Unknown,Transcriptomics,Genomics,Proteomics

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Early stages of yeast senescent cells

ABSTRACT: We performed transcriptional and metabolic profiling for yeast at early stages of senescence (4th, 7th and 11th generation), that is, for populations in which most cells are still alive. Transcriptional profiles showed up- and down-regulation for about 20% of the genes profiled after the first 4 generations, few further changes by the 7th generation, and an additional 12% of the genes were up- and down-regulated after 11 generations. Pathway analysis revealed that these 11th generation cells had accumulated transcripts coding for enzymes involved in sugar metabolism, the tricarboxylic acid (TCA) cycle and amino acid degradation, and showed decreased levels of mRNAs coding for enzymes involved in amino acid biosynthetic pathways. Stationary phase-induced genes were highly expressed after 11 generations even though the growth medium contained adequate levels of nutrients, indicating deterioration of the nutrient sensing and/or signaling pathways by the 11th generation. These changes are presumably early indications of replicative senescence. Cells of yeast strain BY4742 were labeled with biotin and cultivated in YPD medium at 30M-BM-0C. The labeled cells at early stages of senescence (4th, 7th and 11th generation) were isolated using Dynabeads Biotin Binder (Invitrogen, Carlsbad, CA, USA). Total RNA samples were prepared from the senescent cells. Antisense RNA was synthesized and labeled with Cy3 for the 4th, 7th, and 11th generation and with Cy5 for the 1st generation. Each mixture of Cy3- and Cy5-labeled antisense RNA was hybridized on a 3D-Gene Yeast Oligo Chip 6K (Toray, Kanagawa, Japan). The hybridized array was scanned with a 3D-Gene Scanner 3000 (Toray).

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Yukio Mukai

PROVIDER: E-GEOD-59797 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Maintenance of CG methylation (mCG) patterns is essential for chromatin-mediated epigenetic regulation of transcription in plants and mammals. Using successive generations of an Arabidopsis thaliana mutant deficient in maintaining mCG, we found that mCG loss triggered genome-wide activation of alternative epigenetic mechanisms. However, these mechanisms involving RNA-directed DNA methylation, inhibiting expression of DNA demethylases, and retargeting of histone H3K9 methylation act in a stochastic and uncoordinated fashion. As a result, new and aberrant epigenetic patterns were progressively formed over several plant generations in the absence of mCG. Interestingly, the unconventional redistribution of epigenetic marks was necessary to ârescueâ the loss of mCG, since mutant plants impaired in rescue activities were severely dwarfed and sterile. Our results provide evidence that mCG is a central coordinator of epigenetic memory that secures stable transgenerational inheritance in plants. Experiment Overall Design: Per entry, two biological replicates of leaf tissue (approx. 12th true leaf) from 9-12 plants were frozen in liquid nitrogen. DNA was CTAB extracted and isopropanol precipitated, RNase A and Proteinase K treated, re-extracted and ethanol precipitated. Two replicate 3 Âµg genomic DNA samples of 2nd generation met1-3, 4th generation met1-3 and Col-0 were sheared by sonication and methylated DNA was immunoprecipitated, as previously described (Weber et al., 2005), and amplified following the chromatin immunoprecipitation assay protocol (Affymetrix). Two replicates per entry with 7.5 Âµg DNA were fragmented; end labeled, and hybridized on Affymetrix ATH1 arrays as recommended (Affymetrix). Microarray data analysis was performed using the following procedure. First, background adjustments were made using the gcRMA method available in the Bioconductor package of R. Next, background-adjusted values from the six ATH1 datasets were quantile normalized and average signal intensities were determined in log2 scale per probe set. Experiment Overall Design: Contrast coefficient ratios for all pair-wise comparisons were calculated as follows: met1-3 2nd/Col-0, met1-3 4th/Col-0, and met1-3 4th/met1-3 2nd. Corrections for multiple comparisons at the 5% false discovery rate (FDR) were performed and significant differences were detected using Fisherâs test. Contrast coefficient ratios and p values may be found in the Supplementary file at the foot of this record. For each probe set, three outcomes (significantly hypermethylated, no difference, and significantly hypomethylated) were possible per comparison, creating 27 possible methylation profiles analyzed at the 5% FDR level.

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Early stages of yeast senescent cells

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