Partridge1,2* Abstract Continuous Requirement for the Clr4 Complex But Not RNAi for Centromeric Heterochromatin Assembly in Fission Yeast Harboring a Disrupted RITS Complex Sreenath Shanker1., Godwin Job1., Olivia L. George1, Kevin M. Creamer2, Alaa Shaban1, Janet F. 1 Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America, 2 Integrated Program in Biomedical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America Formation of centromeric heterochromatin in fission yeast requires the combined action of chromatin modifying enzymes and small RNAs derived from centromeric transcripts. Positive feedback mechanisms that link the RNAi pathway and the Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in requirements for H3K9 methylation for full siRNA production and for siRNA production to achieve full histone methylation. Nonetheless, it has been proposed that the Argonaute protein, Ago1, is the key initial trigger for heterochromatin assembly via its association with Dicer-independent ‘‘priRNAs.’’ The RITS complex physically links Ago1 and the H3-K9me binding protein Chp1. Here we exploit an assay for heterochromatin assembly in which loss of silencing by deletion of RNAi or Clr-C components can be reversed by re- introduction of the deleted gene. We showed previously that a mutant version of the RITS complex (Tas3WG) that biochemically separates Ago1 from Chp1 and Tas3 proteins permits maintenance of heterochromatin, but prevents its formation when Clr4 is removed and re-introduced. Here we show that the block occurs with mutants in Clr-C, but not mutants in the RNAi pathway. Thus, Clr-C components, but not RNAi factors, play a more critical role in assembly when the integrity of RITS is disrupted. Consistent with previous reports, cells lacking Clr-C components completely lack H3K9me2 on centromeric DNA repeats, whereas RNAi pathway mutants accumulate low levels of H3K9me2. Further supporting the existence of RNAi–independent mechanisms for establishment of centromeric heterochromatin, overexpression of clr4+ in clr4Dago1D cells results in some de novo H3K9me2 accumulation at centromeres. These findings and our observation that ago1D and dcr1D mutants display indistinguishable low levels of H3K9me2 (in contrast to a previous report) challenge the model that priRNAs trigger heterochromatin formation. Instead, our results indicate that RNAi cooperates with RNAi– independent factors in the assembly of heterochromatin. Citation: Shanker S, Job G, George OL, Creamer KM, Shaban A, et al. (2010) Continuous Requirement for the Clr4 Complex But Not RNAi for Centromeric Heterochromatin Assembly in Fission Yeast Harboring a Disrupted RITS Complex. PLoS Genet 6(10): e1001174. doi:10.1371/journal.pgen.1001174 Editor: Hiten D. Madhani, University of California San Francisco, United States of America Received May 19, 2010; Accepted September 21, 2010; Published October 28, 2010 Copyright: 2010 Shanker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by R01GM084045 (JFP), the NIH/NCI Cancer Center Core Support (5 P30 CA 021765-32), and the American Lebanese Syrian Associated Charities (ALSAC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: janet.partridge@stjude.org . These authors contributed equally to this work. Introduction Eukaryotic genomes are characterized by domains of transcrip- tionally permissive euchromatin and relatively transcriptionally inert heterochromatin. In addition to its important role in transcriptional regulation, heterochromatin plays a critical role in the regulation of genomic stability. In fission yeast constitutive heterochromatin assembles at the centromeres, telomeres and the mating type locus. This heterochromatin is required for high fidelity chromosome transmission, protecting chromosome ends from fusion to form dicentric chromosomes, and preventing co- expression of both sets of mating type information which could lead to haploid meiosis and cell death. A major hallmark of heterochromatin in most eukaryotes is the presence of methyl groups on lysine 9 of histone H3. In fission yeast (Schizosaccharomyces pombe), methylation of H3 K9 is carried out by a single enzyme, Clr4 (the homolog of Suvar39 enzymes in higher eukaryotes), which is responsible for mono, di and tri- methylation of H3K9 [1]. This mark is in turn bound by proteins bearing a chromodomain, including the HP1 homologs Swi6 and Chp2, and importantly, Clr4 itself, leading to models for perpetuation and spreading of heterochromatin [2–5]. A fourth chromodomain protein, Chp1, has high affinity for binding the methyl mark [6]. Chp1 is a component of the RITS complex (RNA-induced initiation of transcriptional silencing complex), which is critical for the accumulation of heterochromatin at centromeres [7,8]. Heterochromatin assembly in several organisms also depends upon the cellular RNA interference (RNAi) pathway [9]. RNAi is triggered by double-stranded RNA (dsRNA), which is processed by the RNAseIII-like activity of Dicer into short interfering (si) RNAs. siRNAs are loaded into RNA –induced silencing complexes (RISC), where they associate with Argonaute proteins, and base-pair with and promote the sequence-dependent destruc- PLoS Genetics | www.plosgenetics.org 1 October 2010 | Volume 6 | Issue 10 | e1001174