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. 2020 Jun 22;16(6):e1008905. doi: 10.1371/journal.pgen.1008905

Fig 3. Transcription is required for recruitment of Pch2.

Fig 3

A. Schematic of the anchor away system and Rpo21 within RNAPII. B. Dilution series of wild type and rpo21-FRB anchor away strains, grown on YPD or YPD + rapamycin. C. Schematic of treatment regimens used for anchor away experiments in D-I, and S5G Fig. D. Representative images of immunofluorescence of meiotic chromosome spreads in the rpo21-FRB anchor away cells treated with DMSO or rapamycin. Chromosome synapsis was assessed by α-Zip1 staining. E. ChIP-qPCR analysis of active transcription (α−Rpo21-phospho-Ser5) in rpo21-FRB anchor away cells treated with DMSO or rapamycin at PPR1 (primer pairs: GV2390/GV2391) and GAP1 (primer pairs: GV2597/GV2598). Error bars represent standard error of the mean of at least three biologically independent experiments performed in triplicate. F. Western blot analysis of 3XHA-Pch2 in rpo21-FRB anchor away cells treated with DMSO or rapamycin. G. ChIP-qPCR analysis of 3XHA-Pch2 in rpo21-FRB anchor away cells treated with DMSO or rapamycin at PPR1 (negative control) (primer pair: GV2390/GV2391), GAP1 (primer pair: GV2597/GV2598), HOP1 (primer pair: GV2607/GV2608), TDH3 (primer pair: GV2591/GV2592), and SSA1 (primer pair: GV2587/GV2588). Error bars represent standard error of the mean of at least three biologically independent experiments performed in triplicate. H. Representative images of immunofluorescence of meiotic chromosome spreads in the 3XHA-Pch2 expressing rpo21-FRB anchor away cells, treated with DMSO or rapamycin. Chromosome synapsis was assessed by α-Gmc2 staining. White line and asterisk indicate the position of the nucleoli. I. Quantification of non-nucleolar Pch2 intensity per spread nucleus for the immunofluorescences shown in H, treated with DMSO or rapamycin and from the immunofluorescence shown in S5H Fig (ndt80Δ and ndt80Δzip1Δ cells collected at 4 hours after induction into the meiotic program.) * indicates a significance of p = 0.01–0.05, Mann-Whitney U test. See S3 Table for exact p-values.