In eukaryotes, heterochromatin mediates diverse processes including gene 
silencing and regulation of long-range chromatin interactions. The formation of 
heterochromatin involves a conserved array of histone modifications; in 
particular, methylation of histone H3 at Lys 9 (H3K9me) is essential for 
recruiting HP1/Swi6 proteins. In fission yeast, the Clr4 methyltransferase is 
responsible for H3K9me across all heterochromatic domains. However, the 
mechanism of Clr4 recruitment to these loci is poorly understood. We show that 
Clr4 associates with Cul4, a cullin family protein that serves as a scaffold for 
assembling ubiquitin ligases. Mutations in Cul4 result in defective localization 
of Clr4 and loss of silencing at heterochromatic loci. This is accompanied by a 
severe reduction in H3K9me and Swi6 levels, and accumulation of transcripts 
corresponding to naturally silenced repeat elements within heterochromatic 
domains. Moreover, heterochromatin defects in Cul4 mutants could not be rescued 
by expression of Cul4 protein lacking Nedd8 modification, which is essential for 
its ubiquitin ligase activity. Rik1, a protein related to DNA damage binding 
protein DDB1 and required for H3K9me, also interacts with Cul4, the association 
of which might serve to target Clr4 to heterochromatic loci. These analyses 
uncover a role for Cul4-based protein ubiquitination in regulating H3K9me and 
heterochromatin formation.