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. 2010 Jan 1;285(1):188-96.
doi: 10.1074/jbc.M109.046565. Epub 2009 Nov 9.

RNA polymerase II C-terminal heptarepeat domain Ser-7 phosphorylation is established in a mediator-dependent fashion

Stefan Boeing  1 , Caroline Rigault, Martin Heidemann, Dirk Eick, Michael Meisterernst
Affiliations

RNA polymerase II C-terminal heptarepeat domain Ser-7 phosphorylation is established in a mediator-dependent fashion

Stefan Boeing et al. J Biol Chem. .

Abstract

The largest subunit of RNA polymerase II (RNAPII) C-terminal heptarepeat domain (CTD) is subject to phosphorylation during initiation and elongation of transcription by RNA polymerase II. Here we study the molecular mechanisms leading to phosphorylation of Ser-7 in the human enzyme. Ser-7 becomes phosphorylated before initiation of transcription at promoter regions. We identify cyclin-dependent kinase 7 (CDK7) as one responsible kinase. Phosphorylation of both Ser-5 and Ser-7 is fully dependent on the cofactor complex Mediator. A subform of Mediator associated with an active RNAPII is critical for preinitiation complex formation and CTD phosphorylation. The Mediator-RNAPII complex independently recruits TFIIB and CDK7 to core promoter regions. CDK7 phosphorylates Ser-7 selectively in the context of an intact preinitiation complex. CDK7 is not the only kinase that can modify Ser-7 of the CTD. ChIP experiments with chemical inhibitors provide evidence that other yet to be identified kinases further phosphorylate Ser-7 in coding regions.

