Genome-wide gene expression responses to experimental manipulation of Saccharomyces cerevisiae Repressor Activator Protein 1 (Rap1) expression level
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ABSTRACT:
PROVIDER: PRJNA938450 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23611954.fastq.gz | Fastqsanger.gz | |||
| SRR23611955.fastq.gz | Fastqsanger.gz | |||
| SRR23611956.fastq.gz | Fastqsanger.gz | |||
| SRR23611957.fastq.gz | Fastqsanger.gz | |||
| SRR23611958.fastq.gz | Fastqsanger.gz |
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Project description:We constructed the inducible H3-depleted and FLAG-tag of the repressor activator protein 1 (Rap1) yeasts to investigate the depleted effect of nucleosomal H3 on DNA-binding profiles of Rap1 and resulting transcriptional changes. The H3 inducible shut-off strain (named “DGS200.1” or “H3i” strain) was prepared from the wildtype YEF473A strain by knocking out the HHT1 gene (encoding H3), and replacing the native promoter of the HHT2 gene (the other H3-encoding gene) with an inducible Gal1 promoter. Conditional depletion of H3 was induced by culturing the H3i strain in the YPGal liquid medium (1% yeast extract, 2% peptone, 2% galactose) and transferring to the YPD medium (1% yeast extract, 2% peptone, 2% dextrose) for three hours. WT yeast were also cultured and switched the growth media in the same way as controls. DNA-binding profiles of H3 and Rap1 were determined using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq), and the changes in transcriptional levels were investigated using RNA-seq.
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Project description:We constructed the inducible H3-depleted and FLAG-tag of the repressor activator protein 1 (Rap1) yeasts to investigate the depleted effect of nucleosomal H3 on DNA-binding profiles of Rap1 and resulting transcriptional changes. The H3 inducible shut-off strain (named “DGS200.1” or “H3i” strain) was prepared from the wildtype YEF473A strain by knocking out the HHT1 gene (encoding H3), and replacing the native promoter of the HHT2 gene (the other H3-encoding gene) with an inducible Gal1 promoter. Conditional depletion of H3 was induced by culturing the H3i strain in the YPGal liquid medium (1% yeast extract, 2% peptone, 2% galactose) and transferring to the YPD medium (1% yeast extract, 2% peptone, 2% dextrose) for three hours. WT yeast were also cultured and switched the growth media in the same way as controls. DNA-binding profiles of H3 and Rap1 were determined using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq), and the changes in transcriptional levels were investigated using RNA-seq.
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Project description:We constructed the inducible H3-depleted and FLAG-tag of the repressor activator protein 1 (Rap1) yeasts to investigate the depleted effect of nucleosomal H3 on DNA-binding profiles of Rap1 and resulting transcriptional changes. The H3 inducible shut-off strain (named “DGS200.1” or “H3i” strain) was prepared from the wildtype YEF473A strain by knocking out the HHT1 gene (encoding H3), and replacing the native promoter of the HHT2 gene (the other H3-encoding gene) with an inducible Gal1 promoter. Conditional depletion of H3 was induced by culturing the H3i strain in the YPGal liquid medium (1% yeast extract, 2% peptone, 2% galactose) and transferring to the YPD medium (1% yeast extract, 2% peptone, 2% dextrose) for three hours. WT yeast were also cultured and switched the growth media in the same way as controls. DNA-binding profiles of H3 and Rap1 were determined using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq), and the changes in transcriptional levels were investigated using RNA-seq.
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Project description:To confirm that Rap1-depletion resulted in decreased binding of Rap1 to its target sites, we performed compared Rap1 ChIP-chip experiments in respiratory cells (pre-meiotic/YPA 20 hours) depleted (plus doxycycline for 12 hours) or not depleted (minus doxycycline) of Rap1. There is growing recognition that the binding of a transcription factor near a gene does not always indicate regulatory function, and further that a single factor may function to either activate or repress its targets depending on the cellular context. We examined these issues through a series of experiments involving the S. cerevisiae transcription factor Rap1, and its function throughout critical metabolic and developmental transitions between vegetative growth, respiratory growth, meiosis and sporulation. We simultaneously monitored the expression of all genes and the genomic binding locations of Rap1 throughout the timecourse. Genes downstream of Rap1 binding were activated and repressed dynamically, but a change - or lack of change - in Rap1 binding status was not predictive of activation, repression, or no change in regulation. Despite this, we show that Rap1 is required, at a given point in time, for both activation and repression of different gene targets, within the same cell. Specification of the transcriptional consequences of Rap1 binding is thus highly promoter-specific. The presence of other transcription factor binding motifs, the subtype of Rap1 motif, and the underlying chromatin structure of the promoter cannot fully account for the observed transcriptional outcomes. There is a better accounting for the dynamic binding behavior of Rap1 including specification of an expanded meiotic target set through a Tup1- dependent nucleosome-loss mechanism. The variable and dynamic association between binding and transcription in this simple unicellular system portends a similarly volatile relationship in more complex eukaryotes. Biological interpretations of transcription factor occupancy should be made cautiously and in conjunction with supporting data obtained under the precise condition of interest.
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