A Rik1-associated, cullin-dependent E3 ubiquitin ligase is essential for heterochromatin formation

Heterochromatin is critical for proper centromere and telomere function, and it plays a key role in the transcriptional silencing of specific genomic loci. In fission yeast, the Rik1 protein functions with the Clr4 histone methyltransferase at an early step in heterochromatin formation. Here, we use mass spectrometry and tandem affinity purification of a Rik1-TAP fusion protein to identify Rik1-associated proteins. These studies identify two novel proteins, Raf1 and Raf2, which we find are required for H3-K9 methylation and for transcriptional silencing within centromeric heterochromatin. We also find that subunits of a cullin-dependent E3 ubiquitin ligase are associated with Rik1 and Clr4, and Rik1-TAP preparations exhibit robust E3 ubiquitin ligase activity. Furthermore, expression of a dominant-negative allele of the Pcu4 cullin subunit disrupts regulation of K4 methylation within heterochromatin. These studies provide evidence for a novel Rik1-associated E3 ubiquitin ligase that is required for heterochromatin formation.