Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain

Abstract

The C-terminal domain (CTD) of the RNA polymerase II (Pol II) largest subunit is hyperphosphorylated during transcription. Using an in vivo cross-linking/chromatin immunoprecipitation assay, we found previously that different phosphorylated forms of RNA Pol II predominate at different stages of transcription. At promoters, the Pol II CTD is phosphorylated at Ser 5 by the basal transcription factor TFIIH. However, in coding regions, the CTD is predominantly phosphorylated at Ser 2. Here we show that the elongation-associated phosphorylation of Ser 2 is dependent upon the Ctk1 kinase, a putative yeast homolog of Cdk9/P-TEFb. Furthermore, mutations in the Fcp1 CTD phosphatase lead to increased levels of Ser 2 phosphorylation. Both Ctk1 and Fcp1 cross-link to promoter and coding regions, suggesting that they associate with the elongating polymerase. Both Ctk1 and Fcp1 have been implicated in regulation of transcription elongation. Our results suggest that this regulation may occur by modulating levels of Ser 2 phosphorylation, which in turn, may regulate the association of elongation factors with the polymerase.

Figure 1

(A) Phosphorylation of RNA Pol II at Ser 2 and Ser 5 are differentially affected by Fcp1 CTD phosphatase. Yeast strains grown at 30°C (OD₆₀₀ = 0.8) were further incubated at 37°C (even lanes) or 30°C (odd lanes) for 55 min. Whole-cell extracts were prepared from each strain. Extract protein (80 μg) was assayed by immunoblotting with 8WG16 (CTD, recognizing the nonphosphorylated CTD of Rpb1), H14 and H5 (CTD-S5-P and CTD-S2-P, phosphorylated CTD on Ser 5 and Ser 2 position, respectively), B3 (phosphorylated CTD at either Ser 5 or Ser 2), G₂ (recognizing Rpb1 outside of CTD) and polyclonal antibodies against Fcp1, Ceg1, and Cet1. Yeast strains used are YMK16α, shuffled with plasmids expressing wild-type FCP1 (pFK1; lanes 1,2), fcp1-1 (pFK4; lanes 3,4) and fcp1-2 (pFK7; lanes 5,6). (B) Specificity of CTD antibodies. Biotinylated peptides (100 ng or 1 μg) containing four CTD repeats with the indicated phosphorylations were coupled to the wells of streptavidin-coated 96-well plates. The indicated antibodies were used to probe the peptides and binding was detected by indirect chemiluminescence.

Figure 2

Ser 2 and Ser 5 phosphorylations on the ADH1 gene are differentially affected by the Fcp1 CTD phosphatase during transcription. Chromatin immunoprecipitation was performed with YSB763 shuffled with the same Fcp1 plasmids used in Figure 1 (pFK1, pFK4, and pFK7). To monitor the presence of each protein along the ADH1 gene, chromatin was immunoprecipitated with various antibodies and PCR amplified with primer pairs recognizing promoter (P) and coding (C) regions (see schematic at bottom). Each PCR reaction contained a second primer pair that amplifies a region of chromosome V devoid of ORFs, thus providing an internal control for background (*). Each panel shows a different immunoprecipitation with respective antibodies as follows: α-CTD-S5-P (H14), α-CTD-S2-P (H5), α-CTD (8WG16), α-Rpb3HA (12CA5, recognizing an HA epitope on RNA polymerase subunit Rpb3), and α-Cet1 (a polyclonal antibody recognizing the triphosphatase subunit of capping enzyme). Input shows the signal from the chromatin before immunoprecipitation. Signals were quantitated by PhosphorImager and normalized as described previously (Komarnitsky et al. 2000). The signals for the promoter with wild-type chromatin solution were assigned as 1, except for the CTD-S2-P immunoprecipitation, in which the signal from the coding region was taken as 1. A zero indicates that the signal was <0.005. The primer pairs used are (P) ADH1₋₂₃₅ and ADH1₋₁₃, (C) ADH1₈₄₄ and ADH1₁₀₁₃, (−) Intergenic V −1 and Intergenic V −2.

Figure 3

Ser 2 and Ser 5 phosphorylations on the PMA1 gene are differentially affected by the Fcp1 CTD phosphatase during transcription. The same chromatin immunoprecipitates shown in A were used for PCR amplification with primer sets that amplify DNA throughout the PMA1 gene (see schematic diagram at bottom). Promoter; PMA1-370 and PMA1-90, Coding region 1 (CD1): PMA1₁₆₈ and PMA1₃₇₆; Coding region 2 (CD2): PMA1₅₈₄ and PMA1₈₀₇; Coding region 3 (CD3): PMA1₁₀₁₀ and PMA1₁₂₅₀; Coding region 4 (CD4): PMA1₂₀₁₈ and PMA1₂₂₉₀.

Figure 4

The TFIIH kinase Kin28 is not required for CTD Ser 2 phosphorylation of elongating polymerase. Chromatin IP/PCR was carried out with kin28-16 mutants combined with FCP1, fcp1-1, or fcp1-2 (YMK223, YMK224, and YMK225, respectively). Strains were also transformed with pY3AtURA to provide an HA epitope-tagged Rpb1 subunit. Immunoprecipitating antibodies are indicated to the left of the autoradiographs and PMA1 primer pairs are as in Figure 3.

Figure 5

The Srb10 kinase component of RNA Pol II holoenzyme is not required for CTD Ser 2 phosphorylation of elongating polymerase. Chromatin IP/PCR was carried out with Srb10(D290A) combined with FCP1, fcp1-1, or fcp1-2 (YMK162, YMK164, YMK166, respectively). Strains were also transformed with pY3AtURA to provide an HA epitope-tagged Rpb1 subunit. Srb10 is the kinase associated with RNA Pol II holoenzyme. Srb10 (D290A) is catalytically inactive but successfully incorporated into the holoenzyme. Immunoprecipitating antibodies and PMA1 primer pairs are as in previous figures.

Figure 6

The CTDK-I kinase Ctk1 is required for CTD Ser 2 phosphorylation in vivo. Chromatin IP/PCR was carried out with a ctk1 deletion mutant combined with FCP1, fcp1-1, or fcp1-2 (YSB762 shuffled with pFK1, pFK4, and pFK7, respectively). Strains were also transformed with pY3AtURA to provide an HA epitope-tagged Rpb1 subunit. Ctk1 is the catalytic kinase subunit of CTDK-I. Immunoprecipitating antibodies and PMA1 primers are as in previous figures.

Figure 7

Ctk1and Fcp1 cross-link to both promoter and coding regions. Chromatin IP/PCR was carried out on strains YSB772 containing triple HA-tagged Ctk1 and YMK210 carrying a 13×Myc epitope-tagged Fcp1. 12CA5 monoclonal and 9E10 antibodies were used for immunoprecipitation, respectively. Primers are as in previous figures.