Biology subjects

Fox, C. A.

Publications and source records attributed to Fox, C. A..

6 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↗

bioRxiv2022

Conserved and diverged mechanisms for RNA target-site recognition by the multi KH-domain Bicaudal C protein

The Bicaudal-C (Bicc1) multi-KH domain RNA binding protein controls cell fates by binding and repressing the translation of specific target mRNAs. To provide new insights into Bicc1-RNA binding we used quantitative assays to analyze interactions between Xenopus Bicc1 protein and the 32-nt Bicc1 RNA target site from XlCripto-1 mRNA. High-affinity binding by XlBicc1(Kd=34nM) required both a 5 leader sequence and a 3loop sequence. Bicc1 proteins from humans (HsBicc1), zebrafish (DrBicc1) and fruit flies (DmBicc1) also bound the XlCripto-1 RNA with high-affinity via an interaction requiring the 5 leader sequence, providing evidence for a conserved mechanism of RNA recognition by Bicc1. However, in contrast to XlBicc1, each of the other species Bicc1s showed a significantly relaxed requirement for the 3 loop sequence. Analyses of mutant forms of XlBicc1, revealed that the KH1 domain promoted Bicc1s requirement for the loop sequence via a GXXG-independent mechanism. In addition, a fusion protein comprised of the XlKH1 and the DmBicc1-KH2-KH3 subdomains was sufficient to establish high-affinity, loop-sensitive Bicc1-binding to XlCripto-1 RNA. These data support a model wherein the KH2 domain interacts with the leader sequence via a GXXG-mediated KH-RNA interface, defining the conserved core of the metazoan Bicc1-RNA interaction, while the KH1 domain is required for the formation of a more flexible, secondary protein-RNA interface that allows recognition of the 3 loop sequence. This secondary interface differs between species and possibly between target mRNAs, establishing the potential for a range of translational repression activities for Bicc1 target mRNAs.

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↗

bioRxiv2019

A Meier-Gorlin Syndrome Mutation in Orc4 CausesTissue-Specific DNA Replication Defects in Drosophila melanogaster

Meier-Gorlin syndrome is a rare recessive disorder characterized by a number of distinct developmental defects, including primordial dwarfism, small ears, and small or missing patella. Genes encoding members of the origin recognition complex (ORC) and additional proteins essential for DNA replication (CDC6, CDT1, GMNN, CDC45, and MCM5) are mutated in individuals diagnosed with MGS. The primary role of ORC is to license origins during the G1 phase of the cell cycle, but it also plays roles in cilia development, heterochromatin formation, and other cellular processes. Because of its essential role in DNA replication, ORC is required for every cell division during development. Thus, it is unclear how the Meier-Gorlin syndrome mutations in ORC lead to the tissue-specific defects associated with the disease. To address this question, we have used Cas9-mediated genome engineering to generate a Drosophila melanogaster model of individuals carrying a mutation in ORC4. Like the people with Meier-Gorlin syndrome, these flies reach adulthood, but have several tissue-specific defects. Genetic analysis revealed that this allele is a hypomorph and that mutant females are sterile. We demonstrated that this sterility is caused by a failure in DNA replication. By leveraging the well-studied Drosophila system, we showed that a disease-causing mutation in orc4 disrupts DNA replication, and we propose that in individuals with MGS defects arise preferentially in tissues with a high-replication demand.

developmental biology↗

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↗