-link to assess total input (No-IP, lane 3) or used for immunopreciptation. Immunoprecipitation was performed by either nonrelevant antibody (NR-anti-THTR1 lanes 4 and 6) or anti-CHOP10 polyclonal antibody (lanes 5 and 7). Jun primers used for PCR corresponded to 200 bp adjacent the Jun-TRE sequence within the Jun-luciferase reporter plasmid (upper panel). Luciferase control primers located 1000-bp downstream to the luciferase translation start site corresponding to 320-bp fragment (bottom panel). DNA was extracted and used as template for PCR (23 cycles 63°C and 30 cycles 62°C, respectively). Control PCR reaction with either no DNA (−, lane 1) or with −79-Jun-Luciferase reporter plasmid (+, lane 2) were performed. The resulting PCR reaction was separated on 2% agarose gel, ethidium bromide stained and photographed.

Figure 6.

The basic domain of CHOP10 is essential for transcription activation by JDP2–CHOP10. (A) NIH3T3 cells were co-transfected with expression plasmids encoding the indicated JDP2-CHOP chimeras, together with a –79-Jun-luciferase reporter plasmid. JDP2–CHOP chimeras used encoded for the wild type and mutants lacked either the basic domain (JDP2–CHOPΔBasic) or the leucine zipper motif (JDP2–CHOPΔLZ). JDP2 and CHOP10 expression vectors were used as controls. Twenty-four hours following transfection, cells were harvested and luciferase activity was determined. The activity of the reporter in the presence of the vehicle expression plasmid was designated 1.0, and all other activities were calculated relative to this. The results represent the average ± SEM of three independent experiments. The expression levels of CHOP10 and the JDP2–CHOP chimeras were analyzed by western blotting using antibodies against CHOP10. The level of α-tubulin serves as loading control. (B) Reticulocyte lysates programmed with the indicated expression plasmids were used in EMSA with ³²P-labeled Jun-TRE as the DNA probe. The binding reaction was performed with 70 mM NaCl. The gel was dried and exposed to autoradiography. Retic lysate programmed with the corresponding plasmids used for EMSA in the presence of S³⁵ methionine is shown to ensure that similar expression levels of the JDP2–CHOP wild type and mutants were used in the EMSA assay. Programmed and unprogrammed retic lysate were separated on 12.5% SDS–PAGE dried and exposed to autoradiography.

Figure 7.

Ectopic expression of JDP2 reduced the effects of Tg-induced ER stress. (A) Western blot analysis of NIH3T3 stably expressing: vector (Babe), JDP2 (JDP2) and CHOP10 (CHOP). Cells were either untreated (lanes 1–3) or treated with 200 nM Tg for 6 h (lanes 4–6). Nuclear cell lysate was subjected to 12.5% SDS–PAGE followed by western blotting. The nitrocellulose membrane was probed with antibodies against CHOP10 and JDP2. The level of α-tubulin serves as loading control. (B) NIH3T3 pBabe vehicle-transfected cells (blue), NIH3T3 cells overexpressing CHOP10 (orange) and NIH3T3 overexpressing JDP2 (light blue) were exposed to the indicated concentrations of Tg for 24 h. Cell viability was measured in triplicate as described in the Materials and methods section. The results represent the mean ± SEM of three independent experiments. (C) Parental (squares) CHOP10^−/− (circles) MEF cells stable cell lines expressing the indicated proteins treated with Tg as described in (A) The migration of α-tubulin, JDP2, CHOP10 and JDP2-CHOP is indicated. (D) Cells were exposed to different concentration of Tg for 24 h and cell viability was measured as described in (B) The results represent the mean ± SEM of three independent experiments. Parental MEF cell lines used: pBabe vehicle-transfected (blue), JDP2 expressing cells (light blue), JDP2–CHOP expressing cells (yellow). CHOP10^−/− MEF cells used: pBabe vehicle-transfected (green), JDP2 expressing cells (red) and CHOP10 expressing cells (brown).