Cyclin-dependent kinase 8 positively cooperates with Mediator to promote thyroid hormone receptor-dependent transcriptional activation

Abstract

Mediator is a multisubunit assemblage of proteins originally identified in humans as a coactivator bound to thyroid hormone receptors (TRs) and essential for thyroid hormone (T3)-dependent transcription. Cyclin-dependent kinase 8 (CDK8), cyclin C, MED12, and MED13 form a variably associated Mediator subcomplex (termed the CDK8 module) whose functional role in TR-dependent transcription remains unclear. Using in vitro and cellular approaches, we show here that Mediator complexes containing the CDK8 module are specifically recruited into preinitiation complexes at the TR target gene type I deiodinase (DioI) together with RNA polymerase II (Pol II) in a TR- and T3-dependent manner. We found that CDK8 is essential for robust T3-dependent Dio1 transcription and that CDK8 knockdown via RNA interference decreased Pol II occupancy, and also the recruitment of the Pol II kinase CDK9, at the DioI promoter. Chromatin immunoprecipitation revealed CDK8 occupancy at the DioI promoter concurrent with active transcription, thus suggesting CDK8 involvement in transcriptional reinitiation. Mutagenesis assays showed that CDK8 kinase activity is necessary for full T3-dependent DioI activation, whereas in vitro kinase studies indicated that CDK8 may contribute to Pol II phosphorylation. Collectively, our data suggest CDK8 plays an important coactivator role in TR-dependent transcription by promoting Pol II recruitment and activation at TR target gene promoters.

FIG. 1.

Purification of CDK8-Mediator complex from HeLa cells. (A) Purification scheme for isolation of human CDK8-Mediator from nuclear extract prepared from HeLa cells stably expressing f:MED17. (B) Silver staining of purified human CDK8-Mediator. (C) Immunoblot analyses of purified CDK8-Mediator complexes isolated directly by using an anti-FLAG affinity column (M2 IP) or purified via P11 phosphocellulose chromatography followed by anti-FLAG affinity purification (CDK8 MED).

FIG. 2.

CDK8-Mediator facilitates TR-dependent transcription in vitro. (A and B) Immunodepletion of Mediator from HeLa cell nuclear extracts, as shown by results of immunoblot analysis of HeLa cell nuclear extracts incubated with either anti-MED1/anti-MED6 antibodies or preimmune serum (mock). Specific antibodies used for immunoblotting are indicated to the right of the panels. (C) Mediator depletion abrogates TR-dependent transcription. Transcription was measured in vitro by incubating the TRE₃Δ53 reporter gene in Mediator-depleted (ΔMED) or mock-depleted nuclear extract together with purified baculovirus-expressed TRα, RXRα, and T3 (10⁻⁷ M) as described previously (4). (D) Purified CDK8-Mediator restores TR-dependent transcription in a concentration-dependent manner. In vitro transcription assays were performed exactly as described for panel C, except that increasing concentrations of purified CDK8-Mediator were added to the reaction mixtures as indicated.

FIG. 3.

TR-dependent recruitment of CDK8-Mediator and Pol II into a functional PIC at the DioI promoter in vitro. (A) Schematic representation of the DioI promoter, showing the locations of TREs and biotin-conjugated PCR primers. (B) Single-round immobilized PIC assembly and transcriptional initiation assay. (C) TR-dependent recruitment of CDK8-Mediator and Pol II. Biotinylated immobilized DioI template was incubated with or without purified baculovirus-expressed TRα, RXRα, or T3 (10⁻⁷ M) in HeLa cell nuclear extract for 40 min. The PICs were then isolated by using streptavidin beads, washed, fractionated by SDS-PAGE, and then probed by immunoblotting using the specific antibodies indicated to the right of each panel. (D and E) CDK8 and Pol II dissociate from the PIC upon transcription initiation. PIC assembly on immobilized DioI promoter templates was carried out as described for panel C. The PICs were then isolated by using streptavidin beads, washed, and then resuspended in transcription buffer containing NTPs (100 μM) for the times indicated (D) or containing ATP or NTPs (100 μM) for 2 min (E). The streptavidin conjugates were then precipitated, washed, fractionated by SDS-PAGE, and then probed by immunoblotting using the specific antibodies indicated to the right of each panel.

FIG. 4.

T3-dependent recruitment of CDK8-Mediator to the DioI promoter in vivo coincides with active transcription. (A) Schematic representation of the human DioI promoter. TREs and the specific location of the PCR primers are indicated. (B) T3-dependent recruitment of CDK8-Mediator to the DioI promoter in T3-responsive cells. Chromatin was prepared from α-2 cells cultured with or without T3 (10⁻⁷ M) for 1 h and then immunoprecipitated using the specific antibodies indicated on the right. The immunoprecipitates were subjected to semiquantitative PCR using specific primers spanning the TREs in the promoter region (shown in panel A). (C and D) T3-dependent activation of DioI mRNA expression. Total RNA was extracted from α-2 cells cultured with or without T3 (10⁻⁷ M) for 1 h and then assayed by RT-PCR semiquantitatively (C) or in real time (D) using primers specific for DioI or for β-actin as a control.

