Abstract
The assembly of heterochromatin in fission yeast and metazoans requires histone H3-lysine 9 (-K9) methylation by the conserved Clr4/Suv39h methyltransferase. In fission yeast,H3-K9 methylation requires components of the RNAi machinery and is initiated by the RNAInducedTranscriptional Silencing (RITS) complex. Here we report the purification of a novelcomplex that associates with the Clr4 methyltransferase. By affinity purification of the Clr4-associated protein Rik1, we show that, in addition to Clr4, Rik1 is associated with the fissionyeast E3 ubiquitin ligase Cullin4 (Cul4, encoded by cul4+), the ubiquitin-like protein, Ned8, andtwo previously uncharacterized proteins, designated Cmc1 and Cmc2. In addition, the complexcontains substochiometric amounts of histones H2B and H4, and the 14-3-3 protein, Rad24.Deletion of cul4+, cmc1+, cmc2+, and rad24+ results in a complete loss of silencing of a ura4+ reporter gene inserted within centromeric DNA repeats or the silent mating type locus. Each ofthe above deletions also results in accumulation of noncoding RNAs transcribed fromcentromeric repeats and telomeric DNA regions. Based on these results and extensive sequencesimilarity between Rik1 and Ddb1, a DNA-damage binding protein, previously shown to alsoassociate with Cul4, we propose that RNAi-mediated heterochromatin formation involves therecruitment of Clr4-Rik1-Cul4 complex through the putative nucleic acid binding domain ofRik1.
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