Background: Heat‑ shock molecular chaperone proteins (Hsps) promote the loading of small interfering RNA (siRNA) onto RNA interference (RNAi) effector complexes. While the RNAi process is coupled with heterochromatin assembly in several model organisms, it remains unclear whether the Hsps contribute to epigenetic gene regulation. In this study, we used the fission yeast Schizosaccharomyces pombe as a model organism and investigated the roles of Hsp90 and Mas5 (a nucleocytoplasmic type‑ I Hsp40 protein) in RNAi‑ dependent heterochromatin assembly. Results: Using a genetic screen and biochemical analyses, we identified Hsp90 and Mas5 as novel silencing fac‑ tors. Mutations in the genes encoding these factors caused derepression of silencing at the pericentromere, where heterochromatin is assembled in an RNAi‑ dependent manner, but not at the subtelomere, where RNAi is dispensable. The mutations also caused a substantial reduction in the level of dimethylation of histone H3 at Lys9 at the pericen‑ tromere, where association of the Argonaute protein Ago1 was also abrogated. Consistently, siRNA corresponding to the pericentromeric repeats was undetectable in these mutant cells. In addition, levels of Tas3, which is a protein in the RNA‑ induced transcriptional silencing complex along with Ago1, were reduced in the absence of Mas5. Conclusions: Our results suggest that the Hsps Hsp90 and Mas5 contribute to RNAi‑ dependent heterochromatin assembly. In particular, Mas5 appears to be required to stabilize Tas3 in vivo. We infer that impairment of Hsp90 and Hsp40 also may affect the integrity of the epigenome in other organisms. Keywords: RNAi, Heterochromatin, Fission yeast, Schizosaccharomyces pombe, Heat‑ shock molecular chaperons, Hsp90, Mas5 Background pombe to humans. Studies using S. pombe as a model Assembly of heterochromatin, a dense chromatin struc- organism have established the concept that the RNA ture that represses the expression of embedded genes, interference (RNAi) pathway contributes to the assembly is vital for the establishment and maintenance of cell of heterochromatin (reviewed in [1–4]). In fission yeast, identity. A hallmark of heterochromatin is methylation the RNAi pathway is required predominantly at the peri- of histone H3 at Lys-9 (H3K9me), a modification that is centromeric regions, while the pathway is dispensable for conserved from the fission yeast Schizosaccharomyces the maintenance of the heterochromatin assembled at the subtelomeric regions and the mating-type locus [5–7]. Notably, defects in the RNAi pathway lead to great loss of H3K9me and derepression of silencing at the pericen- *Correspondence: email@example.com‑u.ac.jp Department of Biochemistry, Shimane University School of Medicine, tromeric regions but not at the subtelomeric regions or 89‑1 Enya‑cho, Izumo, Shimane 693‑8501, Japan the mating-type locus [5–8]. These regional differences Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 2 of 15 in dependence on the RNAi pathway have provided among various species. For instance, Hsp90-mediated researchers with clues to ascertain whether the factors of ATP hydrolysis is required for siRNA duplex loading interest act specifically in the RNAi pathway or act more in animal cells, but is instead required for passenger generally in the assembly of heterochromatin [9–11]. strand removal in plant cells [23, 24, 31]. Similarly, the In the S. pombe RNAi pathway, formation of the small formation of small RNA-containing complexes does not interfering RNA (siRNA)-containing effector complex necessarily require Hsp70-family proteins. An Hsp70 is coupled to heterochromatin assembly [1–4]. siRNA is protein is essential for complex formation in the fruit generated, by the Dicer family endoribonuclease Dcr1, fly Drosophila melanogaster, but not in the ciliated pro- from double-stranded non-coding RNA that is comple- tozoan Tetrahymena thermophila [22, 29]. Therefore, mentary to heterochromatin. The siRNA duplex is loaded the differences between species should be acknowl - onto a non-chromatin-associated complex called Argo- edged in examining how such chaperones act in RNAi- naute small interfering RNA chaperone (ARC), which dependent heterochromatin assembly. contains the Argonaute family endoribonuclease Ago1. The S. pombe genome encodes six Hsp70 proteins. The loading of the siRNA duplex onto the Ago1 subunit These Hsps show high sequence similarity to their coun - requires the two ARC-specific subunits Arb1 and Arb2, terparts in the budding yeast Saccharomyces cerevisiae which also inhibit the release of the passenger strand (Additional file 1: Fig. S1), where the