Grant GD, Gamsby J, Martyanov V, Brooks L 3rd, George LK, Mahoney JM, Loros JJ, Dunlap JC, Whitfield ML.
We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S, or a basal promoter with six Forkhead DNA binding sites (6xDB) with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across 3 - 4 synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle-dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. ChIP-seq analysis identified 4329 genomic loci bound by FOXK1; 83% of which contained a FOXK1 binding motif. We validated that a subset of these loci are activated by wild type FOXK1 but not by a FOXK1 (H35A) DNA-binding mutant.