ABSTRACT
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-Ddb1(Cdt)² 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-Ddb1(Cdt)² 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.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA-Binding Proteins/metabolism , Heterochromatin/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Nuclear Proteins/metabolism , Schizosaccharomyces/enzymology , Schizosaccharomyces/genetics , Signal TransductionABSTRACT
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 RNAi-independent silencing at the mat3M or tel2R loci. ers1Delta 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.