cellular roles of . This complex then changes its subunit composition Hsp70 s have been thoroughly examined . Among the to form a chromatin-associated effector complex called six S. pombe Hsp70 proteins, Ssa1 and Ssa2, which show RNA-induced transcriptional silencing (RITS) [12, 13]. high sequence similarity to each other (identity: 94%), are The RITS complex is composed of Ago1, now binding recognized as nucleocytoplasmic Hsp70 proteins . single-stranded siRNA as a guide for target recognition, Ssa1 and Ssa2 also exhibit the strongest sequence similar- and the two RITS-specific subunits Chp1 and Tas3 [12, ity to the D. melanogaster Hsp70 protein Hsc70-4 (iden- 13]. Chp1 uses a chromodomain to recognize H3K9me tity: 75% each), which is essential for the formation of a , whereas Tas3 bridges Ago1 and Chp1 [15, 16]. small RNA-containing complex in that organism [22, 30]. With the ability to interact with both H3K9me and tar- The S. pombe genome encodes 26 Hsp40 family pro- get RNA, RITS plays a central role in the self-enforcing teins, all of which harbor a characteristic DnaJ domain cycle of RNAi-dependent heterochromatin assembly (Additional file 1: Fig. S2). These Hsp40 proteins can be [1–4]. RITS’ function depends on two major interactions. divided into three classes: types I, II, and III . Type-I On the one hand, RITS interacts with the RNA-depend- proteins are also found in S. cerevisiae (Additional file 1: ent RNA polymerase complex, which synthesizes dou- Fig. S3), and have the same names in the two yeast spe- ble-stranded RNA for secondary siRNA generation [17, cies [32, 35]. Mdj1 and Scj1 localize in mitochondria 18]. On the other hand, RITS interacts (via bridging by and the lumen of the ER, respectively . In contrast, the linker protein Stc1 ) with the Clr4 histone meth- Mas5 (also known as Ydj1 in S. cerevisiae) and Xdj1 local- yltransferase-containing complex that methylates the H3 ize in the cytosol and nucleus  and are categorized histone to create the H3K9me epigenetic marker [20, 21]. as nucleocytoplasmic type-I Hsp40 proteins. Among the u Th s, the formation of RITS is crucial for the genera - 26 Hsp40 proteins in S. pombe, Mas5 shows the greatest tion of siRNA and for the assembly of RNAi-dependent sequence similarity to the D. melanogaster protein Droj2 heterochromatin. (identity: 41%), a protein that promotes the formation of The formation of small RNA-containing effector com - a small RNA-containing complex in vitro [22, 30]. plexes is generally assisted by heat-shock molecular In the present study, we identified the S. pombe molec - chaperones [22–29]. However, the heat-shock molecular ular chaperone proteins Hsp90 and Mas5 as novel regu- chaperones responsible for the RNAi-dependent het- lators of RNAi-dependent heterochromatin assembly. erochromatin assembly remain unidentified. The candi - Mutations in the genes encoding these proteins caused dates may belong to one or more of the distinct families derepression of transcriptional silencing and decreases of heat-shock proteins 40, 70, and 90 (Hsp40, Hsp70, and in H3K9me at the pericentromeric heterochromatin Hsp90, respectively) [22–24, 30]. region, while having little effect on the subtelomeric het - Among the three Hsp families, the proteins belong- erochromatin, which can be maintained in the absence of ing to the Hsp90 family promote the in vitro forma- RNAi. Hsp90 and Mas5 were required in vivo for siRNA tion of small RNA-containing complexes in all species generation and chromatin localization of Ago1. In addi- that have been tested [22–24, 29]. Notably, however, tion, the protein level of Tas3 was substantially reduced Hsp90-family proteins appear to act in species-spe- in the absence of Mas5, suggesting that Mas5 is respon- cific manners. For example, the steps that require ATP sible for the stability of Tas3, and thus of the RITS RNAi hydrolysis by Hsp90-family proteins appear to differ effector complex. Therefore, we propose that these Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 3 of 15 molecular chaperones contribute to the assembly of the The hsp90-A4 mutant gene had a cytosine-to-thymine RNAi-dependent heterochromatin in S. pombe. base substitution at position 97 (from the start of the ORF) (Fig. 1b), causing a deduced arginine to cysteine Results substitution at amino acid 33 (R33C). The Arg-33 residue Identification of Hsp90 and Mas5 as silencing factors is located in the ATPase domain of Hsp90 and is highly In a forward genetic screen for factors that affect pericen - conserved from bacteria to humans (Figs. 1c, d). In S. cer- tromeric silencing , we isolated a missense mutation evisiae, the