Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA Polymerase mapping during stress responses reveals widespread nonproductive transcription in yeast

ABSTRACT: The use of genome-wide RNA abundance profiling by microarrays and deep sequencing has spurred a revolution in our understanding of transcriptional control. However, changes in mRNA abundance reflect the combined effect of changes in RNA production, processing, and degradation, and thus, mRNA levels provide an occluded view of transcriptional regulation. To partially disentangle these issues, we carry out genome-wide RNA Polymerase II (“Pol2”) localization profiling in budding yeast in two different stress response time courses. While mRNA changes largely reflect changes in transcription, there remains a great deal of variation in mRNA levels that is not accounted for by changes in Pol2 abundance. We find that genes exhibiting “excess” mRNA produced per Pol2 are enriched for those with overlapping cryptic transcripts, indicating a pervasive role for nonproductive or regulatory transcription in control of gene expression. Finally, we characterize changes in Pol2 localization when Pol2 is genetically inactivated using the rpb1-1 temperature-sensitive mutation. We find that Pol2 is lost from chromatin after roughly an hour at the restrictive temperature, and that there is a great deal of variability in the rate of Pol2 loss at different loci. Together, these results provide a global perspective on the relationship between Pol2 and mRNA production in budding yeast. The array studies consisted of 4 time courses in which the genome-wide location of RNA Polymerase II was mapped via chromatin immunoprecipitation (ChIP) in BY4741 S. cerevisiae cells or in such cells bearing the temperature sensitive mutation rpb1, in response to heat shock at 37 degrees C and/or 1.5 mM diamide. All samples were hybridized as the ChIP sample (Cy3 labeled, channel 1) versus its cognate ChIP input sample (Cy5 labeled, channel 2). The first time course was a heat shock in wild-type BY4741 cells. The second time course was a heat shock in BY4741 rpd1 cells. The third time course was a diamide treatment of BY4741 rpd1 cells. The fourth time course was a 10 minute heat shock in BY4741 rpd1 cells followed by treatment from 15-60 minutes with diamide (3 samples). In the first three time courses the time range is 0-120 minutes (5 samples). A total of 18 arrays were used in this study. No additional replicates or dye-flip experiments were performed.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Oliver Rando

PROVIDER: E-GEOD-22675 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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RNA polymerase mapping during stress responses reveals widespread nonproductive transcription in yeast.

Kim Tae Soo TS Liu Chih Long CL Yassour Moran M Holik John J Friedman Nir N Buratowski Stephen S Rando Oliver J OJ

Genome biology 20100716 7

<h4>Background</h4>The use of genome-wide RNA abundance profiling by microarrays and deep sequencing has spurred a revolution in our understanding of transcriptional control. However, changes in mRNA abundance reflect the combined effect of changes in RNA production, processing, and degradation, and thus, mRNA levels provide an occluded view of transcriptional regulation.<h4>Results</h4>To partially disentangle these issues, we carry out genome-wide RNA polymerase II (PolII) localization profili ...[more]

PMID: 20637075

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Project description:Histone modifications affect DNA-templated processes ranging from transcription to genomic replication. In this study, we examine the cell cycle dynamics of the trimethylated form of histone H3 lysine 4 (H3K4me3), a mark of active chromatin that is viewed as â??long-livedâ?? [1], and that is involved in memory during cell state inheritance in metazoans [2]. We synchronized yeast using two different protocols, then followed H3K4me3 patterns as yeast passed through subsequent cell cycles. While most H3K4me3 patterns were conserved from one generation to the next, we found that methylation patterns induced by alpha factor or high temperature were erased within one cell cycle, during S phase. Early-replicating regions were erased before late-replicating regions, implicating replication in H3K4me3 loss. However, incomplete H3K4me3 erasure occurred at the majority of loci even when replication was prevented, suggesting that most erasure results from an active process. Indeed, deletion of the demethylase Jhd2 slowed erasure at most loci. Together, these results indicate overlapping roles for passive dilution and active enzymatic demethylation in erasing ancestral histone methylation states in yeast. References: [1] Ng HH, Robert F, Young RA, Struhl K (2003) Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. Mol Cell 11: 709-719. [2] Ringrose L, Paro R (2004) Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu Rev Genet 38: 413-443. The overall design of the experiment consists of two cell cycle experiments, each consisting of the following subsets: gene expression, ChIP with anti-H3K4Me3 antibody, and ChIP input. The experiments are as follows: CCA - BY4741 bar1- cells synchronized by alpha factor arrest; CCTS - BY4741 cdc28(ts) cells synchronized by arrest at non-permissive temperature. The individual time courses are enumerated as follows: CCA gene expression (array title "Synchronized cells, xxx min, cell cycle CCA"), 18 arrays; CCA ChIP for anti-H3K4Me3 (array title "H3K4Me3 ChIP cell cycle CCA, xxx min"), 17 arrays, 1 replicate; CCA ChIP input (array title "ChIP Input cell cycle CCA, xxx min"), 18 arrays, 1 replicate; CCTS gene expression (appears in a different dataset as biological replicate "I"; array title "Synchronized cells, xxx min, biological replicate I"), 18 arrays; CCTS ChIP for anti-H3K4Me3 (array title "H3K4Me3 cell cycle CCTS, xxx min"), 2 technical replicates, 18 arrays per replicate; CCTS ChIP input (array title "ChIP Input cell cycle CCTS, xxx min"), 2 technical replicates, 18 arrays per replicate. Gene expression arrays were run against a reference of unsynchronized cells, ChIP-chip anti-H3K4Me3 samples were run against an IP (of the same epitope) of unsynchronized cells, and ChIP input samples were run against either a pool of all the time points in the time course (CCTS) or against sonicated DNA isolated from unsynchronized cells (CCA). Four additional experiments have been performed. They are referred to as series X in the title. Series 1: anti H3K4me3 CHIP time course of BY4741 bar1 delete cells (wt) synchronized with alpha factor and released in the cell cycle, 9 arrays, 1 replicate. Series 2: anti H3K4me3 CHIP time course of BY4741 bar1 jhd2 delete cells (jhd2 delete) synchronized with alpha factor and released in the cell cycle, 7 arrays, 1 replicate. Series 3: anti H3K4me3 CHIP time course of CYM36 cells (cdc7ts) synchronized with alpha factor and released in the cell cycle at the permissive 24C or at the restrictive 37C temperature, 22 arrays, 2 replicates. Series 4: anti H3K4me3 CHIP time course of BY4741 bar1 delete cells (wt) grown in galactose and synchronized with alpha factor and either released in the cell cycle in glucose media or alpha factor arrested and switched to glucose media, 6 arrays, 1 replicate.

