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The budding yeast Fkh1 Forkhead associated (FHA) domain promotes a G1-chromatin state and the activity of chromosomal DNA replication origins.

ABSTRACT: 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 ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 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.

SUBMITTER: Hoggard T

PROVIDER: S-EPMC11326605 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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The budding yeast Fkh1 Forkhead associated (FHA) domain promotes a G1-chromatin state and the activity of chromosomal DNA replication origins.

Hoggard Timothy T Chacin Erika E Hollatz Allison J AJ Kurat Christoph F CF Fox Catherine A CA

PLoS genetics 20240805 8

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) domai ...[more]

PMID: 39102423

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Project description: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 that Fkh1 uses to promote early origin activity are undefined. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module that binds 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 either 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, with ORC contributing 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 Fkh1FHA-dependent. Thus, the Fkh1-FHA domain performed an important post-DNA binding role of the Fkh1 protein in promoting hallmarks of origin-associated protein-DNA architecture in G1-phase and the activity of early origins in S-phase.

Project description:The Saccharomyces cerevisiae Fkh1 protein has roles in cell-cycle regulated transcription as well as a transcription-independent role in recombination donor preference during mating-type switching. The conserved FHA domain of Fkh1 regulates donor preference by juxtaposing two distant regions on chromosome III to promote their recombination. A model posits that this Fkh1-mediated long-range chromosomal juxtaposition requires an interaction between the FHA domain and a partner protein(s), but to date no relevant partner has been described. In this study, we used structural modeling, 2-hybrid assays, and mutational analyses to show that the predicted phosphothreonine-binding FHA domain of Fkh1 interacted with multiple partner proteins. The Fkh1 FHA domain was important for its role in cell-cycle regulation, but no single interaction partner could account for this role. In contrast, Fkh1's interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. Using 2-hybrid assays, co-immunoprecipitation, and fluorescence anisotropy, we mapped a discrete peptide within the regulatory Mph1 C-terminus required for this interaction and identified two threonines that were particularly important. In vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA) specifically abolished the Fkh1-Mph1 interaction in vivo and altered donor preference during mating-type switching to the same degree as mph1Δ. Notably, the mph1-2TA allele maintained other functions of Mph1 in genome stability. Deletion of a second Fkh1-interacting protein encoded by YMR144W also resulted in a change in Fkh1-FHA-dependent donor preference. We have named this gene FDO1 for Forkhead one interacting protein involved in donor preference. We conclude that a phosphothreonine-mediated protein-protein interface between Fkh1-FHA and Mph1 contributes to a specific long-range chromosomal interaction required for mating-type switching, but that Fkh1-FHA must also interact with several other proteins to achieve full functionality in this process.

Dataset Information

The budding yeast Fkh1 Forkhead associated (FHA) domain promotes a G1-chromatin state and the activity of chromosomal DNA replication origins.

Publications

The budding yeast Fkh1 Forkhead associated (FHA) domain promotes a G1-chromatin state and the activity of chromosomal DNA replication origins.

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