Investigating mechanisms of RNAi-dependent heterochromatin establishment in Schizosaccharomyces pombe

Heterochromatin is a condensed conformation of eukaryotic DNA, which plays an essential role in genome homeostasis. In depth research across various species showed that RNA interference (RNAi) is one of the pathways that is important for heterochromatin formation. To date RNAi-driven heterochromatin assembly has been extensively studied in the fission yeast Schizosaccharomyces pombe. At centromeres in S. pombe, RNAi is required for heterochromatin establishment and maintenance. Unlike heterochromatin maintenance, RNAi-driven heterochromatin establishment at the centromeres is not very well understood. The interconnectedness between the RNAi and chromatin modification pathways that are required to establish heterochromatin makes it difficult to elucidate which pathway acts first. Recent studies also showed that heterochromatin establishment requires additional factors dispensable for maintenance. Thus exploring novel factors required for the de novo heterochromatin formation can help us to understand the order and the mechanism of heterochromatin establishment. In this study I developed and tested two assays ⎯ plasmid-based and cross-based assays ⎯ designed to identify novel establishment-specific factors genome-wide. While the plasmid based assay proved unreliable, the cross-based establishment assay was shown to effectively identify establishment-specific factors. It also provided new insights into heterochromatin establishment dynamics.