A Swi4 positive feedback loop that regulates timing of start revealed by two photon scanning number and brightness microscopy

The irreversible commitment to division in Saccharomyces cerevisiae takes place at the G1/S cell cycle transition (Start). While many Start regulators are known, the mechanisms controlling its timing, and hence cell size, remain to be determined. Start is activated by the SBF and MBF transcriptional factor complexes which bind to target sites in G1/S promoters and activate the transcription of ∼200 genes in the G1/S regulon. SBF and MBF contain the same activator subunit, Swi6 but a distinct DNA binding subunit, Swi4 or Mbp1, respectively. SBF is repressed during G1 phase by Whi5. Cyclin dependent kinases produced as part of the G1/S regulon in a positive feedback loop phosphorylate Whi5, leading to its dissociation from SBF and export from the nucleus. We previously reported that G1/S transcription factors, particularly Swi4, are sub-saturating with respect to their promoters in newborn daughter cells. Their increasing copy numbers throughout G1 phase gradually titrate their target promoters, representing a critical determinant of the timing of Start. The positive phosphorylation feedback loop ensures a sharp Start transition once sufficient SBF is present at G1/S promoters. A possible positive feedback mechanism of Swi4 transcription has been suggested and could contribute to the strong Swi4 copy number increase in G1 phase. However, the existence of this feedback loop had never been established. In this study, we demonstrate the existence of this positive feedback loop using two photon scanning number and brightness fluorescence microscopy on yeast strains expressing Swi4-GFP from its natural locus and deleted for key features of the putative feedback loop or ectopically expressing another copy of Swi4. These results are interpreted within the framework of a model for cell size control.