RNA-seq of total RNA in various anchor away strains with and without the addition of rapamycin.
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE97915 | GEO | 2018/01/10
SECONDARY ACCESSION(S): PRJNA383312
REPOSITORIES: GEO
Similar Datasets
Project description:We obtained RNA polymerase II occupancy profile across the genome of S.cerevisiae in several strains, Wild type at 25 and 37 degrees C, RNA15-1 at 25 and 37 degrees C, anchor-away parental strain plus rapamycin, Reb1 anchor-away plus rapamycin, Rap1 anchor-away plus rapamycin, Nrd1-AID plus auxin, and nrd1-AID without auxin. This allowed us to compare polymerase profiles when these proteins are depleted from the nucleus.
2018-01-10 | GSE97913 | GEO
Project description:We obtained RNA polymerase II occupancy profiles across the genome of S.cerevisiae strains: Abf1 anchor-away cells after addition of rapamycin for different time points (including no addition of rapamycin) and Rap1-AID auxin-degron cells after addition of auxin for different time points (including no addition of auxin) . This allowed us to compare polymerase occupancy profiles when these proteins are depleted from the nucleus or degraded.
2018-12-20 | GSE114589 | GEO
Project description:During lagging-strand synthesis, strand-displacement synthesis by DNA polymerase delta (Pol ∂), coupled to nucleolytic cleavage of DNA flap structures, combine to produce a nick translation reaction that replaces the DNA at the 5’ end of the preceding Okazaki fragment. Previous work following depletion of DNA ligase I in Saccharomyces cerevisae suggests that DNA-bound proteins, principally nucleosomes and the transcription factors Abf1/Rap1/Reb1, pose a barrier to Pol ∂ synthesis and thereby limit the extent of nick translation in vivo. However, the extended ligase depletion required for these experiments could lead to ongoing, non-physiological nick translation. Here, we investigate nick translation by analyzing Okazaki fragments purified after transient nuclear depletion of DNA ligase I in synchronized or asynchronous S. cerevisiae cultures. We observe that, even with a short ligase depletion, Okazaki fragment termini are enriched around nucleosomes and Abf1/Reb1/Rap1 binding sites. However protracted ligase depletion leads to a global change in the location of these termini, moving them towards nucleosome dyads from a more upstream location and further enriching termini at Abf1/Reb1/Rap1 binding sites. Additionally, we observe an under-representation of DNA derived from DNA polymerase alpha – the polymerase that initiates Okazaki fragment synthesis – around the sites of Okazaki termini obtained from transient ligase depletion. Our data suggest that, while nucleosomes and transcription factors do limit strand-displacement synthesis by Pol ∂ in vivo, post-replicative nick translation can occur at unligated Okazaki fragment termini such that previous analyses represent an overestimate of the extent of nick translation occurring during normal lagging-strand synthesis.
2021-04-22 | GSE173065 | GEO
Project description:Seven temperature sensitive Saccharomyces cerevisiae BY4741 mutants (rsc3-1, abf1-101, reb1-212, rap1-1, mcm1, tbf1 and cep3-1) were grown at restrictive temperatures until a difference in OD600 was observed relative to a wild-type control. Nucleosomal DNA, whole genomic DNA and total RNA were isolated and hybridized onto yeast whole-genome tiling arrays.<br>
2009-01-15 | E-MEXP-1754 | biostudies-arrayexpress
Project description:Monitoring the location of transcription factors (TFs) binding to DNA is key to understanding transcriptional regulation. The main tool for mapping TF binding is ChIP-seq and its variants. However, current ChIP-based methods are hampered by at least one of the following limitations: large input requirements, low spatial resolution, and limited compatibility with high-throughput automation. Here, we describe SLIM-ChIP (Short fragment enriched, Low input, Indexed, MNase ChIP), which overcomes these challenges by combining enzymatic fragmentation of chromatin and on-bead indexing of immobilized TF-DNA complexes. We show that SLIM-ChIP reproduces high resolution binding map of yeast Reb1 similarly to the high-resolution TF mapping methods ChIP-exo and ORGANIC. Yet, SLIM-ChIP requires substantially less input material, and is fully compatible with high-throughput procedures. We further demonstrate the robustness and flexibility of SLIM-ChIP by probing Abf1 and Rap1 in yeast and CTCF in mouse embryonic stem cells. Finally, we show that the unique combination of high resolution and preservation of DNA protection patterns by SLIM-ChIP provide an additional layer of information on the chromatin landscape surrounding the bound TF. We used this information to identify a class of Reb1 sites in which the proximal -1 nucleosome tightly interacts with Reb1 and unlike in most Reb1 sites is refractory to remodeling by the RSC complex. Importantly, the interaction of Reb1 with the -1 nucleosome prevents transcription initiation and can serve as a more general mechanism for maintaining unidirectional transcription. Altogether, SLIM-ChIP is an attractive solution for mapping DNA binding proteins in a more informative context regarding their surrounding chromatin occupancy landscape at a single cell level.
