title: Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.
abstract: 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.
authors: Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA.
authors: Gavin D
authors: GD
authors: Grant
authors: Joshua
authors: J
authors: Gamsby
authors: Viktor
authors: V
authors: Martyanov
authors: Lionel
authors: L
authors: Brooks
authors: Lacy K
authors: LK
authors: George
authors: J Matthew
authors: JM
authors: Mahoney
authors: Jennifer J
authors: JJ
authors: Loros
authors: Jay C
authors: JC
authors: Dunlap
authors: Michael L
authors: ML
authors: Whitfield
mesh_terms: D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D016000:Cluster Analysis; D016384:Consensus Sequence; D050687:E2F1 Transcription Factor / Q000235:genetics; D051858:Forkhead Transcription Factors / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D015870:Gene Expression; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D028044:Phosphate-Binding Proteins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D034622:RNA Interference; D059010:Single-Cell Analysis; D059467:Transcriptome; D000094524:Gasdermins
chemical_list: D018797:Cell Cycle Proteins; D050687:E2F1 Transcription Factor; D051858:Forkhead Transcription Factors; D047908:Intracellular Signaling Peptides and Proteins; D008156:Luciferases; D009363:Neoplasm Proteins; D028044:Phosphate-Binding Proteins; C493753:E2F1 protein, human; C495879:FOXK1 protein, human; C577011:FoxJ3 protein, human; C498366:GSDMD protein, human; D000094524:Gasdermins