FIG. 2.

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.