Raf1 Is a DCAF for the Rik1 DDB1-Like Protein and Has Separable Roles in siRNA Generation and Chromatin Modification

摘要

Non-coding transcription can trigger histone post-translational modifications forming specialized chromatin. In fission yeast, heterochromatin formation requires RNAi and the histone H3K9 methyltransferase complex CLRC, composed of Clr4, Raf1, Raf2, Cul4, and Rik1. CLRC mediates H3K9 methylation and siRNA production; it also displays E3-ubiquitin ligase activity in vitro . DCAFs act as substrate receptors for E3 ligases and may couple ubiquitination with histone methylation. Here, structural alignment and mutation of signature WDxR motifs in Raf1 indicate that it is a DCAF for CLRC. We demonstrate that Raf1 promotes H3K9 methylation and siRNA amplification via two distinct, separable functions. The association of the DCAF Raf1 with Cul4-Rik1 is critical for H3K9 methylation, but dispensable for processing of centromeric transcripts into siRNAs. Thus the association of a DCAF, Raf1, with its adaptor, Rik1, is required for histone methylation and to allow RNAi to signal to chromatin. Author Summary Heterochromatin is a specialized form of chromatin which is frequently assembled on DNA sequences with little or no coding potential. Heterochromatin formation involves specific post-translational modifications of histone tails (e.g. methylation of histone H3 on lysine 9). In fission yeast, Schizosaccharomyces pombe , heterochromatin is found at centromeres, telomeres, and the mating type locus. Heterochromatin integrity at centromeres is important for normal chromosome segregation. The heterochromatin associated repeats at fission yeast centromeres are known to be transcribed, and these non-coding transcripts are processed into siRNAs. siRNA production is required for establishment and maintenance of H3K9 methylation. But H3K9 methylation itself is required for siRNA production. It is not known how these two processes are coupled. In this study we use structural modelling and genetic analyses to demonstrate that the heterochromatin component Raf1 plays an essential role in coupling H3K9 methylation and siRNA production. Our analyses show that the heterochromatin factors Rik1 and Raf1 can be structurally aligned with Cul4-E3 ubiquitin ligase components DDB1 and DDB2, respectively. We show that specific mutations impair the association of Raf1 with Rik1 and prevent H3K9 methylation but not siRNA production. These functional studies provide mechanistic insights into how siRNA production and chromatin modification are integrated.