A Cullin E3 Ubiquitin Ligase Complex Associates with Rik1 and the Clr4 Histone H3-K9 Methyltransferase and is Required for RNAi-Mediated Heterochromatin Formation

Abstract

Previously published online as a RNA Biology E-publication: http://www.landesbioscience.com/journals/rnabiology/abstract.php?id=2131 KEY WORDSRNAi, heterochromatin, Clr4, Rik1, Cul4, histone H3-K9 methylation, epigenetics ACKNOWLEDGEMENTSWe thank Karl Ekwal and Shiv Grewal for strains, members of the Moazed lab for advice and encouragement, André Verdel for construction of the Rik1-TAP strain, and Diana Libuda and Mark Elenko for early characterization of Rik1-TAP expression. This work was supported by NIH grants GM72805 (D.M.) and GM67945 (S.P.G. ABSTRACTThe assembly of heterochromatin in fission yeast and metazoans requires histone H3-lysine 9 (-K9) methylation by the conserved Clr4/Suv39h methyltransferase. In fission yeast, H3-K9 methylation requires components of the RNAi machinery and is initiated by the RNA-Induced Transcriptional Silencing (RITS) complex. Here we report the purification of a novel complex that associates with the Clr4 methyltransferase, termed the CLRC (CLr4-Rik1-Cul4) complex. By affinity purification of the Clr4-associated protein Rik1, we show that, in addition to Clr4, Rik1 is associated with the fission yeast E3 ubiquitin ligase Cullin4 (Cul4, encoded by cul4 + ), the ubiquitin-like protein, Ned8, and two previously uncharacterized proteins, designated Cmc1 and Cmc2. In addition, the complex contains substochiometric amounts of histones H2B and H4, and the 14-3-3 protein, Rad24. Deletion of cul4 + , cmc1 + , cmc2 + and rad24 + results in a complete loss of silencing of a ura4 + reporter gene inserted within centromeric DNA repeats or the silent mating type locus. Each of the above deletions also results in accumulation of noncoding RNAs transcribed from centromeric repeats and telomeric DNA regions, and a corresponding loss of small RNAs that are homologous to centromeric repeats, suggesting a defect in the processing of noncoding RNA to small RNA. Based on these results, we propose that the components of the Clr4-Rik1-Cul4 complex act concertedly at an early step in heterochromatin formation.

Epigenetic analysis reveals a euchromatic configuration in the FMR1 unmethylated full mutations

“…If and how siRNAs localize histone-modifying activities, in particular HMTs, remains to be determined. 46,47 In the unmethylated full mutations we may hypothesize the presence of a defect in the RNAi machinery, causing the abnormal/absent production of siRNAs specific for FMR1 gene during early embryogenesis. It is also possible that individuals with unmethylated full mutations might have escaped a narrow time window in the course of embryogenesis, during which the epigenetic marks are established.…”

Studies on the Mechanism of RNAi-dependent Heterochromatin Assembly

“…We chose to purify Rik1 because, unlike Clr4, cells carrying an affinity-tagged version of Rik1 were phenotypically wild type for silencing. Purification of Rik1 shows that, in addition to Clr4, it is associated with the Cullin 4 (Cul4) E3 ubiquitin ligase and two proteins of unknown function, Cmc1 (also called Raf1, Dos1, and Clr8) and Cmc2 (also called Raf2, Dos2, and Clr7) in a complex that we have named CLRC (Clr4-Rik1-Cul4) (Hong et al 2005;Horn et al 2005;Jia et al 2005;Li et al 2005;Thon et al 2005). The CLRC complex is architecturally similar to other Cullin ubiquitin ligase complexes, but its composition does not provide a ready answer for how it is recruited to chromatin by the RNAi pathway (Fig.…”

“…The Ddb1 and CPSF-A proteins are involved in the recognition of damaged DNA and polyadenylation signals, respectively (Barabino et al 2000;Hu et al 2004). An intriguing possibility is that Rik1 recognizes a nucleic acid species that is generated during the initial steps in RNAi targeting of the genome; for example, the siRNA-nascent pre-mRNA duplex, or the dsRNA generated by the RDRC, or perhaps a poly(A) tail generated by Cid12 (Motamedi et al 2004;Hong et al 2005). Alternatively, the CLRC complex may be directly recruited to chromatin through physical interactions with one or more subunits of the RITS or RDRC complexes, but these interactions may not be stable enough to withstand biochemical isolation.…”

“…Rik1 is a beta propeller WD repeat protein that belongs to a subfamily of WD repeat proteins that includes the Ddb1 DNA damage protein, the CPSF-A subunit of the poly(A) polymerase complex, and some splicing factors (Fig. 6) (Hong et al 2005). The Ddb1 and CPSF-A proteins are involved in the recognition of damaged DNA and polyadenylation signals, respectively (Barabino et al 2000;Hu et al 2004).…”

Silencing in trans: position matters in fission yeast

“…Rdp1 effectively amplifies the Dcr1 dsRNA substrate. The RNAi machinery subsequently recruits the histone methyltransferase (Clr4)-containing complex CLRC to target loci, thereby promoting H3 Lys 9 methylation (Hong et al, 2005). In turn, this forms a platform for hetero chromatin binding proteins Swi6 (HP1 homologue), Chp1 and Chp2, creating a nucleation site from where heterochromatin can spread to adjacent regions.…”