corresponding amino acid (Arg-32) has been of the hsp90 gene, which encodes the sole Hsp90-family implicated in the modulation of ATP hydrolysis [38, 39]. protein in S. pombe (see “Methods” section). In parallel Although Arg-32 in the S. cerevisiae homolog does not with the genetic screen, we conducted immunoaffinity directly contact ATP, the residue forms hydrogen bonds purification of proteins that interact either with RNA pol - with the adjacent catalytic residue Glu-33 (Glu-34 in the ymerase II or Spt6 and identified an Hsp40-family pro - S. pombe protein) and with a residue in the Hsp90 mid- tein, Mas5, as a silencing factor (see “Methods” section). dle domain . Through these intramolecular contacts, With the same monitoring system, we evaluated the Arg-32 is thought to be involved in ATP hydrolysis and silencing state of mutant cells by monitoring the expres- conformational changes of the Hsp90 protein [38, 39]. sion of ade6 and ura4 marker genes embedded in the Replacement of the S. pombe Arg-33 with cysteine is pericentromere regions  (Additional file 1: Fig. S4). expected to disrupt these intramolecular contacts, imply- In the absence of mutations, this screening strain did ing that the derepression of the pericentromeric silenc- not appreciably express ade6 or ura4; thus, cells without ing in the mutant is caused by a decrease in the ATPase a silencing defect formed red colonies on a plate with a activity of Hsp90. limited amount of adenine (due to the accumulation of an intermediate of adenine biosynthesis) and grew health- Hsp40 protein Xdj1, as well as Hsp70 proteins Ssa1 ily on a plate containing 5-fluoroorotic acid (5-FOA) and Ssa2, is not required for pericentromeric silencing (a pyrimidine precursor analog that is toxic to ura4- Identification of Hsp90 and Mas5 as silencing factors expressing cells)  (Fig. 1a). In contrast, cells bear- prompted us to examine whether other molecular chap- ing mutations in the genes encoding the H3K9 histone erones also contribute to pericentromeric silencing. methyltransferase Clr4 (clr4∆ ), the Dicer family endori- Mas5 is classified as a nucleocytoplasmic type-I Hsp40 bonuclease Dcr1 (dcr1∆), or the Argonaute protein Ago1 protein. There is another protein that falls into this clas - (ago1∆) formed pink colonies (i.e., decreased accumula - sification: Xdj1 (Additional file 1: Figs. S2 and S3). There - tion of red pigment) and were sensitive to 5-FOA. This fore, we tested the involvement of Xdj1 in the silencing result indicated that heterochromatic silencing at the of the pericentromeric ade6 marker gene. As shown in pericentromere requires each of these factors, in agree- Fig. 2a, null mutations in the gene encoding Xdj1 (xdj1∆) ment with the literature [1–4]. Importantly, cells harbor- did not lead to the formation of pink colonies, suggesting ing a mutation in the genes encoding Hsp90 (hsp90-A4) that Xdj1 does not have a major role in the silencing. or Mas5 (mas5∆) formed near-white colonies and exhib - Mas5 has a DnaJ domain, which has been implicated in ited sensitivity to 5-FOA, suggesting that Hsp90 and the regulation of the ATPase activity of Hsp70 proteins Mas5 are also required for pericentromeric silencing. [40, 41]. There are two nucleocytoplasmic Hsp70 proteins The formation of near-white colonies could be caused in S. pombe: Ssa1 and Ssa2 (Additional file 1: Fig. S1). by a defect in red pigment formation. However, as hsp90- Both Ssa1 and Ssa2 physically interact with Mas5  A4 and mas5∆ mutant cells not harboring the pericen - and (among the S. pombe Hsp70 proteins) show the high- tromeric marker genes formed red colonies (Additional est sequence similarity to the D. melanogaster Hsc70- file 1: Fig. S5), Hsp90 and Mas5 appeared not to con- 4. This observation raised the possibility that these two tribute to pigment formation. In addition, hsp90-A4 proteins might also act with Mas5 to silence pericentro- and mas5∆ mutant cells harboring the pericentromeric meric transcription. Nonetheless, as shown in Fig. 2b, marker genes grew faster than wild-type cells on Edin- single- and double-null mutations in the genes encoding burgh minimal medium with supplements (EMMS) lack- Ssa1 and Ssa2 (ssa1∆, ssa2∆, and ssa1∆ ssa2) did not lead ing adenine (Additional file 1: Fig. S6A), suggesting that to the formation of pink colonies, indicating that peri- the ade6 marker gene was indeed expressed by these centromeric silencing remained intact in the absence of mutations. EMMS medium lacking uracil was not suit- these nucleocytoplasmic Hsp70 proteins. able to monitor the expression level of the pericentro- To confirm that the colony color