SUB1 deletion increases Kin28 and Rpb1 Ser5P cross-linking to gene promoters. ChIP analysis. ChIPs were performed on wt and sub1Δ Kin28 HA-tagged strains. (A) Kin28 binding to the promoters of four constitutively expressed genes, ADH1-P, ACT1-P, PYK1-P, and PMA1-P, was examined by qRT-PCR, and quantifications (see Materials and Methods) were graphed. Numbers on the y axis represent the percentages of Kin28 cross-linked to gene promoters in sub1Δ cells relative to the levels in wt cells, where the level of cross-linking is considered to be 100%. (B) Kin28-HA/Rpb1 ratio. Percentages of Kin28-HA and Rpb1 cross-linking for sub1Δ cells relative to the levels in wt cells were independently quantified, and the ratio was calculated and then graphed. (C) ChIP analysis of Rpb1 and CTD Ser5P was performed in wild-type (wt) and sub1Δ cells using 8WG16 (anti-Rpb1) and CTD4H8 (anti-CTD Ser5P) antibodies. Rpb1 and Rpb1 CTD Ser5P binding to promoters of ACT1, PYK1, and PMA1 genes was analyzed by qRT-PCR, and the results graphed. Numbers on the y axis represent the percentages of Rpb1 and Rpb1 CTD Ser5P cross-linked to gene promoters in sub1Δ cells relative to the levels in wt cells, where the level of cross-linking is considered to be 100%. (D) Occupancy of Rpb1 and Ceg1-HA at promoters of ACT1, PYK1, and PMA1 genes was determined by ChIP in wt and sub1Δ cells using 8WG16 and HA antibodies. Numbers on the y axis represent the percentages of Rpb1 and Ceg1-HA cross-linked to gene promoters in sub1Δ cells relative to the levels in wt cells, where the level of cross-linking is considered 100%. Error bars show standard deviations.

Sub1 influences recruitment and kinase activity of the elongation kinase Ctk1 during transcription. (A) sub1Δ and ctk1Δ are synthetically lethal. A diploid yeast strain heterozygous for both SUB1 and CTK1 (sub1Δ::URA3/SUB1 ctk1Δ::kanMX/CTK1) was sporulated, and the meiotic progeny were separated by tetrad dissection and allowed to grow for 3 days. Thirteen tetrads were dissected, with nine showing a tetratype segregation pattern (eight of which are shown), and two showing a paternal ditype segregation pattern. The genotype of the resulting colonies was inferred by growth or lack of growth on selective medium. Cells with a deletion of CTK1 alone show a slow-growth phenotype, as reported previously. (B) SUB1 deletion causes increased Ctk1 kinase activity. Whole-cell extracts were prepared from wt and sub1Δ strains with an HA-tagged Ctk1, and in vitro kinase assay was performed as described in the Fig. 1 legend to analyze CTD Ser2 phosphorylation, using anti-CTD Ser2P. (C) Schematic representation of the ACT1, PYK1, and PMA1 genes. Numbers are nucleotide positions relative to start codon (+1), and black bars represent PCR products analyzed by ChIP. (D) Increased Ctk1-HA association with chromatin in cells lacking SUB1. ChIP for Ctk1-HA was performed in wt and sub1Δ cells. Ctk1-HA association with PMA1, PYK1, and ACT1 genes was analyzed by qRT-PCR, and quantifications were graphed (see Materials and Methods). (E) Ctk1-HA/Rpb1 ratio. Ctk1-HA and Rpb1 cross-linking were independently quantified in wt and sub1Δ cells, and then Ctk1/Rpb1 ratio was calculated and graphed. Error bars show standard deviations.

