Ers1, a Rapidly Diverging Protein Essential for RNA Interference-dependent Heterochromatic Silencing in Schizosaccharomyces pombe

Shankar, Smita; Braun, Sigurd; Rowley, Margot; Madhani, Hiten D.

doi:10.1074/jbc.c800140200

Cited by 20 publications

(30 citation statements)

References 13 publications

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“…Because no conserved protein motifs were found in Ers1 (26), it was difficult to infer the likely functional involvement of the Ser234 residue. The phenotype of C62 is similar to that of the ers1Δ null mutant; the C62 mutation resulted in derepression of a ura4 + marker gene integrated into the outermost (otr) pericentromeric repeat of chromosome 1 (otr1R::ura4 + ) (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Hayashi

Ishida

Kawaguchi

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

“…One of the mutants, denoted as C62, was found to contain an allele of ers1 (also known as rsh1). Ers1 was previously identified in a candidate KO approach and epistasis mapping as being required for RNAidirected heterochromatic silencing (25,26), but its function in this process was poorly understood.…”

Section: Resultsmentioning

confidence: 99%

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

Hayashi

Ishida

Kawaguchi

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…We demonstrated that Ers1 is required for silencing at centromeres, but not at the mating type locus and telomeres, consistent with a role in RNAi-dependent heterochromatin formation (9). Ers1 is needed for proper levels of H3K9 methylation as well as the recruitment of the RITS complex to centromeres (9). Deletion of ers1…”

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confidence: 92%

“…+ also triggers accumulation of noncoding centromeric transcripts and a defect in siRNA production (9). High-throughput analysis demonstrated that ers1 + behaves genetically in a manner similar to silencing factors (10).…”

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Ers1 links HP1 to RNAi

Rougemaille

Braun

Coyle

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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 (...

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RNA as a Regulator of Chromatin Structure

Murakami

2014

Encyclopedia of Molecular Cell Biology and Molecular Medicine

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Ers1, a Rapidly Diverging Protein Essential for RNA Interference-dependent Heterochromatic Silencing in Schizosaccharomyces pombe

Cited by 20 publications

References 13 publications

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

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

Ers1 links HP1 to RNAi

RNA as a Regulator of Chromatin Structure

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