reflected the tran - meric ura4 marker gene, because of its leaky repression scription of the ade6 marker gene, we performed in the wild-type cells and the slow growth phenotype of strand-specific reverse transcription followed by quan - the Hsp mutants (Additional file 1: Fig. S6A). titative polymerase chain reaction (RT-qPCR) analysis Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 4 of 15 Fig. 1 Identification of Hsp90 and Mas5 as silencing factors. a Cells were serially diluted, spotted on normal YES plates (YES) and YES containing limited amount of adenine (low adenine) or 0.1% 5‑FOA (5‑FOA), and incubated at the indicated temperature for 3 days. Marker integration sites are shown in Additional file 1: Figure S4. b Results of sequencing of the antisense strand of the hsp90 gene. Arrows indicate the position of the base substitution. The wild‑type cytosine at position 97 (with respect to the ORF start), which is guanine in the antisense strand, was replaced with thymine (adenine in the antisense strand) in the mutant. c Domain structure of Hsp90 family proteins. The N‑terminal ATPase domains (ATPase), middle domains (M), and C‑terminal domains (CT ) are indicated. The names of proteins are associated with two ‑letter abbreviations indicating the species: “sp” for Schizosaccharomyces pombe (UniProt id: P41887), “sc” for Saccharomyces cerevisiae (P02829), “hs” for Homo sapience (P07900), and “ec” for Escherichia coli (P0A6Z3). The amino acid length (a.a.) of the proteins is shown. The position of R33C substitution is indicated with an asterisk. d Alignment of protein sequences around the R33C mutation. Residue numbers for the first and last amino acid residues for each protein interval are shown. Identical and similar residues are indicated as asterisks and colons, respectively, as seen in a standard ClustalW output. The residues corresponding to the Arg‑33 in the fission yeast Hsp90 are colored in blue. The catalytic glutamate residues corresponding to the Glu‑33 in budding yeast Hsp82 are colored in red (Fig. 2c). Due to the assembly of heterochromatin over nucleocytoplasmic Hsp70 proteins Ssa1 and Ssa2 do the marker gene, the expression level of ade6 was very not appear to be involved in this process. low in wild-type cells. In contrast, ade6 transcription was increased in clr4∆ cells, in which heterochroma- Hsp90 and Mas5 are required for RNAi‑dependent tin was not formed. In agreement with the results of heterochromatin assembly the colony color analyses (Fig. 2a, b), null mutations In the S. pombe pericentromere, silencing of the inserted in other genes encoding Hsp40 (xdj1) and Hsp70 (ssa1 marker genes occurs passively, reflecting the chroma - and ssa2) did not cause substantial increases in the tin state of neighboring pericentromeric repeat regions. transcript level of the marker gene. Thus, although Therefore, we next performed strand-specific RT-qPCR the nucleocytoplasmic Hsp40 protein Mas5 appears to examine the levels of transcripts from the ade6 and to have a role in pericentromeric silencing, the ura4 marker genes as well as those from the dogentai (dg) Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 5 of 15 Fig. 2 Xdj1, Ssa1, and Ssa2 are dispensable for silencing of the pericentromeric marker gene. a, b Silencing assay of Hsp40 mutants (a) and of Hsp70 mutants (b). Cells were serially diluted, spotted on normal YES plates (YES) and YES plates containing limited amounts of adenine (Low adenine), and incubated at 30 °C for 3 days. c Strand‑specific RT ‑ qPCR for the pericentromeric ade6 marker gene. Values are presented as means + SD (n = 3) pericentromeric repeats (Fig. 3a and Additional file 1: Fig. was maintained both in wild-type and RNAi-defective S6B). In wild-type cells, these pericentromeric transcripts mutants (dcr1∆ and ago1∆), but was derepressed in the accumulated at very low levels, as these regions were presence of clr4∆ , as reported previously [6, 7, 11]. Nota- silenced by heterochromatin (Additional file 1: Fig. S6C). bly, silencing of the tlh genes was maintained in hsp90- In contrast, these transcripts accumulated to higher lev- A4 cells and only derepressed by 2–13% in mas5∆ cells, els in dcr1∆, ago1∆, and clr4∆ mutant cells, indicating as compared to the fully depressed state in the clr4∆ cells. that silencing was derepressed by these mutations [8, 20]. Similarly, silencing of the cenH transcript was not mark- Importantly, hsp90-A4 and mas5∆ cells also accumulated edly affected by the hsp90-A4 or mas5∆ mutation. the pericentromeric transcripts. These results suggested These data suggest that Hsp90 and Mas5 are involved that Hsp90 and Mas5 