SUMMARY Partitioning of chromosomes into euchromatic and heterochromatic domains requires mechanisms that specify boundaries. The S. pombe JmjC family protein Epe1 prevents the ectopic spread of heterochromatin and is itself concentrated at boundaries. Paradoxically, Epe1 is recruited to heterochromatin by HP1 silencing factors that are distributed throughout heterochromatin. We demonstrate here that the selective enrichment of Epe1 at boundaries requires its regulation by the conserved Cul4-Ddb1Cdt2 ubiquitin ligase, which directly recognizes Epe1 and promotes its polyubiquitylation and degradation. Strikingly, in cells lacking the ligase, Epe1 persists in the body of heterochromatin thereby inducing a defect in gene silencing. Epe1 is the sole target of the Cul4-Ddb1Cdt2 complex whose destruction is necessary for the preservation of heterochromatin. This mechanism acts parallel with phosphorylation of HP1/Swi6 by CK2 to restrict Epe1. We conclude that the ubiquitin-dependent sculpting of the chromosomal distribution of an antisilencing factor is critical for heterochromatin boundaries to form correctly.

Centromeric silencing and heterochromatin formation in Schizosaccharomyces pombe require the RNA interference (RNAi) machinery. Three factors that mediate this mechanism have been identified: 1) the RNA-dependent RNA polymerase complex RdRC, 2) the Argonaute-containing RITS (RNA-induced initiation of transcriptional silencing) complex, and 3) the endoribonuclease Dicer ortholog Dcr1. S. pombe mutants lacking a new factor described here, Ers1, are completely defective in RNAi-dependent silencing of centromeric regions but, importantly, not in RNAiindependent silencing at the mat3M or tel2R loci. ers1⌬ cells likewise fail to convert centromeric pre-small interfering RNA transcripts into small interfering RNAs, are defective in histone H3 Lys 9 methylation, and are unable to recruit the RITS complex to centromeric sequences. Surprisingly, Ers1 lacks obvious orthologs outside of the genus Schizosaccharomyces. Within this group, it is diverging rapidly, raising the possibility that it is coevolving with target RNA elements.Heterochromatin formation and gene silencing in many eukaryotes require a core system of a histone H3 Lys 9 (H3-K9) methyltransferase and a family of proteins (the HP1 family) that recognize this modification. A link between RNAi 4 and transcriptional silencing was first suggested from studies of doublestranded RNA-dependent DNA methylation in plants (1). Landmark studies in the fission yeast Schizosaccharomyces pombe demonstrated that heterochromatic silencing at centromeres requires three canonical RNAi components, the Dicer endoribonuclease Dcr1, the RNA-dependent RNA polymerase Rdp1, and the Argonaute protein Ago1 (2). Mutation of these components, like mutations in the histone methyltransferase Clr4, results in a loss of centromeric silencing, accumulation of centromeric transcripts, a defect in histone H3 Lys 9 methylation, and a defect in the formation of centromere-coded siRNAs (2). A role for RNAi in heterochromatin has also been found in Drosophila and mammals (3, 4). In S. pombe, Ago1 functions as part of a protein complex called RITS that contains an HP1 homolog, Chp1 (5). The recruitment of this complex to chromatin requires Dcr1, suggesting that Ago1 needs to be loaded with siRNAs to be targeted (5). Rdp1 likewise is part of a complex called RdRC that interacts with the RITS complex (6). Despite these important advances, how the RNAi machinery is recruited to specific sites, how it controls histone methylation, and how it mediates silencing are not clear. Since the initial studies, no factors essential for and specific to RNAi-dependent heterochromatin formation in S. pombe have been reported besides those polypeptides identified in the RITS and RdRC complexes. EXPERIMENTAL PROCEDURESS. pombe Genetic Methods-Homologous replacement of DNA was accomplished by lithium acetate transformation of PCR products containing 100 bp of targeting homology. YS medium (5 g/liter Difco yeast extract ϩ 250 mg/liter each L-histidine, L-leucine, adenine, uracil, and L-lysine and 3% glucose) was used for...