FIG. 5.

CDK8 is required for T3-dependent activation of DioI mRNA expression. (A) RNAi knockdown of Mediator subunits in α-2 cells. Whole-cell extract was prepared from α-2 cells transfected with siRNAs specific for MED1, MED17, CDK8, cyclin C, or a nonspecific scrambled control siRNA and then probed with the specific antibodies indicated on the right. The immunoblots were then stripped and reprobed with antibodies against α-tubulin. (B and C) Loss of CDK8 or cyclin C inhibits T3-dependent gene expression. α-2 cells transfected with either control or Mediator-specific siRNAs were treated with or without T3 (10⁻⁷ M) for 1 h (B). Total RNA was then extracted processed for quantitative RT-PCR in real time using primers specific for DioI. Alternatively, α-2 cells were transfected with 2×TRE-tk-Luc along with either control or Mediator-specific siRNAs for 48 h and then treated with or without T3 (10⁻⁷ M) for 16 h (C). The cells were then harvested and assayed for luciferase activity, which was normalized against expression from a cotransfected β-galactosidase control vector. In the lower panels, equal amounts of cellular lysate from the harvested cells were probed by immunoblotting using the antibodies indicated on the right.

FIG. 6.

Loss of CDK8 expression reduces Pol II occupancy at T3-responsive gene promoters and decreases recruitment of CDK9 at the DioI gene. (A) Schematic representation of the DioI gene, showing the locations of PCR primers in the proximal promoter and downstream intragenic regions. (B, C, and D) α-2 cells were transfected with CDK8 siRNA or a control siRNA for 72 h and then treated with or without T3 (10⁻⁷ M) for 1 h. The cells were then processed for ChIP analyses using the antibodies shown to the right of the panels and specific PCR primer sets indicated below the panels. (E and F) ChIP was carried out exactly as described above except that PCR primers specific for the T3-responsive FAS and ADRB2 gene promoters were used.

FIG. 7.

CDK8 kinase activity is required for full T3-dependent DioI mRNA expression. α-2 cells were transfected with either scrambled or CDK8-specific siRNAs along with either a wild-type CDK8 expression vector or a mutant CDK8 expression vector (D151A) deficient in kinase activity. At 72 h posttransfection, the cells were treated with or without T3 for 1 h and then harvested. Total RNA was processed by quantitative RT-PCR in real time using primers specific for DioI, while whole-cell lysate was analyzed by immunoblotting using antibodies specific for CDK8 and α-tubulin.

FIG. 8.

CDK8/cyclin C specifically phosphorylates the Pol II CTD in vitro. (A) Recombinant human CDK8 and cyclin C expressed in Sf9 cells via baculovirus were purified and stained with Coomassie blue. (B) Purified recombinant CDK8/cyclin C phosphorylates GST-Pol II-CTD in a concentration-dependent manner (see Materials and Methods for further information on the in vitro kinase assay). (C to E) H7 inhibits CDK8/cyclin C kinase activity. (C) GST-Pol II-CTD was preincubated with various concentrations of H7 prior to incubation with CDK8/cyclin C in the presence of [γ³²]ATP. (D and E) GST-Pol II-CTD was incubated with CDK8/cyclin C together with unlabeled ATP in the presence or absence of H7 (10 μM). The reaction mixtures were resolved by SDS-PAGE and then probed by immunoblotting using antibodies specific for the Pol II CTD, phosphorylated Pol II CTD Ser-2P, or phosphorylated Pol II CTD Ser-5P.

FIG. 9.

H7 inhibits both T3-dependent DioI expression and Pol II phosphorylation at the DioI promoter. (A) α-2 cells were cultured with or without T3 (10⁻⁷ M) in the presence or absence of H7 (10 or 25 μM, as indicated) for 1 h and then harvested. Total RNA was processed by quantitative RT-PCR in real time using primers specific for DioI. (B) H7 inhibits Pol II CTD phosphorylation at the DioI promoter in vitro. PICs were assembled on biotinylated DioI promoter templates in the presence of RXR/TR/T3 and HeLa nuclear extract essentially as described for Fig. 3 except that H7 (10 μM) was added to the reaction mixtures as indicated (lanes 4 and 6). The PICs were then isolated by using streptavidin beads and washed, and in selected reactions, transcription was initiated upon the addition of NTPs (100 μM) for 2 min (lanes 5 and 6). The streptavidin conjugates for all reactions were then precipitated, fractionated by SDS-PAGE, and then probed by immunoblotting using antibodies specific for CDK8, Pol II, and phosphorylated Ser-5 Pol II CTD. In reaction 5 (lane 5), the transcription initiation reaction eluate was trichloroacetic acid precipitated and processed for immunoblotting as described above. (C) H7 inhibits Pol II CTD phosphorylation at the DioI promoter in vivo. α-2 cells were cultured with or without T3 in the presence or absence of H7 (10 μM) and then processed for ChIP analyses exactly as described in the Fig. 4 legend.