are involved in the silencing of both in the RNAi-dependent assembly of heterochromatin. the inserted marker genes and the native pericentromeric To test this hypothesis, we performed chromatin immu- repeats. noprecipitation followed by quantitative PCR (ChIP- Derepression of pericentromeric silencing can be qPCR) to monitor the level of dimethylation of histone caused either by a mutation in the factors that are gener- H3 at Lys-9 (H3K9me2) at the ade6, dg, and tlh regions ally required for heterochromatin assembly, such as those (Fig. 3c). In wild-type cells, the three tested regions exhib- directly involved in H3K9 methylation, or by a mutation ited strong enrichment of H3K9me2 compared to that at in the RNAi factors, which direct H3K9me formation the euchromatic gene act1, which encodes actin. In the in a locus-specific manner. In the former case, silencing absence of the histone methyltransferase Clr4 (clr4∆ ), the in the subtelomere regions and the mating-type locus, H3K9me2 mark was abolished. When the RNAi pathway which can be maintained in the absence of RNAi fac- was defective (dcr1∆ and ago1∆), H3K9me2 was com - tors [5–7], should also be derepressed; in the latter case, pletely abolished at the ade6 gene, but was only moder- silencing in those non-pericentromeric regions should ately decreased (i.e., 32–55% compared to the wild-type not be affected. To test the possibility that Hsp90 and control) at the pericentromeric dg repeats and was main- Mas5 act as general heterochromatin factors, we exam- tained at the subtelomeric tlh genes. These observations ined the expression levels of the subtelomeric telomere- are consistent with the results of previous reports [6, 8, linked helicase (tlh) genes and the centromere homology 42]. In agreement with the results of RT-qPCR (Fig. 3a, (cenH) transcript from the mating-type locus by strand- b), hsp90-A4 and mas5∆ mutations caused reductions in specific RT-qPCR (Fig. 3b and Additional file 1: Fig. S7). the level of H3K9me2 at the pericentromeric regions but The silencing of the tlh genes and of the cenH transcript not at the subtelomeres. These results suggest that Hsp90 Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 6 of 15 Fig. 3 Hsp90 and Mas5 are required for the assembly of heterochromatin at the pericentromere. a, b Strand‑specific RT ‑ qPCR for the pericentromeric transcripts from the ade6 gene and dg repeats (a), and for the subtelomeric transcripts from the tlh genes (b). For the dg repeats and tlh genes, transcripts matching forward (Fw) and reverse (Rv) strands were analyzed. Values are normalized to that of the sense strand of ribosomal 28S RNA. c ChIP‑ qPCR using an antibody against H3K9me2 for the pericentromeric ade6 gene, dg repeats, and tlh genes. Color keys under the graphs in a–c correspond to the key shown in a. The euchromatic gene act1 was used as an internal control locus. d ChIP‑ qPCR of Myc‑Ago1 using an antibody against Myc for the pericentromeric ade6 gene, dg repeats, and tlh genes. For all panels, values are presented as means + SD (n = 3) and Mas5 act like RNAi factors in the assembly of hetero- internal control act1 gene. This result indicated that the chromatin at the pericentromeres. Myc tagging did not perturb the chromatin localiza- If Hsp90 and Mas5 are involved in RNAi-dependent tion of Ago1. In accordance with a previous report , heterochromatin assembly, mutations in these proteins the enrichment of Myc-Ago1 in dcr1∆ cells was as low may affect chromatin localization of Ago1. Therefore, as that of the untagged control in the pericentromeric we constructed mutant strains that also express amino ade6 and dg regions. Similarly, the chromatin localiza- (N)-terminally Myc-tagged Ago1 (Myc-Ago1) from its tion of Myc-Ago1 in the pericentromeric regions was own promoter  and examined the chromatin locali- abrogated in hsp90-A4 and mas5∆ cells. Therefore, zation level of Myc-Ago1 by ChIP-qPCR (Fig. 3d). In Hsp90 and Mas5 appeared to be required for the locali- wild-type cells, Myc-Ago1 was enriched at the ade6 zation of Ago1 in the pericentromere. and dg regions when compared to the level at the Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 7 of 15 Hsp90 and Mas5 are required for the formation of the RNAi pericentromeric siRNA signal detected in the immuno- effector complex precipitate from wild-type cells expressing FLAG-Ago1) As molecular chaperone proteins, Hsp90 and Mas5 may (Fig. 4c, FLAG-Ago1, cen siRNA). As expected, the sig- contribute to effector complex formation in the RNAi nal intensity of Ago1-bound siRNA from dcr1∆ cells was pathway. To examine this possibility, we first tested