Project description:Significant insight about biological networks arises from the study of network motifs âoverly abundant network subgraphs, but such wiring patterns do not specify when and how potential routes within a cellular network are used. To address this limitation, we introduce activity motifs, which capture patterns in the dynamic use of a network. Using this framework to analyze transcription in Saccharomyces cerevisiae metabolism, we find that cells use different timing activity motifs to optimize transcription timing in response to changing conditions: forward activation to produce metabolic compounds efficiently, backward shutoff to rapidly stop production of a detrimental product and synchronized activation for co-production of metabolites required for the same reaction Measuring protein abundance over a time course reveals that mRNA timing motifs also occur at the protein level. Timing motifs significantly overlap with binding activity motifs, where genes in a linear chain have ordered binding affinity to a transcription factor, suggesting a mechanism for ordered transcription. Finely timed transcriptional regulation is therefore abundant in yeast metabolism, optimizing the organism's adaptation to new environmental conditions. We generated a set of 13 time courses by measuring gene expression after a metabolic change. Yeast strain KCN118 (MATalpha ade2) was grown at 28 Â°C in 400 ml of synthetic complete media with 2% dextrose (SCD) to an OD600 of 0.6. Synthetic complete was prepared using the standard recipe, except 75 uM inositol was included. At OD600 of 0.6, 100 ml of cells were collected by centrifugation and frozen as a reference sample, and the remaining cells were rapidly collected by filtration, washed with distilled water and resuspended in 300 ml of one of the following media: SCE (SC + 2% ethanol), SCG (SC + 2% galactose), SM1 (SCD lacking amino acids A, R, N, C, Q, G, K, P, S, F and T), SM2 (SCD lacking amino acids L, I, V, W, H and M), S0 (SCD lacking all amino acids), S0G (no amino acids, 2% galactose) or S0E (no amino acids, 2% ethanol). The data appears in Figures 2 and 4 of the manuscript, as it relates to the global analysis of all the arrays used in the dataset. All time courses consist of the following time points (in min): 15, 30, 60, 120, 240, and were hybridized against the t = 0 time point of cells grown in SCD. Each time course was performed as one single biological replicate and one technological replicate, except where noted below. Specifically, the 13 time courses break down into the following groups: Media key: SCD (synthetic complete, not including inositol) SCE (SC + 2% ethanol) SCG (SC + 2% galactose) SM1 (SCD lacking amino acids A, R, N, C, Q, G, K, P, S, F and T) SM2 (SCD lacking amino acids L, I, V, W, H and M) S0 (SCD lacking all amino acids) S0G (no amino acids, 2% galactose) S0E (no amino acids, 2% ethanol). ino = inositol aa = all amino acids supplemented The following group descriptions are the media as described above, followed by the description as indicated in the long title of the individual arrays: 1. S0 (5 arrays) = SD 2. SCD (6 arrays, t = 240 min has 2 tech replicates) = SD+aa 3. SM2 (5 arrays) = SD+aa:ARNCQGKPSDEFTY+ino 4. SM1 + ino (5 arrays) = SD+aa:LIVWHM+ino 5. S0 + ino (6 arrays, t = 240 min has 2 tech replicates) = SD+ino 6. S0E (5 arrays) = SEtOH 7. S0E + aa (7 arrays, t = 240 min and 60 min each have 2 tech replicates) = SEtOH+aa 8. S0E + aa + ino (5 arrays) = SEtOH+aa+ino 9. S0E + ino (8 arrays, t = 240 min, 15 min and 30 min each have 2 tech replicates) = SEtOH+ino 10. S0G (5 arrays) = Sgal 11. S0G + aa (5 arrays) = Sgal+aa 12. S0G + aa + ino (5 arrays) = Sgal+aa+ino 13. S0G + ino (6 arrays, t = 60 min has 2 tech replicates) = Sgal+ino

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RNA Polymerase mapping during stress responses reveals widespread nonproductive transcription in yeast

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RNA polymerase mapping during stress responses reveals widespread nonproductive transcription in yeast.

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