Project description:The inhibitors hydroxymethylfurfural (HMF) and furfural were added to the feed-medium of carbon-limited anaerobic chemostat cultures. Samples were taken for transcriptome analysis at steady-state from cultures with inhibitors and without inhibitors.
Project description:Background Microorganisms adapt their transcriptome by integrating multiple chemical and physical signals from their environment. Shake-flask cultivation does not allow precise manipulation of individual culture parameters and therefore precludes a quantitative analysis of the (combinatorial) influence of these parameters on transcriptional regulation. Steady-state chemostat cultures, which do enable accurate control, measurement and manipulation of individual cultivation parameters (e.g. specific growth rate, temperature, identity of the growth-limiting nutrient) appear to provide a promising experimental platform for such a combinatorial analysis. Results A microarray compendium of 170 steady-state chemostat cultures of the yeast Saccharomyces cerevisiae is presented and analyzed. The 170 microarrays encompass 55 unique conditions, which can be characterized by the combined settings of 10 different cultivation parameters. By applying a regression model to assess the impact of (combinations of) cultivation parameters on the transcriptome, most S. cerevisiae genes were shown to be influenced by multiple cultivation parameters, and in many cases by combinatorial effects of cultivation parameters. The inclusion of these combinatorial effects in the regression model led to higher explained variance of the gene expression patterns and resulted in higher function enrichment in subsequent analysis. We further demonstrate the usefulness of the compendium and regression analysis for interpretation of shake-flask-based transcriptome studies and for guiding functional analysis of (uncharacterized) genes and pathways. Conclusions Modeling the combinatorial effects of environmental parameters on the transcriptome is crucial for understanding transcriptional regulation. Chemostat cultivation offers a powerful tool for such an approach. Keywords: chemostat steady state samples
Project description:The inhibitors hydroxymethylfurfural (HMF) and furfural were added to the feed-medium of carbon-limited anaerobic chemostat cultures. Samples were taken for transcriptome analysis at steady-state from cultures with inhibitors and without inhibitors. Three biological replicates from each condition (inhibitors, no inhibitors) were analyzed.
Project description:We combined the nuclear run-on (NRO) assay which labels and captures nascent transcripts with high throughput DNA sequencing to examine transcriptional activity in Saccharomyces cerevisiae. Examination of nascent transcripts and steady-state transcripts in exponentially growing and heat-shock treated yeast.
Project description:Genomic events including gene regulation and chromatin status are controlled by transcription factors. Here we report that the Hsp90 molecular chaperone broadly regulates the transcription factor protein family. Our studies identified a biphasic use of Hsp90 in which early inactivation (15 min) of the chaperone triggered a wide reduction of DNA binding events along the genome with concurrent changes to chromatin structure. Long-term loss (6 h) of Hsp90 resulted in a decline of a divergent yet overlaying pool of transcription factors that produced a distinct chromatin pattern. Although both phases involve protein folding, the early point correlated with Hsp90 acting in a late folding step that is critical for DNA binding function whereas prolonged Hsp90 inactivation led to a significant decrease in the steady-state transcription factor protein levels. Intriguingly, despite the broad chaperone-impact on a variety of transcription factors, the operational influence of Hsp90 was at the level of chromatin with only a mild effect on gene regulation. Thus, Hsp90 selectively governs the transcription factor process overseeing local chromatin structure.