approximately 96% weaker than that from wild-type cells. whether the protein level of Ago1 is altered in the mutant Notably, the Ago1-bound siRNA signals from hsp90-A4 cells (Fig. 4a). Yeast strains that did (FLAG-Ago1) or and mas5∆ cells were comparable to that from dcr1∆ did not (untagged) express N-terminally FLAG-tagged cells. The amounts of FLAG-Ago1 protein in the immu - Ago1 from its own promoter were used for this analysis noprecipitates differed among samples (Fig. 4c, FLAG- . The amount of FLAG-Ago1, Hsp90, and α-tubulin Ago1); however, this magnitude of difference did not in cell extracts were examined by western blotting. The appear to explain the observed decrease in siRNA. These α-tubulin signals indicated that equal amounts of sam- data suggested that Hsp90 and Mas5 are required for the ples were loaded on the gel. Interestingly, the amount formation of functional, siRNA-containing effector com - of Hsp90 itself was not altered in hsp90-A4 cells. This plex in vivo. observation suggested that the function, rather than the As the loading of siRNA onto Ago1 depends on the quantity, of the Hsp90 protein is affected by the R33C formation of the ARC complex , we next investi- mutation. As the FLAG-Ago1 signal appeared to be gated the interaction between Ago1 and Arb1 in the decreased in the mutant cells, we conducted western mutant cells. We constructed strains that co-expressed blotting with twofold serial dilutions (Additional File 1: carboxy (C)-terminally Myc-tagged Arb1 (Arb1-Myc) Fig. S8). The results indicated that the signal intensity of and FLAG-Ago1 from the respective native promoters. FLAG-Ago1 in hsp90-A4 or mas5∆ cells was less than a We subjected extracts of the resulting strains to immu- half of that in wild-type cells. These results suggest that noprecipitation of Arb1-Myc, and examined the amounts Hsp90 and Mas5 are required to maintain the proper of Arb1-Myc and FLAG-Ago1 in the immunoprecipi- amount of Ago1 protein in cells. tates by western blotting (Fig. 4d). In the soluble extracts Next, we examined the amount of pericentromeric (Input) from wild-type strains, comparable amounts of siRNA by northern blotting (Fig. 4b). In total RNA FLAG-Ago1 were detected irrespective of the Myc tag- extracts from wild-type cells, we detected 21–24-nt ging of Arb1. This result demonstrated that the double siRNA that were complementary to the pericentro- tagging did not affect the bulk amount of Ago1 in the meric repeats; U6 small nuclear RNA was used as a wild-type background. In the soluble extracts (Input) loading control. Comparable amounts of siRNA were from hsp90-A4 and mas5∆ cells, the signal intensity of detected irrespective of the FLAG tag, indicating that (as Arb1-Myc was slightly lower than that from the wild- described previously ) the epitope tagging of Ago1 type cells. This observation suggests that both Hsp90 did not affect siRNA generation. In dcr1∆ cells, in which and Mas5 are required to maintain Arb1 levels in the siRNA generation should be abolished, the siRNA was cell. It is possible that Hsp90 regulates Arb1 at the RNA not detected, again consistent with previous results . level, as the expression of arb1 mRNA was decreased in Remarkably, siRNA was undetectable in hsp90-A4 or hsp90-A4 mutant cells (Additional file 1: Fig. S9). None- mas5∆ cells, suggesting that Hsp90 and Mas5 have major theless, comparable amounts of Arb1-Myc were detected roles in siRNA generation in S. pombe. in the immunoprecipitates (Myc-IP) from Arb1-Myc- The above data suggested that Ago1 does not bind expressing cells, permitting further analysis of the inter- appropriate amounts of pericentromeric siRNA in action between Arb1 and Ago1. Notably, in the wild-type hsp90-A4 and mas5∆ cells. To confirm this hypothesis, background, FLAG-Ago1 was detected in an Arb1-Myc- we attempted to detect siRNA in FLAG-Ago1-contain- dependent manner, indicating that double tagging did ing immunoprecipitates by northern blotting (Fig. 4c). not disrupt the interaction between Ago1 and Arb1. In Specifically, we subjected extracts from cells expressing contrast, in the immunoprecipitates from hsp90-A4, FLAG-Ago1 to immunoprecipitation with anti-FLAG mas5∆, and arb2∆ cells, Arb1-associated Ago1 signals antibody. Cells that did not express FLAG-Ago1 were were lower than that in the wild-type sample (Fig. 4d, used as negative controls. Successful immunoprecipita- Myc-IP). These observations suggested that Hsp90 and tion was confirmed by detecting FLAG-Ago1 by means Mas5 contribute to the formation of the ARC complex of western blotting (Fig. 4c, FLAG-Ago1). In this analysis, in vivo, as previously reported