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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 [1]

['Timothy Hoggard', 'Department Of Biomolecular Chemistry', 'School Of Medicine', 'Public Health', 'University Of Wisconsin', 'Madison', 'Wisconsin', 'United States Of America', 'Erika Chacin', 'Biomedical Center Munich']

Date: 2024-10

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.

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.

Funding: This work was supported by the NIH (R35GM141641 to CAF), including salary support for TH, AJH and CAF, and by the Deutsche Forschungsgemeinschaft (DFG) (the German Research Foundation) (project ID 213249687—SFB 1064 to CFK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this report, the Fkh1-FHA domain’s impact on chromosomal origin activity and ORC-origin interactions was addressed at the genome scale using S-phase SortSeq (henceforth SortSeq), ORC ChIPSeq and MNaseSeq experiments to compare FKH1 and fkh1-R80A mutant yeast. The SortSeq data provided evidence that the Fkh1-FHA domain was required for the normal activity of approximately half of all yeast chromosomal origins, acting as a positive regulator of most origins that typically fire in early S-phase (early origins; termed FHA-SORT-positive origins), and a negative regulator of many late origins (FHA-SORT-negative origins). A significant number of FHA-SORT-positive origins were also identified previously in a BrdU ChIP-chip experiment as Fkh1/2-activated origins [ 10 ], but there were also distinct origins in the FHA-SORT-positive group. Specifically, while Cen-associated origins were not defined as Fkh1/2-activated origins, they were identified as FHA-SORT-positive origins, suggesting that features of Cen-associated origin control remain undefined [ 5 , 17 , 24 ]. ORC ChIPSeq data provided evidence that normal levels of ORC-origin binding at FHA-positively regulated origins required the Fkh1-FHA domain. MNaseSeq experiments provided evidence that the Fkh1-FHA domain promoted the stability of nucleosomes adjacent to both origins and promoters in G1-arrested cells while reducing their stability in proliferating cells, providing evidence for a global, cell-cycle regulated role of the Fkh1-FHA domain in normal nucleosome behavior within key genomic regulatory regions. In contrast, higher-resolution analyses of nucleosome and smaller ORC-sized DNA fragments provided evidence that the Fkh1-FHA domain promoted chromatin architectural features distinct to G1-specific origin-associated chromatin. Thus, the yeast Fkh1-FHA domain controlled origin activity, normal ORC-origin interactions and G1-specific hallmarks of origin-associated chromatin at a substantial fraction of yeast chromosomal origins.

However, some evidence indicates that Fkhs may also regulate the G1-phase licensing step of the origin cycle. For example, Fkhs interact with ORC and/or the MCM complex, and Fkh-origin association is enhanced in G1-phase [ 10 , 11 , 19 ]. A specific motivation for this study was the identification of a subset of yeast origins, named FHA-dependent origins, which require the forkhead associated (FHA) domain of Fkh1 for normal levels of ORC binding [ 12 ]. FHA domains are conserved protein-binding modules with a distinctive specificity for peptides that contain a phosphorylated threonine (pT) [ 20 ]. FHA domains also recognize other residues adjacent to the pT within the peptide target, but their identities are distinct for each FHA domain. Thus, pT recognition is the defining functional characteristic of FHA domains. A key conserved arginine within FHA domains is critical for pT recognition, and its substitution abolishes FHA-dependent protein-protein interactions. In yeast, fkh1-R80A is the relevant allele, and inactivates the Fkh1-FHA domain [ 21 , 22 ]. The fkh1-R80A mutant reduces the activity of FHA-dependent origins, while the activity of origins within a control group (referred to as FHA-independent) are unaffected or even enhanced [ 12 ]. As a group, FHA-dependent origins are characterized by a FKH site in a T-rich orientation positioned 5’ of the origin’s essential ORC site (5’ FKH-T). Mutation of the 5’ FKH-T motif within several selected FHA-dependent origins abolishes their Fkh1-FHA domain-dependent stimulation, providing genetic evidence that the Fkh1-FHA domain performs its role at these origins through a single, discrete FKH site [ 12 ]. This 5’ FKH-T site requirement distinguishes FHA-dependent origins from the Fkh1/2-activated origins that have been examined to date that require FKH motifs in the opposite orientation and positioned 3’ of the essential ORC site (3’ FKH-A). These different motif requirements raise the possibility that Fkhs can promote origin activity through multiple mechanisms. FHA-dependent origins were defined parsimoniously by the ability to experimentally assign them a distinct ORC-origin recognition mechanism [ 23 ]. As such, this origin group constitutes <5% of yeast genomic origins. Thus, it was unclear whether the impact the Fkh1-FHA domain had on FHA-dependent origins would apply to other yeast origins.