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Figures

FIGURE 1.
FIGURE 1.
Gradual phosphorylation of Ser-7 residues on RNAPII bound to DNA templates. A, shown is a reaction scheme. NEX, nuclear extract. B, preinitiation complexes were formed on ML-promoter templates in the presence of the activator GAL-VP16 and the indicated concentrations of ATP (lanes 3–10). As a control for the presence of residual ATP, the kinase inhibitor H-8 was used in lane 2 in the absence of exogenously added ATP. In, 20% input of the PIC formation reaction. Two exposures are shown to better illuminate the different CTD forms with gradually increasing mobility. Note that minor amounts of phosphorylated RNAPII (Ser-5-P-long exposure) present in nuclear extracts migrate with mobility different from the hypophosphorylated forms generated at low ATP levels.
FIGURE 2.
FIGURE 2.
CTD Ser-7 phosphorylation requires Mediator. A, PICs were formed on pGL2-MRG5 promoter templates in the presence of 1 μm ATP and washed and either analyzed directly by immunoblot (lanes 3 and 4) or subjected to a second incubation step in the presence of 100 μm concentrations of each ATP, CTP, GTP, and UTP (lanes 5 and 6). Mock-treated (ΔIso) or Mediator-depleted (ΔMED) Jurkat nuclear extract was used. B, preinitiation complexes were formed in the presence of 1 μm ATP either with mock-treated (ΔIso), Mediator-depleted (ΔMED), or Mediator-depleted extract supplemented with Mediator affinity purified on IP beads as indicated. Mediator IPs were washed stringently at 800 mm KCl before add-back. PICs were either analyzed by immunoblot or probed in an in vitro transcription assay (TXN). NEX, nuclear extract. C, PICs were formed under basal conditions on major-late promoter DNA templates with either mock-treated (ΔIso) or Mediator-depleted (ΔMED) extracts in the presence of 1 μm ATP (lanes 4–13). Before PIC formation, immobilized promoter templates were incubated with recombinant human TBP and recombinant (rec (r)) human TFIIB as indicated. Basal PICs were either analyzed directly by immunoblot or probed in an in vitro transcription assay.
FIGURE 3.
FIGURE 3.
CDK7 is a CTD Ser-7 kinase. A, shown is an immobilized template assay on the pGL2-MRG5 promoter template in the presence of the activator GAL-VP16. Mock-treated or CDK7-, CDK8-, and CDK9-depleted nuclear extracts (lanes 5–8) were used in the PIC formation reactions. Reactions were carried out under standard in vitro transcription conditions in the presence of 1 μm ATP. Lanes 1–4 show 20% of reaction input. B, shown is an immobilized template assay on the ML-promoter template in the presence of GAL-VP16 using mock-treated (ΔIso), core-TFIIH (Δp62), CDK7 (ΔCDK7)- or CDK8-depleted (ΔCDK8) Jurkat nuclear extract. Lanes 1–4 show 20% reaction input. TXN, in vitro transcription analysis. C, shown is an immobilized template assay on ML-promoter DNA templates under basal conditions at physiological salt concentrations. PICs were formed in the presence of the indicated amounts of ATPγS, washed, and then analyzed by immunoblot. D, shown is an immobilized template assay on the ML-promoter DNA template using mock-treated (ΔIso), Mediator-depleted (ΔMED), and CDK7-depleted (ΔCDK7) 0.5 m P11 fractions together with GAL4-VP16, recombinant yeast TBP, Toa, and recombinant human TFIIB as indicated. In lane 6 a high salt-washed MED15-IP was added. PIC formation was carried out under physiological conditions in the presence of 1 μm ATP and 10 μm ATPγS. NEX, nuclear extract. E, immobilized template assay on ML-promoter DNA templates using either Jurkat nuclear extract (lane 1) or a recombinant transcription system consisting of yeast TBP, Toa, human TFIIB, TFIIE, and TFIIF) in combination with a high salt-washed Mediator-IP (which also provides RNAPII; lanes 2 and 3) is shown. In lane 3 a stringently washed CDK7 IP was also added to the PIC formation reactions. PIC formation in D and E was carried out under physiological conditions in the presence of 1 μm ATP and 10 μm ATPγS.
FIGURE 4.
FIGURE 4.
CTD Ser-7 phosphorylation is established in the context of a functional preinitiation complex. A, shown is an in vitro kinase assay using GST-CTD as a substrate. As sources of kinase activity, the indicated immunoprecipitations or Jurkat nuclear extract (NEX) were used. B, shown is an immobilized template assay on ML-DNA templates in the presence of the activator GAL-VP16. Jurkat nuclear extracts were either mock-treated (ΔIso) or depleted for RNAPII (ΔRNAPII). ΔRNAPII extracts in lanes 3 and 4 were supplemented with immunoprecipitated Mediator complexes (high salt-washed MED15 IPs). PICs were washed and either analyzed by immunoblotting or probed in an in vitro transcription assay (TXN). C, shown is an immobilized template assay on pGL2-MRG5 DNA templates in the presence of the activator GAL-VP16. Jurkat nuclear extracts were either mock-treated (ΔIso) or depleted for TFIIB (ΔIIB). D, shown is an immobilized template assay on ML-DNA templates using mock-treated (ΔIso), TBP-depleted (ΔTBP), or TBP-depleted extracts supplemented with recombinant human TBP. PIC formation was carried out under basal conditions in the presence of 1 μm ATP. All assays in this figure were carried out under in vitro transcription conditions.
FIGURE 5.
FIGURE 5.
Evidence for another Ser-7 kinase. A, shown is an immobilized template assay using mock-treated (ΔIso) or CDK7-depleted (ΔCDK7) nuclear extracts. PICs were formed in the presence of the indicated amounts of ATP and roscovitine. PICs in lanes 1 and 2 as well as 5 and 6 were formed under basal conditions, and PICs in lanes 3 and 4 as well as in lanes 7–10 were formed in the presence of the activator GAL4-VP16. Physiological salt conditions were used. B, PICs were formed on ML-promoter DNA templates in the presence of the indicated amounts of ATP or ATPγS under physiological salt conditions. PICs were formed with mock-treated (ΔIso), CDK7-depleted (ΔCDK7), or Mediator-depleted (ΔMED) Jurkat nuclear extracts.
FIGURE 6.
FIGURE 6.
CTD Ser-7 phosphorylation is roscovitine-sensitive in vivo. A, shown is a ChIP experiment on the doxycycline-induced tet-VP16 system. Induction of cells with 1 μg of doxycycline (Dox) per ml medium was carried out for 60 min. B, ChIP analysis is shown of the tet-VP16-inducible gene system 60 min after induction of Luciferase gene expression with doxycycline (1 μg/ml). If indicated, roscovitine (Rosco) was added to the cell culture medium to a final concentration of 30 μm 30 min before induction.
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