The Ulp2 SUMO Protease Promotes Transcription Elongation through Regulation of Histone Sumoylation (2019)
Figure 6

Figure 6. Ctk1 recruitment and CTD S2-P of RNAPII are diminished in ulp2Δ cells.

A The ratio of CTD S2-P relative to Rpb3 at 3' ORF regions was analyzed by ChIP using anti-S2-P and anti-Rpb3 antibodies. The error bars represent the SD from triplicate experiments.

B A ulp2Δ single mutant and three isolates of ulp2Δ ctk1Δ with YCplac33-ULP2 were grown on SD-Ura or SD+5-FOA plates at 30°C for 2-3 days.

C Pie graphs showing percentages of genes in ulp2Δ cells (gray color) with significantly changed expression compared with WT (MHY1379). RNA-seq data were subdivided into groups of genes regulated by SAGA, TFIID, or both factors based on a previous report (Huisinga & Pugh, 2004). Gene numbers are shown in parentheses.

D Schematic diagram of TFIID-dominated genes UTH1 and ATP2. The TATA/promoter region and ORF are represented by black and white boxes, respectively. Lines and numbers below the genes show that the relative positions of the ChIP PCR products used in all later panels.

E ChIP analyses of Ctk1-TAP in the indicated strains. The ORF of TFIID-dominated genes indicates position 2 of PCR fragments from the UTH1 and ATP2 genes shown in (D). Error bars represent the SD from triplicate experiments.

F Co-IP analysis of Ctk1 and histone H2B interaction. WT and ulp2Δ strains expressing the indicated tagged proteins were precipitated with IgG-Sepharose. IP and INPUT proteins were subjected to immunoblotting with PAP (Ctk1-TAP) and anti-Flag (Flag-H2B) antibodies. Protein signals were quantified relative to PGK and then normalized to the WT value.

G ChDIP of Flag-H2B-SUMO in the indicated strains as in Fig 5D. The "Pro" and "ORF" of UTH1 and ATP2 genes indicate positions 1 and 2 of PCR fragments, respectively, as described in Fig 6D. The error bars indicate the SD from three independent ChDIPs.

H Sensitivity to 6-AU of indicated htb1-1 htb2-1 (null alleles for both chromosomal H2B genes) strains carrying pRS314 plasmids that express Flag-H2B or 2SUMO-H2B. Assays were performed as in Fig 2A.

I, J ChIP analyses of the indicated strains used in (H). Occupancy of Rpb3 (I) and the ratio of CTD S2-P relative to Rpb3 (J) at 3' ORF regions of the indicated genes were individually analyzed by ChIP, as described in Fig 1E and (A). Error bars show SD from three independent analyses.

K ChIP analyses of Ctk1-TAP in the indicated strains. The ORF of TFIID-dominated genes indicates position 2 of PCR fragments from the UTH1 and ATP2 genes shown in (D). Error bars represent the SD from triplicate experiments.

L Immunoblot analysis of TAP-tagged Ctk1 proteins in extracts prepared from the strains in (K). Anti-PGK blotting shows similar loading.

M, N ChIP analyses of the indicated strains used in Fig 4B. Occupancy of Rpb3 (M) and the ratio of CTD S2-P relative to Rpb3 (N) at 3' ORF regions of the indicated genes were individually analyzed by ChIP, as described in Fig 1E and (A). Error bars show SD from three independent analyses.

Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01). See Dataset EV2 for qPCR raw data.