The thyroid hormone receptor antagonizes CREB-mediated transcription

Abstract

Combinatorial regulation of transcription involves binding of transcription factors to DNA as well as protein-protein interactions between them. In this paper, we demonstrate the existence of a mutual transcriptional antagonism between the thyroid hormone receptor (TR) and the cyclic AMP response element binding protein (CREB), which involves a direct association of both transcription factors. TR inhibits transcriptional activity of CREB and represses activation of cAMP response element (CRE)-containing promoters. TR does not bind to the CRE in vitro, but in vivo the liganded receptor is tethered to the promoter through protein-protein interactions. In turn, expression of CREB reduces TR-dependent transcriptional responses. The association of TR with CREB inhibits the ability of protein kinase A to phosphorylate CREB at Ser133, and leads to a reduction in the ligand-dependent recruitment of the p160 coactivators by TR. These results indicate the existence of a transcriptional cross-talk between CREB and TR signalling pathways, which can have important functional consequences.

Fig. 1. T3 represses CRE-mediated transactivation, irrespective of cellular CBP levels. (A) GH4C1 cells were used for transient transfection with the indicated CRE-containing constructs. Assays were carried out in cells incubated for 48 h with T3, and with TPA or forskolin (FK) during the last 4 h. CAT activity was quantitated and is expressed as the fold induction over the values obtained in the corresponding untreated cells. (B) The GHF-1 promoter construct (10 µg) was cotransfected into GH4C1 cells, with 10 µg of an expression vector for CBP or with the same amount of a non-coding vector. CAT activity was determined in cells treated as in (A).

Fig. 2. T3 inhibits CREB phosphorylation. (A) Western blot analysis of GH4C1 cell extracts with phospho-CREB (P-CREB) and total CREB antisera after treatment with T3 for the indicated time periods and forskolin (FK) for the last 15 min. The upper panel shows quantitation of the P-CREB blot. (B) The cells were incubated with T3 for 24 h and/or FK during the last 15 min, fixed, and analysed by indirect immunofluorescence with the P-CREB antibody. Background (Bkg) immunofluorescence obtained in the absence of the antibody is also shown. (C) Western blot analysis with P-CREB and total CREB antisera in GH4C1 cells treated with FK for 15 min, or with TPA for the indicated time periods. In (D) the cells were incubated for T3 for 24 h and with TPA for 15 min. Immunofluorescence assays with the P-CREB antibody under the same conditions are shown in (E).

Fig. 3. The thyroid hormone receptor inhibits CREB transcriptional activity. In (A), GH4C1 cells were transfected with 5 µg Gal-DBD alone (Gal), or fused to either native CREB (Gal-CREB) or CREB mutated in Ser133 (Gal-CREB^Ala133). These constructs were cotransfected with 10 µg of the reporter plasmid 4xUAS-LUC, and luciferase activity was determined in cells treated with T3 for 48 h and/or TPA and forskolin (FK) during the last 4 h. Reporter activity is expressed as the fold induction over the values obtained in the corresponding untreated cells. (B) HeLa cells were cotransfected with 3 µg of 4xUAS-LUC and 1.5 µg of Gal-CREB, either alone or in combination with an expression vector for TR (1 µg). The Gal-DBD plasmid was also used as a control. Luciferase activity was determined as in (A), after incubation with T3 and/or FK and TPA.

Fig. 4. Trichostatin A (TSA) modulates the inhibitory effect of T3 on CREB-dependent transcription. In (A), GH4C1 cells were transfected with the GHF-1–CAT construct and incubated for 48 h with T3 in the presence and absence of 200 nM TSA. When indicated, the cells were also treated with TPA and forskolin (FK) during the last 4 h. CAT activity is expressed as the fold induction over the values obtained in the untreated cells. In (B), GH4C1 cells were transfected with Gal-DBD alone (Gal) or with Gal–CREB. Luciferase activity was determined as in (A), after incubation with T3, TSA and/or FK and TPA.

Fig. 5. In vitro interaction of TR with CREB. (A) Pull-down assay performed with 1 µg GST–0 or GST–TR and 10 µl of ³⁵S-labelled CREB in the presence and absence of 1 µM T3. Input represents 20% of the protein used. (B) Schematic representation of TR showing the different functional domains. The wild-type receptor (wt) and the indicated TR mutants were used in pull-down assays with [³⁵S]CREB. (C) Representation of CREB domains that were used in the interaction assay with [³⁵S]TR in the presence and absence of T3. (D) In vivo interaction of TR with CREB. GH4C1 cells were transfected with HA-TR and CREB, and were treated with T3 for 24 h and forskolin for the last 15 min. Immunoprecipitates (1.2 mg) with 1 µg anti-HA antiserum (α-HA) or with a control serum were subjected to western blot analysis together with 10% of the cell extract (input). A non-specific band (n.s.) appearing in the immunoprecipitates is labelled with an asterisk.

Fig. 6. Association with TR inhibits CREB phosphorylation by PKA. (A) Recombinant CREB (50 ng) was incubated with [γ-³²P]ATP in the presence of PKA. Phosphorylation assays were performed in the presence and absence of GST–TRα (100 ng) or the same amount of GST alone. Labelled proteins are shown using arrows. In (B), the wild-type receptor (wt) and the indicated TR mutants were used in the phosphorylation assays.

Fig. 7. In vivo occupancy of the GHF-1 promoter by TR and CREB. (A) Soluble chromatin was extracted from GH4C1 cells treated with T3 for 24 h or with forskolin for the last 15 min, and immunoprecipitated with antibodies against TR, CREB or tubulin. DNA was amplified using primers that cover the GHF-1 promoter. A representative PCR at 23 cycles of amplification is shown. (B) ChIP assays from the chromatin of cells incubated with T3 for the indicated time periods and with forskolin for 15 min were quantified using real-time PCR. PCR signals obtained in the corresponding untreated cells were arbitrarily set to 1 and values were calculated compared with this. Each data point corresponds to two to four independent values.

Fig. 8. Mutant TRC51G that does not interact with CREB does not repress CREB transcriptional activity. Reporter activity was determined in HeLa cells transfected with Gal–CREB in combination with an expression vector for native TR or the C51G mutant. Luciferase activity was determined as in Figure 3, after incubation with T3 and/or FK and TPA.

Fig. 9. CREB reduces coactivator recruitment by T3 and antagonizes T3-mediated transactivation. (A) In the left panel, GH4C1 cells were transfected with 2 µg of the reporter plasmid MMTV-TREpal-LUC, and 2 µg of vectors for CREB or ICERγI. In the right panel HeLa cells were transfected with the reporter plasmid and 2 µg of TRα, in the presence and absence of 2 µg of vectors coding either native CREB or the CREB^Ala133 mutant. Cells were treated with T3 for 24 h and reporter activity was determined. (B) Pull-down assays were performed with in vitro translated TR (5 µl) and GST–ACTR or GST–SMRT (500 ng) in the presence and absence of recombinant CREB, and with and without T3 (1 µM). Quantitation of the radioactivity in each band is shown in the lower panel.