Fig. 2.

Fkh1 and Fkh2 regulate dynamics of mitotic Clb cyclins in a cell cycle-dependent manner. a Quantitative real-time PCR of mitotic CLB transcripts in yeast cells treated with nocodazole (NOC). Total mRNA was isolated from arrested wild type, fkh1Δ, fkh2Δ, and fkh1Δfkh2Δ cells, and CLB1, CLB2, CLB3, and CLB4 mRNA levels were measured. ACT1 and TSA1 genes were used as negative controls, as they are not affected by cell cycle dynamics. Error bars on the histograms represent SDs from the mean of three independent experiments; p-values are indicated on the histograms in Supplementary Fig. S2a. Nocodazole-arrested cells show that fkh2Δ affects both CLB3 and CLB4 transcript accumulation, whereas fkh1Δ affects CLB4 transcript accumulation. b, c Binding of Fkh2 (b) and Ndd1 (c) to mitotic CLB promoter regions. Chromatin immunoprecipitation was performed by precipitating protein/DNA complexes from cells grown in exponential phase using an anti-Myc antibody. ACT1 and TSA1 genes were used as negative controls, whereas CLB1 and CLB2 genes as positive controls. Error bars on the histograms represent SDs from the mean of three independent experiments. d, e Fkh2 controls the timing of Clb3 protein expression. d Time course of Clb2-18Myc, Clb3-TAP, Clb5-HA and Sic1-TAP are shown for wild type (YAN49), fkh1Δ (YAG20), and fkh2Δ (YAG21) strains. Yeast cells were synchronized by centrifugal elutriation in YPD at 30 °C, released into fresh YPD at 30 °C, and sampled for western blot analysis at the indicated times. Clb3 levels were quantified in wild type, fkh1Δ and fkh2Δ strains (e). The experiments were performed at the same time, and the membranes processed identically, with the same antibody aliquots and then exposed to the same levels. fkh1Δ resulted in an decreased amount of Clb3 produced and no time delay as compared to wild type, whereas fkh2Δ resulted in both a decreased amount of Clb3 produced and a time delay as compared to wild type. The result is representative of three independent experiments