(A) Set2 and Set3 are required for repression of NDC80ORF expression during early meiosis. Control (FW1902), set2Δ (FW2929), set3Δ (FW2928) and set2Δset3Δ (FW1922) cells harboring pCUP-IME1/pCUP-IME4 and NDC80-V5 were grown in rich medium, transferred to pre-sporulation medium, and then shifted to SPO medium. After 2 hr, IME1 and IME4 expression were induced, and samples for northern and western blot analyses were taken at the indicated time points. Northern blot membranes were prepared and hybridized with a probe that detects both NDC80luti and NDC80ORF transcripts. As a loading control, membranes were also hybridized with SCR1. Ndc80 protein was detected with anti-V5 antibodies and Hxk1 levels were determined with anti-hexokinase antibodies. (B) Scheme of the synchronous meiosis protocol in which cells were shifted directly from rich medium to SPO medium. Cells were grown in rich medium (YPD) to OD600 of 1–2, shifted to reduced glucose medium (YPD, 1% glucose) grown overnight to saturation, and then transferred to SPO. After 2 hr, IME1 and IME4 were induced. (C) Flow cytometry analysis of DNA content in control (FW1902) and set2Δset3Δ (FW1922) strains. Synchronous meiosis was induced as described in B. Samples were taken at the indicated time points after transfer to SPO and were stained with propidium iodide. (D) Similar to A except that meiosis was induced as described in B. (E) Strains described in A were grown to undergo a synchronous meiosis as described in B, and selective time points were taken for northern blot analysis of NDC80luti and NDC80ORF transcripts on the same membrane. As a loading control, the northern membranes were hybridized with SCR1. The NDC80ORF levels were quantified (right panel) and data from three independent experiments plus the standard error of the mean (SEM) is displayed. One-tailed, unpaired t-tests were conducted to test if the differences in NDC80ORF levels were statistically significant. A single asterisk * denotes p-value<0.05. A double asterisk ** denotes p-value<0.01. ‘n.s.’ means ‘not significant’. To control for technical variation between different northern blots, the NDC80ORF signal from the two hour time point from the control strain of each blot was set to one. (F) NDC80luti transcription requires Set2 and Set3 to establish a repressive chromatin state at the promoter of NDC80ORF. Chromatin structure at the NDC80 locus was determined by ChIP of histone H3 on micrococcal nuclease (MNase) treated extracts in control (FW1902) and set2Δ set3Δ (FW1922) cells as described in A. Samples were taken prior to IME1/IME4 induction at 2 hr in SPO (2 hr, premeiotic) and after induction at 4 hr in SPO (4 hr, S + prophase), fixed with formaldehyde, and mononucleosome fragments were isolated. The recovered DNA fragments were quantified by qPCR using ten different primer pairs directed against the NDC80 locus relative to a no MNase input. The signals from each primer pair were then normalized over a primer pair directed against the PHO5 core promoter. The midpoint position of each primer pair is indicated in the x-axis. The mean signal from three independent experiments plus the standard error of the mean for each primer pair is displayed. (G) Ectopic expression of NDC80luti is lethal in mitosis, but is rescued in a set2Δ set3Δ mutant. Spot assays of control cells, which harbor a wild-type NDC80 locus, with SET2 SET3 (UB1252), set2Δ (UB3545), set3Δ (UB3547), and set2Δ set3Δ (UB3549); as well as cells expressing NDC80luti from the heterologous GAL promoter (pGAL-NDC80luti) with SET2 SET3 (UB1218), set2Δ (UB1236), set3Δ (UB1237), and set2Δ set3Δ (UB1235). These cells also expressed the Gal4 fused to estrogen receptor (Gal4-ER), which translocates to the nucleus in the presence of β-estradiol to activate the GAL1-10 promoter. Cells were grown overnight on YP-glycerol plates, diluted in sterile water, and spotted on YP +raffinose + galactose (YP-RG) plates in the absence or presence of β-estradiol. (H) Ectopic expression of NDC80luti fails to downregulate Ndc80 in the set2Δ set3Δ mutant. Cells expressing NDC80luti from the GAL promoter with SET2 SET3 (UB1217) or set2Δ set3Δ (UB8114) were grown to exponential phase in YP-RG, and they were induced to express NDC80luti with β-estradiol. Samples were taken at the indicated time points. Ndc80 protein levels were determined by western blot using anti-V5 antibodies. Hxk1 levels were detected with anti-hexokinase antibodies. Ndc80 and Hxk1 were quantified and the relative expression (Ndc80/Hxk1) with respect to the 0 hr time point is displayed.