Model showing how Sub1 might function to regulate RNAP II CTD phosphorylation. In wild-type cells, nonphosphorylated Sub1 joins the promoter (PROM) (possibly via TFIIB [see references 28, 37, and 60]), contacting the promoter via its DNA binding domain. At that point, Sub1 interacts with the CDK8 (Srb10) Mediator complex, helping to maintain the PIC in a stable but inactive conformation. Sub1 is then phosphorylated (possibly by the action of kinases at the PIC, similarly to PC4), losing its DNA binding capacity and promoting clearance of TFIIB (26, 35). The PIC next changes conformation such that Kin28 can be activated and, with the help of Srb10, promotes PIC dissociation into the scaffold complex, as well as the recruitment of elongating kinases Ctk1 and Bur1. In contrast, in sub1Δ cells, Srb10 activity and recruitment are decreased, while Kin28 recruitment and activity increase, in agreement with TFIIH being negatively regulated by CDK8-containing Mediator complexes (4, 51). As a result, Ser5P levels are increased, and consequently, Bur1 and Ctk1 association with chromatin is also enhanced (19, 57). Furthermore, in sub1Δ cells, there is a reduction in Fcp1 phosphatase levels and its association with chromatin, which induces an additional increase in Ser2P, impairing RNAP II recycling after transcription termination. Thus, a decrease in RNAP II recruitment is observed in cells lacking Sub1 (11). Different font sizes in the figure labels indicate the increase or decrease of the corresponding CTD-modifying enzymes in sub1Δ versus wt cells.

SUB1 deletion increases Kin28 CTD kinase activity and Kin28 cross-linking to gene promoters. (A) In vitro kinase assay. Whole-cell extracts were prepared from wild-type (wt) and sub1Δ strains expressing HA-tagged Kin28. The epitope-tagged kinase complexes were immunoprecipitated with 12CA5-protein A beads, and kinase activity was assayed with 2.5 mM ATP and recombinant GST-CTD as substrate. SDS-PAGE and immunoblot analysis were performed to analyze CTD phosphorylation, using the following antibodies: CTD4H8 (anti-CTD Ser5P), 8WG16 (anti-CTD), 12CA5 (anti-HA, for Kin28-HA), and PGK (anti-PGK, as total protein level control). (B) Whole-cell extracts were prepared from the following strains: tagged Kin28-HA (wt and kin28-K36A mutant), Sub1-HA, and Cdc5-HA strains and two nontagged strains (wt and sub1Δ). In vitro kinase assays were conducted as described for panel A, and CTD Ser5 phosphorylation analyzed with CTD4H8 antibody. (C) Whole-cell extracts were prepared from wt and sub1Δ strains expressing HA-tagged Cdc5, and in vitro kinase assays performed to analyze CTD Ser5P as described above. (D) In vitro kinase assay to analyze CTD Ser5 phosphorylation was performed using immunoprecipitated Kin28-HA and Rpb3 from wt and sub1Δ Kin28-HA tagged strains. (E) Kin28-HA sub1Δ cells were transformed with an empty plasmid (vector) or with a plasmid bearing SUB1 (pSUB1) under the control of its own promoter. Kin28-HA was immunoprecipitated, kinase assays performed, and CTD Ser5P analyzed as described for panel B.