The DDK phosphorylates the MCM complex, an early step in the conversion of this complex into the two active helicases that unwind DNA. In the simplest version of this model, early origins exist in chromatin regions enriched for FKH sites thus promoting higher concentrations of Fkh proteins that in turn recruit the DDK [ 11 , 14 , 18 ]. Thus, early origins are exposed to a higher local concentration of DDK compared to late origins at the start of S-phase, and hence are more likely to fire.

In Saccharomyces cerevisiae (budding yeast), the forkhead transcription factors (Fkhs), specifically the paralogs encoded by FKH1 and FKH2, have emerged as important non-histone chromatin regulators of this organism’s DNA replication origins [ 5 ]. In contrast to the core origin-control proteins, the Fkhs are not essential for cell division [ 6 ]. Instead, Fkhs1/2 act in a partially redundant manner to promote cell-cycle regulated transcription and to enhance the activity of early S-phase origins (referred to as Fkh1/2-activated origins) [ 7 – 10 ]. The Fkh proteins are posited to promote the activity of many early origins within the yeast genome via a direct mechanism, requiring that Fkhs bind to origin-adjacent Fkh binding sites (FKH sites). Thus, the origin-regulatory roles of the Fkh proteins are viewed as distinct from and not an indirect consequence of their roles in gene transcription [ 10 – 14 ]. Multiple non-mutually exclusive mechanisms have been proposed for how Fkhs promote early origin activity. The most definitive molecular explanation to date is that the Fkh proteins physically interact with the essential S-phase kinase DDK (Dbf4 dependent kinase) that is present in limiting levels relative to licensed origins, though the mechanism of this interaction remains incompletely defined [ 14 – 18 ].

Efficient and accurate duplication of the eukaryotic genome requires that multiple independent DNA replication origins, the loci that initiate DNA replication during S-phase, are distributed both spatially and temporally across each chromosome. Biochemical and structural progress have provided the field with a clear picture of the core proteins and steps required to form an origin [ 1 ]. Briefly, the origin cycle can be divided into two cell-cycle restricted phases. First, in G1-phase, the ORC (origin recognition complex) and the Cdc6 protein form a complex on the DNA that directs the loading of the DNA replicative helicases in an inactive form called the MCM (mini chromosome maintenance) complex (origin licensing phase) (reviewed in [ 2 ]). Second, in S-phase, additional origin-control proteins associate with the inactive MCM complex and promote its remodeling into two active helicases that bidirectionally unwind DNA (origin activation phase). These steps occur at every DNA locus that acts as an origin. However, the local chromatin composition of the locus has an impact on the probability that either the G1-phase licensing and/or S-phase activation reactions will proceed to completion (reviewed in [ 3 ]). This chromatin-influenced variation in origin-reaction probabilities contributes to the stochasticity of origin-use, such that each individual S-phase uses a distinct cohort of origins to replicate the cellular genome. Another result is the generation of a characteristic spatial and temporal pattern of genome replication that can be observed at the cell population level and that is linked to both genome stability and cell identity [ 4 ]. While the essential mechanics of the origin cycle are now understood in molecular detail, the mechanisms by which local origin-adjacent chromatin alters origin activity are not.

