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Investigation of Rap1 binding occupancy in global H3-depletion condition [RNA-seq]

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE232684 | GEO | 2023/10/31

REPOSITORIES: GEO

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Investigation of Rap1 binding occupancy in global H3-depletion condition [ChIP-Seq]

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 understand the biological relevance of the role played by the HHT1 histone H3 variant in C. albicans, we performed a transcriptome analysis of the mutant by global gene expression array analysis. We analyzed gene expression profile of the mutant in strains LR107 and LR108 (hht1∆/hht1∆) and the parent wild type (SC5314) strain grown in YPD liquid medium at 30°C. In the microarray analysis, a total of 1222 genes were found to be expressed significantly different (fold change < 1.5 at p-value < 0.05) between wild type and two hht1 null mutants. Out of the 1222 differentially expressed genes, 670 genes were up-regulated whereas 552 genes were down-regulated. Gene ontology (GO) analysis of up-regulated and down-regulated genes suggests that biofilm, Hap43, transcription, filamentation, white-opaque/mating and invasive growth are the major pathways altered in minor H3 null mutant. Transcription profiling in planktonic YPD condition suggests that minor H3 represses biofilm genes in the planktonic mode of growth. Therefore we performed a genome-wide expression array study after growing the wild type strain and hht1/hht1 mutant strains in Spider induced biofilm condition. This experiment was carried out with two biological replicates of hht1 null mutants. The biofilm induced microarray data revealed that a total of 1010 genes were differentially expressed (at p-value <0.05 and fold change <1.5) between the wild type and hht1 mutants. Out of 1016 differentially expressed genes, 572 genes were up-regulated whereas 444 genes were down-regulated.

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