In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of approximate to 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting approximate to 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase. DNA replication must be regulated both spatially and temporally to insure the accurate and efficient duplication of the eukaryotic genome. Altering this spatiotemporal control can cause mistakes in genome copying and/or deficiencies in cell proliferation that promote disease. Therefore, the proteins and mechanisms underlying the normal spatiotemporal progression of eukaryotic genome duplication are of keen interest. The Fkh1 protein, a type of DNA binding protein that regulates eukaryotic cell proliferation, contributes to the spatiotemporal control of genome duplication in budding yeast. We learned that a single amino acid change within one region of the Fkh1 protein, named the FHA domain, altered the spatiotemporal progression of budding yeast genome duplication. FHA domains convey molecular information by directly binding to partner proteins that are phosphorylated on threonine residues. This information in turn stimulates specific molecular activities or events required by the cell. Thus, our study revealed that a protein-protein interaction controlled by threonine-phosphorylation is required for the normal spatiotemporal progression of yeast genome duplication.
