RNAi, Histone Modification and the DDB1/CPSF-like Complex Rik1

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DESCRIPTION (provided by applicant): The pericentromeric repeats of the fission yeast Schizosaccharomyces pombe are transcribed and subject to RNA interference (RNAi). This influences heterochromatic histone modification, which silences reporter genes and recruits proteins required for centromere function, including cohesin. This mechanism is conserved in mammals and plants, and has implications both for gene therapy as well as for the epigenetic basis for human disease. In fission yeast, the Rik1 complex is related to both cleavage and polyadenylation specificity factor (CPSF) and to DNA damage binding protein (DDB1) complexes and is required for RNAi and histone modification. It is recruited to heterochromatin by association with the highly conserved, transposase-related protein Cenp-B, in a process that also requires cleavage (slicing) of nascent heterochromatic transcripts by Argonaute. We will investigate how transcripts are initiated by Pol II and processed by RNAi and the exosome. We will determine how Rik1 is recruited by RNAi, how it influences histone methylation and demethylation, and how it interacts with the nuclear envelope. We will use state-of- the-art microarray profiling techniques, as well as ultra high resolution Structured Illumination microscopy. S. pombe is a particularly useful system to study RNAi mediated heterochromatin, because this seems to be the only function of RNAi in fission yeast (there are no known miRNA) and mutants are generally viable. It should be possible therefore to take one or more of these screens to saturation, providing a unique catalog of genes involved in the primitive function of RNAi. This "function appears to be conserved in archaebacteria, fission yeast, protozoans, plants and animals, and may provide insight into the evolution of the chromosome, and its relationship to repetitive or "junk" DNA. Transposable elements and other repeats are common to all of these genomes, and may hold the key to epigenetic mechanisms of gene regulation and chromosome function.