Growth and medium conditions

For time course experiments, cells were grown in YPD (1.0% (wt/vol) yeast extract, 2.0% (wt/vol) peptone, 2.0% (wt/vol) glucose, supplemented with tryptophan (96 mg/l), uracil (24 mg/l), and adenine (12 mg/l), and grown to exponential phase at 30 °C and 300 rpm. Approximately 0.05 OD₆₀₀ units of exponentially growing yeast were inoculated into new flasks containing reduced glucose YPD (1.0% (wt/vol) yeast extract, 2.0% (wt/vol) peptone, 1.0% (wt/vol) glucose), supplemented with uracil (24 mg/l) and adenine (12 mg/l). Cultures reached OD₆₀₀ ≥ 10.0 after 16–18 h. Subsequently, cells were pelleted by centrifugation at room temperature (2000 g, 3 min), washed with sterile miliQ water, centrifuged again (2000 g, 3 min), and suspended in supplemented sporulation media (SPO) (1.0% (wt/vol) potassium acetate, supplemented with adenine/uracil (40 mg/l each), histidine/leucine/tryptophan (20 mg/l each), and 0.02% (wt/vol) raffinose) at OD₆₀₀ of 2.5. For all experiments, cells were incubated in a shaker incubator at 30 °C and 300 rpm.

For the master time course, 50 μM CuSO₄ was added after 2 h in SPO to induce IME1 transcription from the CUP1 promoter, which induces sporulation synchronously. After 6 h in SPO, 1 μM β-estradiol was added to induce NDT80 expression, which in turn induces meiotic divisions and spore formation. To induce re-entry into the mitotic cell cycle, after 6 h in SPO, cells were pelleted by centrifugation at room temperature (2000g, 3 min) and resuspended in an equal volume of pre-warmed YPD.

To induce SPT16-AID depletion, cells expressing SPT16-AID and the pCUP-TIR1 were grown to saturation in YPD as described above. Two hours after shifting to SPO, 50 μM CuSO₄ was added to induce IME1 and TIR1 expression from the CUP1 promoter while 500 μM indole-3-acetic acid (IAA) was added.