Heterochromatin protein 1 homologue Swi6 acts in concert with Ers1 to regulate RNAi-directed heterochromatin assembly

Abstract

In fission yeast, the RNAi pathway is required for centromeric heterochromatin assembly. siRNAs derived from centromeric transcripts are incorporated into the RNA-induced transcriptional silencing (RITS) complex and direct it to nascent homologous transcripts. The RNA-induced transcriptional silencing-bound nascent transcripts further recruit the RNA-directed RNA polymerase complex (RDRC) to promote dsRNA synthesis and siRNA production. Heterochromatin coated with Swi6/Heterochromain Protein 1 is then formed following recruitment of chromatin modification machinery. Swi6 is also required for the upstream production of siRNA, although the mechanism for this has remained obscure. Here, we demonstrate that Swi6 recruits RDRC to heterochromatin through Ers1, an RNAi factor intermediate. An ers1 + mutant allele (ers1-C62) was identified in a genetic screen for mutants that alleviate centromeric silencing, and this phenotype was suppressed by overexpression of either the Hrr1 RDRC subunit or Clr4 histone H3-K9 methyltransferase. Ers1 physically interacts with Hrr1, and loss of Ers1 impairs RDRC centromeric localization. Although Ers1 failed to bind Clr4, a direct interaction with Swi6 was detected, and centromeric localization of Swi6 was enhanced by Clr4 overexpression in ers1-C62 cells. Consistent with this, deletion of swi6 + reduced centromeric localization of Ers1 and RDRC. Moreover, tethering of Ers1 or Hrr1 to centromeric heterochromatin partially bypassed Swi6 function. These findings demonstrate an alternative mechanism for RDRC recruitment and explain the essential role of Swi6/Heterochromain Protein 1 in RNAi-directed heterochromatin assembly.RNA interference | histone H3-lysine 9 methylation I n a eukaryotic cell, the formation of higher order chromatin structure, heterochromatin, is critical for genomic stability, chromosome segregation, and epigenetic gene silencing. Heterochromatic structure is also essential for functional organization of chromosomal domains, such as the centromeres and telomeres, and is defined by specific posttranslational modifications of nucleosome histone tails. The methylation of lysine 9 of histone H3 (H3K9me) is a key marker of heterochromatin and is provided by the methyltransferase Clr4/Suv3-9 (1-3). This H3K9me marker serves as a binding site for Heterochromatin Protein 1 (HP1) family proteins, which provide a platform for recruitment of transacting factors to maintain repressive chromatin structure (4).In the fission yeast Schizosaccharomyces pombe, the assembly of heterochromatin at centromeres also depends on transcription of the centromeric dg and dh repeats by RNA polymerase II during S phase (5-7) and the subsequent recruitment of RNAi machineries (8-10). In this RNAi system, siRNAs derived from the centromeric repeats and the RNA-induced transcriptional silencing (RITS) complex play a central role to establish H3K9me at centromeric regions (11). RITS complex, containing Argonaute (Ago1), the GW-repeat Tas3 protein, and the chromodomain (CD) protein Chp1, targets nasce...

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).…”

Ers1 links HP1 to RNAi

“…Finally, because Ers1, but not Swi6, is required for the recruitment of RITS to chromatin (9), Ers1 likely also plays Swi6-independent roles promoting RNAi. In agreement with this, centromeric silencing triggered by the tethering of Hrr1 can bypass a requirement for Swi6, but not Ers1, indicating that the function of Ers1 is not limited to bridging HP1 to RDRC (23). An additional contact between the histone methylation and RNAi machineries is likely to be provided by the LIM domain protein Stc1.…”

“…This view is particularly consistent with our genetic suppression results. In addition, while this work was under review, an independent study by Hayashi et al (23) showed that tethering of Hrr1 to centromeric regions can bypass the requirement for Swi6 in heterochromatin formation, further highlighting a role for HP1 in recruiting RDRC to chromatin. The Ers1-Swi6 interaction documented here also likely explains the previously described role for Swi6 in RNAi and the observation that Swi6 physically associates with centromeric transcripts in vivo (4).…”

Endogenous Nuclear RNAi Mediates Behavioral Adaptation to Odor

“…A feed-forward silencing loop is established as chromodomain proteins, including the HP1 homolog, and methyl-transferases are nucleated by RITS complexes, and in turn recruit more methyl-transferases. Concurrently, RNA-directed RNA polymerase complexes (RDRCs) are recruited, thus increasing siRNA production (Hayashi et al, 2012; Rougemaille et al, 2012; Yamanaka et al, 2013). A direct link between chromatin, RNAi and RITS was demonstrated when the CSD of pombe HP1 was shown to interact with several members of the RNAi and RITS machinery via the HP1 binding protein, Ers1 (Rougemaille et al, 2012).…”