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

Fig 4. Interplay between Pch2, Orc1 and transcription.

Fig 4

A. Schematic of Pch2 and ORC, including the domain organization of Orc1. B. 3XFLAG-Pch2 and 3XFLAG-Pch2-E399Q binding (ChIP-seq normalized against the input) plotted relative to the center of ARSs. C. ChIP-qPCR analysis of 3XFLAG-Pch2-E399Q at PPR1 (primer pair: GV2390/GV2391), GAP1 (primer pair: GV2597/GV2598), ARS202 (primer pair: GV2583/GV2584) and ARS1116 (primer pair: GV2577/GV2578) during meiotic G2/prophase (t = 4 hours). Error bars represent standard error of the mean of at least three biologically independent experiments performed in triplicate. D. ChIP-qPCR analysis of Orc1-TAP and Orc2-TAP at PPR1 (primer pair: GV2390/GV2391), GAP1 (primer pair: GV2597/GV2598), HOP1 (primer pair: GV2605/GV2606), ARS1116 (primer pair: GV2577/GV2578) during meiotic G2/prophase (t = 4 hours). Error bars represent standard error of the mean of at least three biologically independent experiments performed in triplicate. E. ChIP-qPCR analysis of 3XFLAG-Pch2-E399Q in ORC1 or orc1-161 background at PPR1 (primer pair: GV2390/GV2391) and GAP1 (primer pair: GV2597/GV2598) during meiotic G2/prophase (t = 4 hours). Experiments were performed at 30°C. Error bars represent standard error of the mean of at least three biologically independent experiments performed in triplicate. F. Representative images of immunofluorescence of meiotic chromosome spreads in 3XHA-Pch2 expressing ORC1 or orc1-161 cells collected at 6 hours after induction into the meiotic program. Experiments were performed at 30°C. Chromosome synapsis was assessed by α-Gmc2 staining. Experiments were performed at 30°C. G. Quantification of non-nucleolar Pch2 intensity per spread nucleus for the immunofluorescence analysis shown in F and for immunofluorescence analysis of cells progressing synchronously through meiotic prophase at 4, 5 and hours (S6A and S6B Fig). *** indicates a significance of p≤ 0.001, Mann-Whitney U test. H. ChIP-qPCR analysis of 3XHA-Pch2-E399Q in ORC1 or orc1Δbah background at PPR1 (primer pair: GV2390/GV2391), GAP1 (primer pair: GV2597/GV2598) during meiotic G2/prophase (4 hours). Error bars represent standard error of the mean of at least three biologically independent experiments performed in triplicate.