Results

The Fkh1-FHA domain contributed to normal replication of the yeast genome during an unperturbed cell cycle SortSeq experiments were used to examine genome replication in proliferating yeast populations. In this approach, the yeast cells are harvested, fixed, stained and sorted into S-phase and late G2-phase populations [25] (S1 Fig). The normalized S-phase DNA copy numbers for each 1 kb genomic region were determined and used to generate chromosome replication profiles (Fig 1A). SortSeq data allows visualization of major replication intermediates, with peaks indicating origins, slopes indicating replication forks, and valleys indicating termination zones. Fig 1A shows the replication profile obtained for chromosome II in FKH1 (black) and fkh1-R80A mutant (red) cells. All yeast chromosomes are shown in S2 Fig. The replication profiles obtained for FKH1 cells recapitulated outcomes from published experiments [26,27]. Because biological or technical variables might cause variation in the outcomes of these experiments, data from biologically independent SortSeq replicates of wild-type (FKH1) (n = 3, black) and mutant (fkh1-R80A) (n = 2, red) yeast were generated and processed independently. The chromosomal scan in Fig 1A depicts the mean value for each 1 kb region across the genome of a given genotype as well as the 95% confidence interval for that mean. These values are indicated as solid lines and vertical shading, respectively. The greatest consistent variation between genetically identical independent experiments occurred over origin peaks and at termination valleys, though some chromosomal-end regions also showed substantial variation (Fig 1A, black) [26]. Nevertheless, even taking into account the variation between independent replicate SortSeq experiments, the data revealed that the fkh1-R80A yeast altered the replication of yeast chromosomes. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. The Fkh1-FHA domain contributed to normal replication of the yeast genome during an unperturbed cell cycle. (A) Normalized S-phase copy numbers across chromosome II for two independent fkh1-R80A mutant cell populations (red) and three independent FKH1 populations (black). The solid lines are the mean obtained for each 1 kb region assessed from each experiment, and the shaded vertical lines indicate the 95% confidence interval for that mean. Downward arrows mark origins whose activity is reduced in fkh1-R80A cells, while upward pointing arrows mark origins whose activity is enhanced. The * marks a termination zone whose replication has been altered substantially by the fkh1-R80A allele. (B) The mean value S-phase copy numbers for every 1 kb bin of the fkh1-R80A mutant (y-axis) are plotted against their value in FKH1 cells (x-axis) (C) The approach used to quantify the impact of the fkh1-R80A allele on each origin across the yeast genome is depicted for FHA-positive origin ARS216. First a 10 kb fragment centered on the T-rich start of the defined ORC site was selected (1, boxed region). Next, the mean S-phase value for each 1 kb region across this 10 kb origin fragment for FKH1, (black) or fkh1-R80A (red) cells was determined (2, each dot represents a distinct 1 kb region within the 10 kb origin locus). Next, the median value of the 10 means was determined for each origin and assigned as a distinct S-phase value in either FKH1 or fkh1-R80A cells (3). Finally, the log2 of each origin’s fkh1-R80A/FKH1 S-phase copy number ratio was determined (4) and used in subsequent graphs. (D) The distribution log2(fkh1-R80A/FKH1) values for each origin was summarized by smoothed kernel density estimates (KDE) for the indicated origin groups. KDE plots of origin number (density, y-axis) versus log2(fkh1-R80A/FKH1) ratios (x-axis) are displayed. ‘All origins’ refers to the 393 confirmed origins from the 410 origins defined in [48] for which we could assign a high-confidence ORC site [12]. ‘FHA-ARS’ refers to the 32 origins characterized in a previous study as described in [12]. ‘Trep timing’ refers to the 238 origins assigned a replication time (Trep) as measured for a synchronous S-phase as in [29]. Origins with log2(fkh1-R80A/FKH1) values ≤ -0.1 were considered positively regulated by the Fkh1-FHA domain (FHA-SORT-positive), while those with log2(fkh1-R80A/FKH1) values ≥ +0.1 were considered negatively regulated (FHA-SORT-negative). These cut-offs are indicated on the KDE plots with vertical lines. All origins that failed to meet these cut-offs were placed in the ‘Other’ category. (D) Quantitative analysis of the effect of the fkh1-R80A allele on duplication of the entire yeast genome. The genome was parsed into 11,800 1 kb regions. The mean S-phase copy number for each region in fkh1-R80A cells (y-axis) was plotted against the mean of its S-phase copy number in FKH1 cells (x-axis). The colors indicate the regions that replicate in the first (early, purple), middle (mid, yellow) or last (late, green) third of S-phase. https://doi.org/10.1371/journal.pgen.1011366.g001 Several points were noted. First, the activity of many of the most frequently used (i.e. efficient) origins, defined here as the most highest peaks in FKH1 cells, were reduced in fkh1-R80A cells. Second, euchromatic origins near the chromosomal termini, in particular those defined by less prominent peaks, showed enhanced replication efficiency in fkh1-R80A cells. For example, the activity of at least one origin (in the vicinity of on the right end of chromosome II) was enhanced in fkh1-R80A cells compared to FKH1 cells. Third, a few replication origin zones, defined as three or more ARSs within the same contiguous chromosomal region (within ≤ 30 kbp [24,28]), showed peak broadening in the SortSeq data. For example, the intrinsic probability of ARS201.7 and ARS203 increasing relative to ARS202 could explain the broadening of the peak in this region (Fig 1A). Fourth, the behavior of some termination zones was altered, an expected byproduct of altered origin use within the population [26]. For example, the replication of the termination zone between ARS202 and ARS207.5, which is one of the last regions of chromosome II to be duplicated in FKH1 cells, was enhanced in fkh1-R80A cells, presumably as a consequence of the enhanced activation probability of two inefficient origins, ARS206 and ARS207.5. Substantial alterations in the distribution of origin activity across chromosomes should alter the normal spatiotemporal pattern of genome duplication. To generate a quantitative test of this expectation, the replication of the entire yeast genome in fkh1-R80A mutant cells was plotted against its replication in FKH1 cells (Fig 1B). Specifically, the genome was divided into 11,800 1 kb regions, and the S-phase copy number for each region in fkh1-R80A cells (y-axis) was plotted against its S-phase copy number in FKH1 cells (x-axis). This analysis revealed that the fkh1-R80A allele mitigated the differential between the yeast genome’s earliest and latest replicating regions, flattening the temporal landscape observed in FKH1 cells.

