In all eukaryotic cells, passage through the cell cycle is hallmarked by successive waves of gene expression. A number of transcription factors involved in cell cycle-regulated gene expression have been identified in budding yeast, including SBF in late G1; however upstream factors and pathways that impinge upon these genes remain largely uncharacterized. To identify novel proteins that control the expression of genes during specific phases of the cell cycle either directly or indirectly, we are using a promoter-based reporter system combined with high-throughput genetics. Specifically, we have fused the endogenous promoters of selected cell cycle-regulated genes to GFP and are systematically introducing these reporters into different genetic backgrounds. GFP reporter expression from the resulting strains is measured directly from colonies arrayed on solid agar plates. We are currently screening the yeast haploid deletion array and will expand our studies to include the GAL-ORF overexpression array and the TET-repressible array of essential genes. Deletion mutants that display lower and higher levels of GFP reporter expression compared to wild type represent putative positive and negative transcriptional regulators of the cell cycle gene, respectively, while the converse is likely true for overexpression strains.

As proof-of-principle, we have introduced a construct containing a GFP reporter fused to the well-characterized promoter of the G1 cyclin Cln2 into the haploid deletion array and quantified GFP intensity of approximately 5000 deletion strains. We found that GFP expression was lowest in the swi4Δ strain, which was expected as Swi4 is a master activator of G1-specific CLN2 expression. To demonstrate that the Swi4 effect on CLN2 expression is specific, an additional screen was performed with a GFP reporter driven by the RPL39 promoter, which is constitutively active and independent of cell cycle regulation. GFP expression driven by this promoter was not affected in the swi4Δ deletion strain ruling out Swi4’s role as a general transcriptional activator. Other potential hits from this screen are being confirmed to identify novel regulators of CLN2 transcription.  Encouraged by these results, we are continuing to screen additional promoters specific to other phases of the cell cycle for which little is known in a large effort to map out the cell cycle-transcriptional regulatory network in Saccharomyces cerevisiae.