Live-Cell Monitoring of Periodic Gene Expression in Synchronous Human Cells Identifies Forkhead Genes involved in Cell Cycle Control
Molecular Biology of the Cell
Geisel School of Medicine
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 with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four 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. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.
Grant GD, Gamsby J, Martyanov V, Brooks L 3rd, George LK, Mahoney JM, Loros JJ, Dunlap JC, Whitfield ML. Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control. Mol Biol Cell. 2012 Aug;23(16):3079-93. doi: 10.1091/mbc.E11-02-0170. Epub 2012 Jun 27. PMID: 22740631; PMCID: PMC3418304.
Dartmouth Digital Commons Citation
Grant, Gavin D.; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K.; Mahoney, J. Matthew; Loros, Jennifer J.; Dunlap, Jay C.; and Whitfield, Michael L., "Live-Cell Monitoring of Periodic Gene Expression in Synchronous Human Cells Identifies Forkhead Genes involved in Cell Cycle Control" (2012). Dartmouth Scholarship. 3862.