Ers1 links HP1 to RNAi

Abstract

Pericentromeric heterochromatin formation is mediated by repressive histone H3 lysine 9 methylation (H3K9Me) and its recognition by HP1 proteins. Intriguingly, in many organisms, RNAi is coupled to this process through poorly understood mechanisms. In Schizosaccharomyces pombe, the H3-K9 methyltransferase Clr4 and the heterochromatin protein 1 (HP1) ortholog Swi6 are critical for RNAi, whereas RNAi stimulates H3K9Me. In addition to the endoribonuclease Dcr1, RNAi in S. pombe requires two interacting protein complexes, the RITS complex, which contains an Argonaute subunit, and the RDRC complex, which contains an RNA-dependent RNA polymerase subunit. We previously identified Ers1 (essential for RNAi-dependent silencing) as an orphan protein that genetically acts in the RNAi pathway. Using recombinant proteins, we show here that Ers1 directly and specifically interacts with HP1/Swi6. Two-hybrid assays indicate that Ers1 also directly interacts with several RNAi factors. Consistent with these interactions, Ers1 associates in vivo with the RITS complex, the RDRC complex, and Dcr1, and it promotes interactions between these factors. Ers1, like Swi6, is also required for RNAi complexes to associate with pericentromeric noncoding RNAs. Overexpression of Ers1 results in a dominant-negative phenotype that can be specifically suppressed by increasing levels of the RDRC subunit Hrr1 or of Dcr1, further supporting a functional role for Ers1 in promoting the assembly of the RNAi machinery. Through the interactions described here, Ers1 may promote RNAi by tethering the corresponding enzyme complexes to HP1-coated chromatin, thereby placing them in proximity to the nascent noncoding RNA substrate. Heterochromatin is a specialized form of DNA packaging that plays numerous critical roles in chromosome biology. However, the mechanisms that drive its assembly remain poorly understood. In fission yeast Schizosaccharomyces pombe, heterochromatin is assembled on pericentromeric repeats, subtelomeric regions, and the silent mat2/3 mating type locus (1, 2). Establishment and maintenance of heterochromatin at these sites depend on histone-modifying enzymes, histone marks, and histone-binding proteins. Among the histone-modifying enzymes is the histone methyltransferase, Clr4, the ortholog of Drosophila melanogaster and mammalian SU(VAR)3-9 enzymes (1, 2). Clr4 catalyzes methylation of histone H3 lysine 9 (H3K9me), an essential heterochromatin mark that serves as a docking site for the chromodomain-containing HP1 proteins Swi6 and Chp2 (1, 2). Surprisingly, many sites of heterochromatin formation correspond to sites that serve as templates for RNAi. Pericentromeric dh and dg repeats, for instance, are transcribed by RNA polymerase II, converted into double-stranded RNA by an RNAdependent RNA polymerase (Rdp1), and processed into siRNAs by a Dicer enzyme (Dcr1) (1, 2). Remarkably, RNAi requires the formation of heterochromatin. Loss of Clr4 or of Swi6 results in a loss of siRNAs (3-5). RNAi also promotes, but is not essential for, H3K9Me (...

A conserved ncRNA-binding protein recruits silencing factors to heterochromatin through an RNAi-independent mechanism

“…S1B). This is in contrast to RNAi factors that require H3K9me to display a RIP signal on the dg and dh transcripts (Rougemaille et al 2012).…”

“…S1B). This is in contrast to RNAi factors that require H3K9me to display a RIP signal on the dg and dh transcripts (Rougemaille et al 2012).We next isolated a mutation in seb1 + that was defective in heterochromatic silencing. Because seb1 S2) and transformed this library into a strain harboring an ectopic heterochromatic silencing reporter system that we had developed for other studies.…”

RNAi and Heterochromatin Assembly

“…The Swi6 protein, which binds to methylated H3K9, plays a major role in linking heterochromatic transcription with RNA degradation by promoting the interaction of heterochromatic transcripts with the RNAi and exosome pathways (see Fig. 4, dotted lines) (Motamedi et al 2008;Reyes-Turcu et al 2011;Hayashi et al 2012;Keller et al 2012;Rougemaille et al 2012).…”

“…4A,B) (Motamedi et al 2008). Specifically, Swi6 interacts with an accessory factor called Ers1, which, in turn, interacts with the RDRC complex to promote dsRNA synthesis and RNA degradation (Hayashi et al 2012;Rougemaille et al 2012). In addition, Swi6 has nonspecific affinity for RNA and may help retain heterochromatin-transcribed RNAs on the chromosome until they are degraded by the exosome (Keller et al 2012).…”

The Spatiotemporal Dynamics of Chromatin Protein HP1α Is Essential for Accurate Chromosome Segregation during Cell Division

“…All three isoforms have a conserved N-terminal chromodomain (CD), 3 a non-conserved hinge region, and a C-terminal chromoshadow domain (CSD) (2,3). HP1 is a multifunction protein, involved in many biological progresses, such as heterochromatin formation (4 -6), transcriptional regulation (7,8), DNA recombination and damage repair (9 -11), RNA interference (12,13), kinetochore protein assembly (14), chromatin condensation (15), and sister chromatid cohesion regulation (16 -19). HP1␣ localizes to centromeres in interphase through its CD domain, whereas mitotic centromeric localization depends on the CSD domain (20).…”