Our project is to understand the role of non coding (nc)RNA in the regulation of epigenetic landscape and gene expression. RNA interference pathway is absent in the budding yeast but recent works from our laboratory showed the existence of an original ncRNA-dependent pathway that controls gene expression in S. cerevisiae. We characterized a cryptic unstable ncRNA mediating the transcriptional silencing of the transposon Ty1 through histone methylation. Furthermore, unpublished data suggest the existence of subtelomeric ncRNAs that might control telomere metabolism and promoter-associated ncRNAs that mediate repressive epigenetic marks. We propose that a class of unstable ncRNA mediates genome expression and fluidity through histone modifications. Following 2 directions, our aim is to systematically identify these ncRNAs (A) and further characterize their regulatory mechanisms (B). (A) First, we aim to identify the regulatory ncRNAs by performing genome-wide approaches in strains accumulating these regulatory ncRNAs. We envisage developing protocols to analyze the cryptic transcriptome using deep sequencing technologies. (B) Second, we will further characterize the previously identified regulatory ncRNAs controlling repetitive regions (Ty1 transposon and telomeric repeats) but also gene expression. Through a range of experimental procedures from living cell biology (Fluorescence Immuno Hybridization), biochemical approaches (RNA-TRAP) and genetic, we will determine the dynamics of the regulatory ncRNA within the cell, the associated proteins that regulate their activities and the chromatin defects resulting from their expression. Our aim is to extensively describe the RNAi-like regulation in S. cerevisiae, that we anticipate to be broadly conserved in other eukaryotes.
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