Two cyclin-dependent kinases promote RNA polymerase II transcription and formation of the scaffold complex

Abstract

Three cyclin-dependent kinases, CDK7, -8, and -9, are specifically involved in transcription by RNA polymerase II (Pol II) and target the Pol II C-terminal domain (CTD). The role of CDK7 and CDK8 kinase activity in transcription has been unclear, with CDK7 shown to have variable effects on transcription and CDK8 suggested to repress transcription and/or to target other gene-specific factors. Using a chemical genetics approach, the Saccharomyces cerevisiae homologs of these kinases, Kin28 and Srb10, were engineered to respond to a specific inhibitor and the inhibitor was used to test the role of these kinases in transcription in vivo and in vitro. In vitro, these kinases can both promote transcription, with up to 70% of transcription abolished when both kinases are inhibited together. Similarly, in vivo inhibition of both kinases together gives the strongest decrease in transcription, as measured by chromatin immunoprecipitation of Pol II. Kin28 and Srb10 also have overlapping roles in promoting ATP-dependent dissociation of the preinitiation complex (PIC) into the Scaffold complex. Using the engineered kinases and an ATP analog, specific kinase substrates within the PIC were identified. In addition to the previously known substrate, the Pol II CTD, it was found that Kin28 phosphorylates two subunits of Mediator and Srb10 targets two subunits of TFIID for phosphorylation.

FIG. 1.

Bur1 and Ctk1 are not PIC components. (A) Nuclear extracts (NE) made from Kin28-Flag, Bur1-Flag, and Ctk1-Flag strains were incubated with the immobilized template for 40 min. PICs were isolated and analyzed by Western blotting. The top panel was probed with the anti-Flag M2 antibody. The asterisk indicates the position of Kin28-Flag, Bur1-Flag, or Ctk1-Flag protein. The lower three panels were probed with antibodies directed against known PIC components. The experiment shown in panel B is the same as that in panel A but demonstrates that Srb10 is a stable PIC component (24).

FIG. 2.

Inhibition of Kin28-as and Srb10-as activities by NA-PP1. (A) Growth inhibition of either wild-type or Kin28-as strains. Three microliters of 1 mM NA-PP1 was spotted on a 0.6-cm filter disk placed on a soft agar plate containing the indicated strain and incubated for 16 h at 30°C. The structure of NA-PP1 is shown. (B) Kin28-as or Kin28 wild-type (wt) complexes were purified from yeast WCE as described in Materials and Methods. The effects of NA-PP1 on the kinase activities of these complexes were assayed using GST-CTD as a substrate. Reactions were performed in transcription buffer containing 600 μM ATP-5 μCi of [γ-³²P]ATP. After SDS-PAGE, the phosphorylated GST-CTD was quantitated by phosphorimaging. The activities of Kin28-as (lane 1) and Kin28-wt (lane 7) were normalized to 100%. Shown below is Western analysis of equal amounts of the two Kin28-purified complexes. (C) Immune precipitates from Srb10-as-Flag, Srb10-wt-Flag, and untagged strains were assayed for kinase activities in the presence of increasing amounts of NA-PP1. Reactions and quantitation were performed as described for panel B. Immune precipitate from an untagged strain was used as a control. The lower panel is a Western analysis of equal amounts of the anti-Flag immune precipitates.

FIG. 3.

Both Kin28 and Srb10 can promote transcription. (A) Nuclear extracts (NE) made from wild-type, Kin28-as, Srb10-as, or Kin28-as Srb10-as strains were used as indicated. The transcription reactions were performed as described in Materials and Methods. Quantitation of the results is shown in the lower panel. Transcription activity of each nuclear extract without inhibitor was normalized to 100%. (B) Transcription reaction performed as described for panel A using immobilized templates and NA-PP1 with extracts from the indicated strains. In lane 9, a saturating amount of purified wild-type CAK was added.

FIG. 4.

Inhibition of Kin28 and Srb10 in vivo. (A) Representative chromatin IP data from one experiment. The indicated strains were either treated or not treated with 6 μM NA-PP1 for 12 min and cross-linked with formaldehyde, and sheared DNA was isolated. Pol II cross-linking was assayed by IP with Rpb3 antisera followed by quantitative PCR. For all samples, a series of four different DNA concentrations were used for PCR in the linear range of the assay. Shown are PCR products of representative IN or IP samples. (B) Quantitation of results from two separate chromatin IP experiments. Results are plotted as the ratio of signals seen with and without NA-PP1 treatment.