SUB1 genetically interacts with SRB10, and SUB1 deletion negatively influences Srb10 kinase activity and association with active genes. (A) Genetic interaction between SUB1 and SRB10. SUB1 deletion partially suppresses the slow-growth phenotype of srb10Δ cells and overexpression of SUB1 enhances it, as shown by spot assay. To overexpress SUB1, its open reading frame was cloned under the control of the ADH1 promoter in a plasmid that was transformed into the srb10Δ strain (ADH1SUB1). Yeast strains with the indicated genotypes were spotted onto synthetic complete medium and grown at 30 and 37°C for 2 days. (B) Results of in vitro CTD kinase assay showing that SUB1 deletion causes a decrease in CTD phosphorylation by Srb10. Whole-cell extracts were prepared from the wt and sub1Δ strains expressing MYC-tagged Srb10, and in vitro kinase assay and CTD phosphorylation analysis were performed as described in the Fig. 1 legend. CTD Ser5 and Ser2 phosphorylation was analyzed using the following antibodies: CTD4H8 (anti-CTD Ser5P), ab5095 (anti-CTD Ser2P), 8WG16 (anti-CTD), anti-MYC antibody (Srb10 levels), and anti-PGK antibody (PGK levels, as control). (C) Induction of GAL1 transcription was monitored by RT-PCR. Total RNA was isolated from wt and sub1Δ cells grown under noninducible (2% raffinose [RAF]) and inducible (2% galactose [GAL]) conditions. cDNA was synthesized, and PCR performed using specific primers for GAL1, PYK1, SUB1, and SRB10 genes. (D) ChIP analysis of Srb10 was conducted with wt and sub1Δ cells grown in raffinose- or galactose-containing medium, using anti-MYC antibody. Srb10-MYC cross-linking to the promoter of PYK1 and to the upstream activating sequence of the GAL1 (UAS-GAL1) gene was analyzed by qRT-PCR. Quantifications of the results are shown in the graph, where numbers on the y axis represent the ratio of the values obtained from specific primer products to the value for the negative control (intergenic region of chromosome VII), after normalizing to the results for the input controls. Error bars show standard deviations. (E) SRB10 deletion causes an increase in CTD Ser5 phosphorylation by Kin28, reflecting increased Kin28 levels in the srb10Δ cells. Whole-cell extracts were prepared from wt and srb10Δ strains expressing HA-tagged Kin28, and in vitro kinase assays and CTD phosphorylation analysis were performed. CTD phosphorylation was analyzed using the following antibodies: CTD4H8 (anti-CTD Ser5P) and 8WG16 (anti-CTD). Kin28-HA and PGK levels were analyzed using 12CA5 (anti-HA) and anti-PGK, respectively.

Bur1 kinase recruitment and activity is also enhanced by deletion of SUB1. (A) SUB1 deletion increases the elongation defect of bur1 mutants. Phenotypic analysis of SUB1-BUR1 genetic interaction. SUB1 was deleted in a bur1-2 strain and in the isogenic wt strain. In the case of the bur1-23 mutant, we generated a diploid strain by crossing it with a sub1Δ mutant. The diploid was sporulated, and the tetrads dissected and analyzed. The growth phenotype of one tetrad is shown. Yeast strains with the indicated genotypes were spotted on yeast extract-peptone-dextrose or synthetic complete (SC) medium containing 50 μg/ml of 6-azauracil (6-AU), and plates were incubated for 3 days. As shown, SUB1 deletion increases the growth and elongation defects of bur1-23 cells and the elongation defect of bur1-2 cells. (B) sub1Δ cells are sensitive to 6-AU in liquid medium. Growth curves of wt and sub1Δ strains in SC medium without or with 75 μg/ml of 6-AU. (C) Bur1 kinase activity is increased in sub1Δ mutant. In vitro kinase assay was performed as described in the Fig. 1 legend in wt and sub1Δ cells expressing an HA-tagged Bur1. CTD phosphorylation was analyzed using anti-CTD Ser5P (CTD4H8, top left), anti-Ser2P (ab5095, top right) and anti-nonphosphorylated CTD (8WG16) antibodies. Bur1-HA levels were tested with anti-HA antibody. For both Ser2P and Ser5P, we observe increased phosphorylation of the CTD in the absence of Sub1. (D) SUB1 deletion increases Bur1 autophosphorylation and Bur1/RNAP II-CTD interaction. Whole-cell extracts were prepared from wt and sub1Δ Bur1-HA strains. Epitope-tagged kinase complexes were immunoprecipitated with 12CA5-protein A beads, and kinase activity with or without ATP was assayed. SDS-PAGE and immunoblot analysis were performed to analyze Bur1 autophosphorylation using HA antibody and CTD Ser5P/Bur1-HA coimmunoprecipitation using anti-Ser5P (CTD4H8) antibody. (E) SUB1 deletion slightly influences Bur1 association with coding gene regions compared to its influence on Rpb1 association. Bur1 occupancy at PMA1, PYK1, and ACT1 genes was assayed by ChIP in wt and sub1Δ cells. Graph shows qRT-PCR quantifications performed as described in the Fig. 4 legend.