Fig. 4

Ime1 changes local chromatin via IRT2 and promotes its own expression. a Diploid cells harboring pCUP-IME1 (FW3006) were grown in rich and pre-sporulation medium before shifted to SPO, and were either not treated (−Cu) or treated with copper sulfate (+Cu) after 1 h in SPO. Samples were taken at 0 and 6 h in SPO. Chromatin extracts were treated with micrococcal nuclease (MNase). Mononucleosome DNA fragments were isolated and quantified using 14 primers pairs. The signals were normalized to a no MNase input. Means ± SEM of n = 3. b Diploid cells with pCUP1-IME1 and RME1-H-V5 (FW1366) were induced to enter meiosis and were either not treated (−Cu) or treated (+Cu) after 1 h in SPO. Samples for chromatin immunoprecipitation were taken at 0 or 2 h after induction, and quantified by qPCR. The signals were normalized to the silent mating locus (HMR). Means ± SEM of n = 3. c IRT1, IRT2, and IME1 expression in cells harboring RME1-H and pCUP-IME1 (FW2270). Cells were grown as described in a. Northern blot membranes were hybridized with probes that detects IRT1, IRT2, and IME1. As a loading control, the ribosomal RNA is shown. d IRT1, IRT2, and IME1 expression in diploid cells harboring RME1-H (FW1196) or RME1-H together with the set2Δset3Δ mutant (FW1312) during entry in meiosis. Cells were grown and samples were taken as described in c. Northern blot membrane was probed for IRT1, IRT2, and IME1. SCR1 expression was used as loading control. e Cells containing one copy of pCUP-IME1, one copy of pIME1-GFP-IME1 (pIME1) (FW5291), or pIME1-Δu6-GFP-IME1 (pIME1-Δu6) (FW5295) were induced to enter meiosis in the absence (−Cu) or presence (+Cu) of IME1 expression. Signals were quantified (n = 100 cells) per time point. Means ± error bars represent the 95% confidence interval. f Cells containing RME1-H and one copy of pCUP-IME1, one copy of pIME1-LacZ (FW5337), or a mutant pIME1-EIt-LacZ plasmid lacking part of the IRT2 sequence including the Ume6 motif (FW5341) were induced to enter meiosis. The graph displays the ratio of β galactosidase activity signals from IME1 induced samples versus not-induced samples. Means ± SEM of n = 3. *Treatment with copper sulfate