for Arb2 . we detected faint background RNA signals of unknown Next, we examined whether the formation of the RITS origin even in the immunoprecipitates from untagged complex in vivo is affected by the mutations. We con - strains (Fig. 4c, untagged, cen siRNA). However, these structed strains that co-express C-terminally Myc-tagged RNA signals were very weak (i.e., 6–12% compared to the Tas3 (Tas3-Myc) and FLAG-Ago1 from the respective Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 8 of 15 Fig. 4 Hsp90 and Mas5 are required for the formation of siRNA‑ containing Ago1 complexes. a Detection of proteins in whole‑ cell extracts by western blotting. Antibodies against FLAG epitope, Hsp90, and α‑tubulin were used. b Detection of pericentromeric siRNA. Equal amounts of total RNA extracted from untagged cells or from cells expressing FLAG‑Ago1 were loaded into each lane. Pericentromeric siRNA (cen siRNA) was detected by northern blotting with specific probes (see “Methods ” section). U6 snRNA was used as a loading control. c Detection of pericentromeric siRNA in Ago1 complex. FLAG‑Ago1 complex was immunoprecipitated with an antibody against the FLAG epitope. Pericentromeric siRNA extracted from the immunoprecipitates was detected by northern blotting (cen siRNA). Signal intensities of the pericentromeric siRNA relative to the wild‑type FLAG‑Ago1 sample are indicated. Successful immunoprecipitation was validated by means of western blotting with an antibody against the FLAG epitope (FLAG‑Ago1). Untagged cells were used as negative controls. d, e Detection of FLAG‑Ago1 in Arb1‑Myc (d) and Tas3‑Myc (e) immunoprecipitates. Soluble extracts (Input) and immunoprecipitates (Myc‑IP) were separated on SDS‑PAGE and detected with antibodies against the Myc and FLAG epitopes; the loading control protein was detected with an antibody against α‑tubulin. Asterisks, background signals. Signal intensities of FLAG‑Ago1 normalized with those of Arb1‑Myc are indicated Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 9 of 15 native promoters. Unexpectedly, Tas3-Myc protein was difficult to detect in the mas5∆ background (Fig. 4e, Input; Additional file 1: Fig. S10). RT-qPCR analysis showed that tas3 mRNA accumulated to higher (not lower) levels in the mas5∆ mutant (compared to the wild-type background) (Additional file 1: Fig. S9). This observation suggested that Mas5 is required to stabilize the level of Tas3 protein in S. pombe cells. When wild-type extracts were subjected to immu- noprecipitation with anti-Myc antibody, FLAG-Ago1 was detected in the immunoprecipitates in a Tas3-Myc- dependent manner (Fig. 4e, Myc-IP), indicating that Tas3-Myc formed a complex with FLAG-Ago1 in vivo. FLAG-Ago1 also was detected in the immunoprecipitates Fig. 5 Model. Formation of the siRNA‑ containing complexes from dcr1∆ cells, as reported previously . In arb1∆ ARC and RITS are key steps in the assembly of RNAi‑ dependent and arb2∆ cells, the amount of FLAG-Ago1 interact - heterochromatin at the pericentromere. In ARC and RITS ing with Tas3-Myc was decreased to background levels complexes, the Argonaute protein Ago1 binds double‑stranded (Fig. 4d, Myc-IP long exposure), indicating that the ARC and single‑stranded siRNA, respectively. The subunits of the subunits Arb1 and Arb2 are required for the formation of non‑ chromatin‑associated ARC complex are responsible for the loading of siRNA onto Ago1, which is then incorporated into the RITS, consistent with the previous report . However, RITS complex. RITS acts in the center of the self‑ enforcing loop comparable amounts of FLAG-Ago1 were detected in the of RNAi‑ dependent heterochromatin assembly. RITS recruits the Tas3-Myc immunoprecipitates in wild-type and hsp90- RNA‑ dependent RNA polymerase complex (RDRC) for siRNA A4 cells, suggesting that this hsp90 mutation does not generation, while also recruiting (via Stc1) the Clr4‑ containing impair the interaction between Ago1 and Tas3. methyltransferase complex that methylates H3K9. The generation of siRNA and loading of siRNA onto Ago1 in vivo require Hsp90 and Mas5. Mas5 also maintains the protein level of the RITS subunit Tas3. Thus, Hsp90 and Mas5 are required for RNAi‑ dependent Discussion heterochromatin assembly In this study, we demonstrated that the S. pombe molec- ular chaperones Hsp90 and Mas5 are required for the silencing, heterochromatin assembly, and chromatin for understanding how this chaperone contributes to the localization of Ago1 in the pericentromere (Figs. 1 and epigenetic regulation of chromatin formation. 