Coactivator TIF1beta interacts with transcription factor C/EBPbeta and glucocorticoid receptor to induce alpha1-acid glycoprotein gene expression

Abstract

The transcription of the alpha1-acid glycoprotein gene is induced by inflammatory cytokines and glucocorticoids. C/EBPbeta is a major transcription factor involved in the induction of the agp gene by some cytokines. In this report, we have identified a novel transcriptional intermediary factor, TIF1beta, which could enhance the transcription of the agp gene by the glucocorticoid receptor (GR) and C/EBPbeta. TIF1beta belongs to a subgroup of RING (really interesting new gene) finger proteins that contain a RING finger preceding two B box-type fingers and a putative coiled-coil domain (RBCC domain). Immunoprecipitation experiments showed that the interaction between GR and TIF1beta is ligand independent. The overexpression of the TIF1beta gene enhances GR-regulated expression in a ligand- and glucocorticoid-responsive element (GRE)-dependent manner. TIF1beta can also augment C/EBPbeta-mediated activity on wild-type and GRE-mutated agp genes, but this augmentation is diminished when all three C/EBPbeta-binding elements are mutated. Functional and biochemical characterizations indicated that the bZIP domain of C/EBPbeta and the RBCC domain, plant homeodomain finger, and bromodomain of TIF1beta are crucial for the interactions of these proteins. Taken together, these results suggest that TIF1beta serves as a converging mediator of signal transduction pathways of glucocorticoids and some inflammatory cytokines.

FIG. 1

Protein-protein interactions between C/EBPβ and TIF1β. (A) P388D1 whole-cell extracts were immunoprecipitated with anti-C/EBPβ polyclonal antibody (C/EBPβ) or preimmune serum (PI). The precipitated proteins were subjected to SDS-PAGE followed by Western blotting with anti-C/EBPβ monoclonal or anti-TIF1β polyclonal antibodies. (B) Several recombinant C/EBPβ constructs were incubated with glutathione bead-immobilized GST-TIF1β (lanes 4 to 6) or GST (lanes 7 to 9). After extensive washes, the protein complexes were subjected to SDS-PAGE and immunoblotted with anti-C/EBPβ antibody. Lanes 1 to 3 represent direct loading of different recombinant C/EBPβ constructs. FL, N, and C represent full-length C/EBPβ, C/EBPβ-N, and C/EBPβ-C, which are described in Materials and Methods.

FIG. 2

Stimulation of C/EBPβ-mediated gene activation by TIF1β. (A) BHK cells were cotransfected with 2 μg of AGP (WT)-CAT, 0.5 μg of pCMV-C/EBPβ (C/EBPβ), and increasing amounts of pcDNA3-TIF1β (TIF1β) (1, 5, and 10 μg). (B) In 3.5-cm-diameter petri dishes, BHK cells were cotransfected with 0.5 μg of AGP (WT)-CAT, AGP (C mutant)-CAT, AGP (D mutant)-CAT, AGP (E mutant)-CAT, AGP (CDE mutant)-CAT, or AGP (GRE mutant)-CAT and 0.1 μg of pCMV-C/EBPβ with or without 2 μg of pcDNA3-TIF1β. The data represent the average activity of two independent duplicate experiments. The fold induction by TIF1β is indicated below the panel. mt, mutant. (C) (Left panel) In 3.5-cm-diameter petri dishes, BHK cells were cotransfected with 0.5 μg of AGP (WT)-CAT and 0.05 μg of pCMV-C/EBPα in the presence or absence of 2 μg of pcDNA3-TIF1β. (Right panel) BHK cells were transfected with 0.25 μg of pC/EBPβ-CAT, and 0.1 μg of pCMV-C/EBPβ or both 0.1 μg of pRSV-CREB and 0.1 μg of pCMV-PKAc in the absence or presence of 2 μg of pcDNA3-TIF1β. Error bars indicate standard deviations. −, pcDNA3 vector control.

FIG. 3

Functional and biochemical characterization of C/EBPβ-interacting domains of TIF1β. (A) (Upper panel) Schematic representation of several TIF1β expression vectors; numbers denote amino acid positions. (Lower panel) Transient transfection assays. BHK cells (in 3.5-cm-diameter petri dishes) were transfected with 0.5 μg of AGP (WT)-CAT, 0.1 μg of pCMV-C/EBPβ, and 2 μg of pcDNA3-TIF1β (full length [fl] or from amino acid 1 to 563 or 1 to 372, or full length but with amino acids 80 to 383 deleted). (B) (Upper panel) Schematic representation of several GST-TIF1β fusion proteins. (Lower panel) Protein pull-down assay. Glutathione bead-immobilized recombinant GST-TIF1β (fl or from amino acid 80 to 383, 383 to 563, or 383 to 834) incubated with full-length recombinant C/EBPβ (100 ng). After extensive washes, the protein complex was analyzed by immunoblotting with anti-C/EBPβ antibody. −, pcDNA3 vector control.

FIG. 4

TIF1β potentiates GR-activated gene expression. (A) HeLa cells were transiently transfected with 2 μg of pMMTV-CAT reporter plasmid, 1 μg of pRSV-hGR (GR), and increasing amounts of pcDNA3-TIF1β (TIF1β) (1, 5, and 10 μg). Each assay was done in the presence of 1 μM dexamethasone. (B) BHK cells were transiently transfected with 2 μg of AGP (WT)-CAT reporter plasmid. Other plasmids and conditions of treatment are as described for panel A. Relative CAT activity normalized with an internal control represents an average of two independent duplicate experiments. Error bars indicate standard deviations.

FIG. 5

Protein-protein interactions between GR and TIF1β. (A) Immunoprecipitation of GR by anti-TIF1β antibody. P388D1 whole-cell extracts were immunoprecipitated with anti-TIF1β or control (PI) antibody in the presence (+) or absence (−) of 1 μM dexamethasone (DEX) and subjected to SDS-PAGE and Western blotting with anti-GR polyclonal antibody. (B) Recombinant GST-TIF1β was incubated with recombinant GR in the presence (+) or absence (−) of 1 μM dexamethasone and then immunoprecipitated with anti-TIF1β antibody or preimmune serum. After extensive washes, the protein complex was analyzed by Western blotting with anti-GR antibody. Recombinant GR used for the interaction assay (1/5 input) was included as a control.

FIG. 6

GRE-dependent and C/EBPβ-binding-element-independent augmentation of the activation of AGP-CAT by GR and TIF1β. AGP (WT)-CAT, AGP (CDE mutant)-CAT, or AGP (GRE mutant)-CAT was used as a reporter (see Materials and Methods). In 3.5-cm-diameter petri dishes, BHK cells were cotransfected with one reporter plasmid (1 μg) and 0.5 μg of pRSV-hGR (GR), 5 μg of pcDNA3-TIF1β (TIF1β), or both in the presence or absence of 1 μM dexamethasone (DEX). The fold induction for each experiment is shown. The values are the averages of at least two independent experiments. Error bars indicate standard deviations.

FIG. 7

Activation of the agp gene by C/EBPβ, GR, and TIF1β. BHK cells (grown in 6-cm petri dishes) were transfected with 2 μg of AGP (WT)-CAT and 0.5 μg of pCMV-C/EBPβ (C/EBPβ), 1 μg of pRSV-hGR (GR), or 5 μg of pcDNA3-TIF1β (TIF1β) in various combinations in the absence or presence of 1 μM dexamethasone (DEX). Normalized relative CAT activity represents an average of two independent experiments. Error bars indicate standard deviations.