The budding yeast Fkh1 forkhead-associated (FHA) domain promotes the G1-chromatin status and activity of chromosomal DNA replication origins
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). Fkh1 binds directly to origin-adjacent Fkh1 binding sites (FKH sites), providing evidence that Fkh1 acts directly. However, the post-DNA binding functions Fkh1 uses to promote early origin activity are not defined. Fkh1 contains a conserved FHA (fork head associated) domain, a protein-binding module that binds to 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. The relevance of the Fkh1-FHA to chromosomal replication or ORC-origin interactions at genome scale has not been reported. Here, S-phase SortSeq experiments were used to assess genome replication in proliferating FKH1 and fkh1R80A mutant cells. The data provided evidence that the Fkh1-FHA domain promoted the activity of ~ 100 origins that act in early S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ~ 100 late origins. ORC ChIPSeq data provided evidence that the Fkh1-FHA domain promoted normal ORC-origin binding at FHA-stimulated origins. Origins are associated with a distinctive nucleosome-organization that frames a nucleosome deplected region (NDR) over the origin, and ORC contributes to this architecture. To ask whether the Fkh1-FHA domain had an impact on the ORC- and nucleosome protein-DNA interactions at origins, MNaseSeq data were generated from G1-arrested and proliferating FKH1 and fkh1R80A mutant cell populations. These data provided evidence that origin groups differentially regulated by the Fkh1-FHA domain were characterized by distinct G1-phase ORC- and nucleosome configurations that were Fkh1-FHA domain-dependent. Thus, the Fkh1-FHA domain performed an important post-DNA binding role of the Fkh1 protein in promoting hallmarks of G1-phase origin-associated protein-DNA architecture and the activity of early origins.