Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA400160 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5973473.fastq.gz | Fastqsanger.gz | |||
| SRR5973474.fastq.gz | Fastqsanger.gz | |||
| SRR5973475.fastq.gz | Fastqsanger.gz | |||
| SRR5973476.fastq.gz | Fastqsanger.gz | |||
| SRR5973477.fastq.gz | Fastqsanger.gz |
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Project description:Initiation of eukaryotic chromosome replication follows a spatiotemporal program. Current model suggests that replication origins compete for a limited pool of initiation factors. However, it remains to be answered how these limiting factors are preferentially recruited to early origins. Here, we report that Dbf4 is enriched at early origins through its interaction with forkhead transcription factors Fkh1 and Fkh2. This interaction is mediated by Dbf4 C-terminus and was successfully reconstituted in vitro. An interaction defective mutant dbf4ΔC phenocopies fkh alleles in terms of origin firing. Remarkably, genome-wide replication profiles reveal that the direct fusion of the DNA-binding domain of Fkh1 to Dbf4 restores the Fkh-dependent origin firing, but specifically interferes with the pericentromeric origin activation. Furthermore, Dbf4 directly interacts with Sld3 and promotes the recruitment of downstream limiting factors. These data suggest that Fkh1 targets Dbf4 to a subset of non-centromeric origins to promote early replication, in a manner that is reminiscent to the recruitment of Dbf4 to pericentromeric origins by Ctf19.
2018-01-19 | GSE103073 | GEO
Project description:Eukaryotic genomes are replicated in spatiotemporal patterns that are stereotypical for individual genomes and developmental profiles. In the model system S. cerevisiae, two primary mechanisms determine the preferential activation of replication origins during early S phase, thereby largely defining the consequent replication profiles of these cells. Both mechanisms are thought to act through specific recruitment of a rate-limiting initiation factor, Dbf4-dependent kinase (DDK), to a subset of licensed replication origins. Fkh1/2 is responsible for stimulation of most early-firing origins, except for centromere (CEN)-proximal origins that recruit DDK via the kinetochore protein Ctf19, which is required for their early-firing. The C-terminus of Dbf4 has been implicated in its recruitment to origins via both the Fkh1/2 and Ctf19 mechanisms. Here, we show that the Zn-finger motif within the C-terminus is specifically required for Dbf4 recruitment to CENs to stimulate CEN-proximal/Ctf19-dependent origins, whereas stimulation of origins via the Fkh1/2 pathway remains largely intact. These findings re-open the question of exactly how Fkh1/2 and DDK act together to stimulate replication origin initiation.
2022-11-10 | GSE215190 | GEO
Project description:The S. cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. As stimulators of early origin activation, we hypothesized that Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, were not well suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advance the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase. 5 total experiments with replicates
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Project description:Transcriptional re-activation of hTERT is the limiting step in tumorigenesis. While mutations in hTERT promoter seen in 19% of all cancers are recognized as key drivers of hTERT reactivation, mechanisms by which the wildtype hTERT (WT-hTERT) promoter is reactivated, as observed in the majority of cancers, remain unknown. We report that unlike with the mutant-hTERT promoters, a T-INT2 (Tert INTeracting region 2) region located ~120kb upstream of the hTERT proximal promoter is essential to uniquely reactivate the WT-hTERT promoter, via long-range chromatin interactions. Unlike mutant-hTERT promoters, which are driven by GABPa/b tetramers tethered between T-INT1 (Tert-INTeracting-region 1) and de-novo ETS sites created by promoter mutations, WT-hTERT promoter firing is initiated by 2 events a) elevated JunD mediated recruitment of CTCF and b) β-catenin and CBP mediated recruitment of Sp1 tetramers between T-INT2 and WT-hTERT proximal-promoter, leading to chromatin-openness, pol2 recruitment and productive transcription.
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Project description:Transcriptional re-activation of hTERT is the limiting step in tumorigenesis. While mutations in hTERT promoter seen in 19% of all cancers are recognized as key drivers of hTERT reactivation, mechanisms by which the wildtype hTERT (WT-hTERT) promoter is reactivated, as observed in the majority of cancers, remain unknown. We report that unlike with the mutant-hTERT promoters, a T-INT2 (Tert INTeracting region 2) region located ~120kb upstream of the hTERT proximal promoter is essential to uniquely reactivate the WT-hTERT promoter, via long-range chromatin interactions. Unlike mutant-hTERT promoters, which are driven by GABPa/b tetramers tethered between T-INT1 (Tert-INTeracting-region 1) and de-novo ETS sites created by promoter mutations, WT-hTERT promoter firing is initiated by 2 events a) elevated JunD mediated recruitment of CTCF and b) β-catenin and CBP mediated recruitment of Sp1 tetramers between T-INT2 and WT-hTERT proximal-promoter, leading to chromatin-openness, pol2 recruitment and productive transcription.
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Project description:The S. cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. As stimulators of early origin activation, we hypothesized that Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, were not well suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advance the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase.
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Project description:The S. cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. As stimulators of early origin activation, we hypothesized that Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, were not well suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advance the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase.
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