The Fkh1-FHA domain promoted the activity of centromere-associated origins Cen-associated origins were a specific example of a difference between this study’s FHA-SORT-positive origins and the Fkh1/2-activated origin collection defined previously [10]. Cen-associated origins fire in early S-phase due to recruitment of the DDK by the Ctf19 kinetochore complex [17,24], and are a small subset of early origins that are not Fkh1/2-activated [5]. In the SortSeq experiments, however, many Cen-associated origins qualified as FHA-SORT-positive (Figs 3B and 3C, and S4 Fig). Indeed, based on KDE analyses, Cen-associated origins were among the the most FHA-SORT-positive origins (Fig 3C). 17 Cen-associated origins were defined as FHA-positive based on a stringent criterion (origins within a 10 kb span 5’ or 3’ of the defined centromere) [24]. Twelve of these were FHA-SORT-positive, while none were called as Fkh1/2-activated (Fig 3D). Current models posit that Fkh proteins promote origin activity by binding near these origins’ ORC sites. Therefore, Fkh1 binding near Cen-associated origins was examined using genomic Fkh1 ChIP datasets (Fig 3E). Fkh1 ChIP signals at FHA-SORT-positive, Cen-associated origins were detected over DNA regions typically required for full origin activity, demarcated by the ORC start site (T-rich strand) and DNA sequence 3’ of that site. In contrast, Fkh1 ChIP signals were relatively depleted at the Cen-associated origins that were not affected by fkh1-R80A.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011366

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