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.
Project description:To characterize the ecological interactions among S. cerevisiae strains coming from the same geographical area, we examined the fitness of two natural isolates from San Giovese grapes, alone or in competition, in synthetic wine must (SWM). We performed genome-wide analyses in order to identify the genes involved in yeast competition and cooperation.
Project description:This project aims to identify novel RNA binding proteins in the baker's yeast, Saccharomyces cerevisiae. Since interactions between RNAs and proteins may be transient, yeast cells were crosslinked with UV light at 254 nm which promotes the covalent link between proteins and RNAs. After this, polyadenylated mRNAs were purified via oligo(dT) coupled to magentic beads under stringet conditions. Finally, samples were subjected to mass spectrometry analysis. To rule out the possibility of RNA-independent binding we also analysed other samples: i) samples digested with RNase one; ii) samples where we performed competition assays with polyadenylic acid.
Project description:The pluripotency control regions (PluCRs) are defined as genomic regions that are bound by Oct4, Sox2, and Nanog in vivo. We utilized a high-throughput binding assay to record more than 270,000 different DNA/protein binding measurements along incrementally tiled windows of DNA within these PluCRs. This high-resolution binding map is then used to systematically define the context of Oct factor binding and reveals patterns of cooperativity and competition in the pluripotency network. The most prominent pattern is a pervasive binding competition between Oct4 and the forkhead transcription factors. Like many transcription factors, Oct4 is co-expressed with a paralog, Oct1, that shares an apparently identical binding specificity. By analyzing thousands of binding measurements we discover context effects that discriminate Oct1 from Oct4 binding. Proximal Nanog binding promotes Oct4 binding, whereas nearby Sox2 binding favors Oct1. We demonstrate by cross-species comparison and by chromatin immunoprecipitation (ChIP) that the contextual sequence determinants learned in vitro are sufficient to predict Oct1 binding in vivo.