FIG. 5.

Inhibition of Kin28 and Srb10 kinases inhibits PIC dissociation. PICs were assembled on the immobilized template using nuclear extracts (NE) made from wild-type, Kin28-as, Srb10-as, and Kin28-as Srb10-as strains. Variable amounts of NA-PP1 along with 600 μM ATP-10 μCi of [γ-³²P]ATP were added for 4 min. (A) Scaffold complexes (Scaf) were washed, isolated by PstI digestion, and analyzed by Western blotting for the indicated factors. Factors in the PIC without ATP addition (PIC) are shown for comparison. (B) Factors released from the PIC upon ATP addition into the supernatant (Sup) (Pol II, IIB, and IIF) were recovered and analyzed by Western blotting.

FIG. 6.

Inhibition of Kin28 and Srb10 kinases inhibits phosphorylation of the Pol II CTD. (A) Phosphorylated Pol II released into the supernatant (Sup) during Scaffold complex formation. Phosphorimager analysis was performed on the membrane containing the supernatants (Fig. 5), and the quantitation of the phosphorylated Pol II CTD is shown below. The phosphorylation signal of each extract without inhibitor (lanes 1, 5, 9 and 13) was normalized to 100% (lower panel). (B) Both Kin28 and Srb10 contribute to hyperphosphorylation of the CTD during Scaffold complex formation. PIC and Scaffold complexes formed from the indicated extracts were fractionated on a 3 to 8% Tris-acetate gel and analyzed by Western blotting using antibody YN-18, which detects Rpb1 independent of the phosphorylation state. IIo* represents a partially phosphorylated Pol II form.

FIG. 7.

Identification of Kin28 and Srb10 substrates in the PIC. (A) The immobilized template assay was performed as described in the legend for Fig. 6, using the indicated nuclear extracts (NE). After PICs were assembled, 600 μM ATP/[γ-³²P]ATP or 600 μM ATP/[γ-³²P]N⁶-benzyl-ATP was used to form Scaffolds. Scaffold complexes (Scaf) and supernatants (Sup) were analyzed on a 4 to 12% NuPAGE gel and visualized by phosphorimaging. Lanes 1 to 6, PIC phosphorylation after adding ATP/[γ-³²P]ATP; lanes 7 to 16, specific labeling of Kin28 or Srb10 substrates after adding ATP/[γ-³²P]N⁶-benzyl-ATP. * and Δ indicate the three major substrates of Kin28 and Srb10, respectively. (B) NA-PP1 inhibited specific labeling of the Kin28 and Srb10 substrates. Wild-type (WT), Kin28-as, and Srb10-as Scaffolds were formed using 600 μM ATP/[γ-³²P]N⁶-benzyl-ATP in the absence or presence of an increasing amount of NA-PP1. Samples were resolved on a 3 to 8% Tris-acetate gel. The positions of Kin28 and Srb10 substrates are indicated by the * and Δ symbols, respectively.

FIG. 8.

Identification of Med4 and Rgr1 as Kin28 substrates in the PIC. (A) Identification of Med4 as the 39-kDa Kin28 substrate. Substrate candidates were Flag tagged in a wild-type strain background (except Kin28, which directly used Kin28-as-Flag). Nuclear extract made from a Flag-tagged strain (2nd NE) was mixed with Kin28-as nuclear extract (1st NE) in equal amounts. Scaffolds were formed as described in the legend for Fig. 7, loaded onto a 4 to 12% NuPAGE gel, and electroblotted to a polyvinylidene difluoride membrane. Phosphorimaging and Western blotting were performed on the same membrane. The * indicates the position of Med4-Flag on both phosphorimaging and Western blotting. (B) Identification of Rgr1 as the 97-kDa Kin28 substrate. The procedure was the same as for panel A, except samples were separated on a 3 to 8% Tris-acetate gel. The * indicates the position of Rgr1-Flag on both phosphorimaging and Western blotting. In addition, Rpb1 was identified as the 191-kDa Kin28 substrate by H14 antibody in both panels A and B.

FIG. 9.

Identification of Bdf1 and TAF2 as Srb10 substrates. The same methods were used as for Fig. 8, except that some of the strains contained both a Flag-tagged gene as well as the Srb10-as mutation. In lane 6, Srb10-as extract was mixed with a Bdf1-Flag extract. The Δ indicates the position of Bdf1-Flag, and the “o” indicates the position of TAF2-Flag. For Western blotting, anti-Flag M2 and the H14 anti-Ser5 antibodies were used as indicated.