2018-03-06 | GSE108948 | GEO
Project description:Several techniques have been developed to study specific gene function in loss-of-function situations. In Drosophila melanoga st er, RNAi and the generation of mutant clones are widely used. However, both techniques have the limitation that there is a significant time lag before gene function is abolished. Given the relatively rapid development of Drosophila, such perdurance is a serious impediment to study gene function. Here we describe the adaptation of the anchor-away technique for use in Drosophila Anchor-away was originally developed in yeast to quickly and efficiently abrogate the function of nuclear proteins by sequestering - anchoring - them away in a different cellular compartment. The required components are present in the cells, and the system is triggered by the addition of rapamycin, resulting in a rapid generation of a loss-of-function situation. We provide here proof of principle for the system by producing loss-of-function situations for two nuclear proteins - Pygopus and Brinker. The system allows to study the requirement of any protein during any time window, and at the same time circumvents difficulties, such as off-target effects or variable phenotypes, which are inherent in other techniques, for example RNAi.
| S-EPMC7202031 | biostudies-literature
Project description:Key messageGPI anchor addition is important for JAGGER localization and in vivo function. Loss of correct GPI anchor addition in JAGGER, negatively affects its localization and function. In flowering plants, successful double fertilization requires the correct delivery of two sperm cells to the female gametophyte inside the ovule. The delivery of a single pair of sperm cells is achieved by the entrance of a single pollen tube into one female gametophyte. To prevent polyspermy, Arabidopsis ovules avoid the attraction of multiple pollen tubes to one ovule-polytubey block. In Arabidopsis jagger mutants, a significant number of ovules attract more than one pollen tube to an ovule due to an impairment in synergid degeneration. JAGGER encodes a putative arabinogalactan protein which is predicted to be anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Here, we show that JAGGER fused to citrine yellow fluorescent protein (JAGGER-cYFP) is functional and localizes mostly to the periphery of ovule integuments and transmitting tract cells. We further investigated the importance of GPI-anchor addition domains for JAGGER localization and function. Different JAGGER proteins with deletions in predicted ω-site regions and GPI attachment signal domain, expected to compromise the addition of the GPI anchor, led to disruption of JAGGER localization in the cell periphery. All JAGGER proteins with disrupted localization were also not able to rescue the polytubey phenotype, pointing to the importance of GPI-anchor addition to in vivo function of the JAGGER protein.
| S-EPMC11377618 | biostudies-literature
Project description:RAP1 is well known as a telomere-binding protein; yet its functions in human stem cells remain unclear. Here we generated RAP1-deficient human embryonic stem cells (hESCs) by CRISPR/Cas9 and obtained RAP1-deficient human mesenchymal stem cells (hMSCs) and neural stem cells (hNSCs) via directed differentiation. In both hMSCs and hNSCs, RAP1 negatively regulated telomere length. In addition, RAP1 acted as a transcriptional regulator of RLEN by tuning the methylation status of its promoter region. Phenotypically, RAP1 deficiency resulted in enhanced self-renewal and delayed senescence in hMSCs rather than in hNSCs, suggesting a complex role of RAP1 in regulating lineage specific stem cell homeostasis. Altogether, these results indicate that RAP1 plays both telomeric and non-telomeric roles in regulating the homeostasis of human stem cells.
2018-11-20 | GSE122654 | GEO
Project description:We analyed the nucleosome positions by using 2 concentrations of micrococcal nuclease on yeast strains that were grown in raffinose or galactose containing media (synthetic complete). Strains contained FRB-tags to Sth1, Swi2, Sth1 & Swi2 or Sth1 and TBP, where TBP was tagged on one of two alleles in a diploid strain. Cells were treated for 60 min either with 7.5 uM rapamycin or the same volume of DMSO.
2023-01-11 | GSE190737 | GEO
Project description:Promoter shutoff of essential genes in the diploid Candida albicans has often been insufficient to create tight, conditional null alleles due to leaky expression and has been a stumbling block in pathogenesis research. Moreover, homozygous deletion of non-essential genes has often been problematic due to the frequent aneuploidy in the mutant strains. Rapid, conditional depletion of essential genes by the anchor-away strategy has been successfully employed in Saccharomyces cerevisiae and other model organisms. Here, rapamycin mediates the dimerization of human FK506-binding protein (FKBP12) and FKBP12-rapamycin-binding (FRB) domain-containing target protein, resulting in relocalization to altered sub-cellular locations. In this work, we used the ribosomal protein Rpl13 as the anchor and took two nuclear proteins as targets to construct a set of mutants in a proof-of-principle approach. We first constructed a rapamycin-resistant C. albicans strain by introducing a dominant mutation in the CaTOR1 gene and a homozygous deletion of RBP1, the ortholog of FKBP12, a primary target of rapamycin. The FKBP12 and the FRB coding sequences were then CUG codon-adapted for C. albicans by site-directed mutagenesis. Anchor-away strains expressing the essential TBP1 gene or the non-essential SPT8 gene as FRB fusions were constructed. We found that rapamycin caused rapid cessation of growth of the TBP-AA strain within 15 minutes and the SPT8-AA strain phenocopied the constitutive filamentous phenotype of the spt8Δ/spt8Δ mutant. Thus, the anchor-away toolbox for C. albicans developed here can be employed for genome-wide analysis to identify gene function in a rapid and reliable manner, further accelerating anti-fungal drug development in C. albicans.ImportanceMolecular genetic studies thus far have identified ~27% open-reading frames as being essential for the vegetative growth of Candida albicans in rich medium out of a total 6,198 haploid set of open reading frames. However, a major limitation has been to construct rapid conditional alleles of essential C. albicans genes with near quantitative depletion of encoded proteins. Here, we have developed a toolbox for rapid and conditional depletion of genes that would aid studies of gene function of both essential and non-essential genes.
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