Figure 2: Periodic transcription in G2-arrested fission yeast cells.

(a) Schematic representation of the experimental procedure. Inhibitor-sensitive minimal cells were synchronized in G2 with 1 μM inhibitor (3-MBPP1) for 2 h 40 min as described¹⁶. Upon washing off the inhibitor, cells resumed their cycle (Release), progressing through mitosis, G1, S and entering the next G2 after ∼70 min (see b and c). The culture was subsequently treated with 1 μM 3-MBPP1 (T0), blocking the onset of the next mitosis. DMSO-treated cells were used as a control. Samples were then taken every 10 min for 120 min, and gene expression changes were assessed. (b) Percentage of binucleated cells in a. Unlike the control culture, cells treated with inhibitor at T0 do not undergo mitosis. Release is as in a. n>100 at each time point. (c) DNA content analysis of cells in a. At T0, cells have undergone DNA replication (black profile, bottom) and have entered G2. Subsequently, control cells undergo a second round of replication after 40 min (black profile, top), while inhibitor-treated cells are blocked in G2. Note that the reduced synchrony in the second cell cycle makes the next S phase more difficult to monitor by flow cytometry (see DMSO profiles). (d–f) Changes in gene expression for the indicated genes in a. C and A refer to cycling (DMSO-treated) and arrested (inhibitor-treated) cells, respectively. Fold changes are normalized to actin RNA levels and represented relative to the values at T0 (set to 1). (g) Heatmaps reflecting the differences in expression levels (ratios between conditions) for periodic genes between cycling (C) and arrested (A) cells at the indicated time points. Included are ∼450 periodic genes as described in Cyclebase (Supplementary Data 1), sorted by induction amplitude^4,12. Coloured boxes on the left show the known targets of PBF, Ace2, MBF and Ams2. Within each cluster, shades of blue indicate three groups according to their transcription amplitude, each containing the same number of genes. Darker blue corresponds to higher fold inductions. See Supplementary Table 1 for the percentages of genes above the cutoffs for the ratios in expression between the cultures.