Biology subjects

Hoggard, T.

Publications and source records attributed to Hoggard, T..

4 recordsLinked to original sources

bioRxiv2024

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). 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.

molecular biology↗

bioRxiv2021

The Fkh1 Forkhead Associated Domain Promotes ORC Binding to a Subset of DNA Replication Origins in Budding Yeast

The pioneer event in eukaryotic DNA replication is binding of chromosomal DNA by the origin recognition complex (ORC), which directs the formation of origins, the specific chromosomal regions where DNA will be unwound for the initiation of DNA synthesis. In all eukaryotes, incompletely understood features of chromatin promote ORC-DNA binding. Here, we uncover a role for the Fkh1 (forkhead homolog) protein, and, in particular, its forkhead associated (FHA) domain in promoting ORC-origin binding and origin activity at a subset of origins in Saccharomyces cerevisiae. The majority of the FHA-dependent origins within the experimental subset examined contain a distinct Fkh1 binding site located 5 of and proximal to their ORC sites (5-FKH-T site). Epistasis experiments using selected FHA-dependent origins provided evidence that the FHA domain promoted origin activity through Fkh1 binding directly to this 5 FKH-T site. Nucleotide substitutions within two of these origins that enhanced the affinity of their ORC sites for ORC bypassed these origins requirement for their 5 FKH-T sites and for the FHA domain. Significantly, direct assessment of ORC-origin binding by ChIPSeq provided evidence that this mechanism affected ~25% of yeast origins. Thus, this study reveals a new mechanism to enhance ORC-origin binding in budding yeast that requires the FHA domain of the conserved cell-cycle transcription factor Fkh1.

molecular biology↗

bioRxiv2020

Sir2 tempers an intrinsic imbalance in origin licensing efficiency between early and late replicating euchromatin

A eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled on chromosomal DNA in G1-phase (a.k.a. origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing established the normal spatial distribution of origins across yeast chromosomes required for normal genome duplication. Significance statementIn eukaryotes, multiple DNA replication origins, the sites where new DNA synthesis begins during the process of cell division, must be adequately distributed across chromosomes to maintain normal cell proliferation and genome stability. This study describes a repressive chromatin-mediated mechanism that acts at the level of individual origins to attenuate the efficiency of origin formation. This attenuation is essential for achieving the normal spatial distribution of origins across the chromosomes of the eukaryotic microbe Saccharomyces cerevisiae. While the importance of chromosomal origin distribution to cellular fitness is now widely acknowledged, this study is the first to define a specific chromatin modification that establishes the normal spatial distribution of origins across a eukaryotic genome.

genetics↗

bioRxiv2018

Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins

Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are robustly suppressed by deletion of either SIR2 or SIR3. While this and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using both MCM ChIP-Seq and MNase-H4K16ac ChIP-Seq data, we show that a SIR2 deletion rescues MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibits a pervasive effect at the majority of euchromatic origins. Importantly, in wild type (i.e. CDC6) cells, origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner. In addition, both Sir2 and Sir3 directly bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin - SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 - correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly bind euchromatic DNA replication origins and modify their local chromatin environment.

genetics↗