3). In contrast, the heterochromatin assembled at the We observed that some H3K9me2 remained at the peri- subtelomeric regions and mating-type locus, which centromeric ade6 marker gene in hsp90-A4 cells, while can be maintained in the absence of RNAi, was not H3K9me2 levels were decreased to background levels in strongly affected by the mutations in Hsp90 or Mas5 mas5∆ cells (Fig. 3c). Given this result, we cannot confi - (Fig. 3 and Additional file 1: Fig. S7). We also showed dently state that Hsp90 is essential for RNAi-dependent that the in vivo generation of siRNA complementary to heterochromatin assembly. The residual H3K9me2 may the pericentromeric repeats required these chaperones reflect residual activity of Hsp90 and residual siRNA, (Fig. 4b, c). Furthermore, we showed that Mas5 contrib- which would be technically difficult to detect as a positive utes to maintenance of protein levels of Tas3 in the cells signal by Northern blotting, in the hsp90-A4 cells. Alter- (Fig. 4e). Together, these results indicated that Hsp90 and natively, Hsp90 may be important, but not essential, for Mas5 are involved in RNAi-dependent heterochromatin RNAi-dependent heterochromatin assembly. The hsp90 assembly in S. pombe (Fig. 5). gene is essential for growth, precluding silencing analysis Hsp90 has been shown to promote the formation of an in a gene deletion mutant. The development of specific RNAi effector complex in plants and animals [22–24, 30]. genetic tools may be necessary to determine whether However, previous studies on the relationship between Hsp90 is essential for RNAi-dependent heterochromatin Hsp90 and effector complexes were based mainly on assembly in vivo. in vitro experiments; few of these studies examined the Hsp40 proteins act as modulators of Hsp70 proteins in vivo effects of Hsp90 inhibition on the chromatin [32, 46, 47]. In D. melanogaster, the Hsp70 protein state. Notably, previous works did not examine whether Hsc70-4 physically interacts with Argonaute proteins and Hsp90 is required for RNAi-dependent heterochroma- is essential for the formation of RNAi effector complexes. tin assembly. In this regard, the discovery of Hsp90 as The D. melanogaster Hsp40 protein Droj2 also is associ- a silencing factor in S. pombe may be an important step ated with Argonaute proteins [22, 30]. Droj2 protein is Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 10 of 15 not essential for the effector complex formation but has mas5∆ mutants grew much more slowly than the wild- been shown to promote the effector complex formation type control, while also exhibiting temperature-sensitive in vitro . Droj2 is categorized as a nucleocytoplasmic growth; these phenotypes were not seen in the other type-I Hsp40 and exhibits higher sequence identity to silencing mutants evaluated here (Fig. 1a). Thus, Hsp90 the S. pombe Mas5 (41%) than to the other S. pombe par- and Mas5 appear to have roles beyond the RNAi path- alog Xdj1 (29%). Therefore, it is possible that Mas5, as a way, affecting cell growth and tolerance to heat stress. conserved Hsp40 protein, promotes the formation of the Colony colors of canonical heterochromatin mutants RNAi effector complex in the S. pombe cells. (i.e., clr4∆ and dcr1∆) harboring the ade6 marker gene Although we identified a nucleocytoplasmic type-I in the pericentromere (otr1R(SphI)::ade6 ) are often Hsp40 (Mas5) as a silencing factor that is essential for described as “white” or “light pink” in the literature [19, RNAi-dependent heterochromatin assembly, we demon- 48, 49]. This is true when the auxotrophic missense allele strated that the double-null mutation in the genes encod- ade6-m210, which can be interallelically complemented ing the nucleocytoplasmic Hsp70 proteins Ssa1 and Ssa2 by another auxotrophic allele ade6-m216 , is located does not cause a detectable defect in pericentromeric in the endogenous ade6 locus on Chromosome III (Addi- silencing in fission yeast (Fig. 2). Ssa1 and Ssa2 each show tional file 1: Fig. S11). However, when the deletion allele 75% identity to D. melanogaster Hsc70-4. Involvement of ade6-DN/N is used instead of ade6-m210, the canonical the remaining four S. pombe Hsp70 proteins in silencing heterochromatin mutants form apparently darker “pink” is difficult to imagine, given that these fission yeast para - colonies  (Additional file 1: Fig. S11). Similarly, col- logs exhibit restricted intracellular locations. Interest- ony colors of trichostatin A-treated cells are dependent ingly, in the ciliated protozoan T. thermophila, inhibition on the endogenous ade6 alleles . Thus, the ade6-DN/ of Hsp70 does not impair the formation of the effector N-driven enhanced pigmentation may help us visually complex in vitro . Therefore, it is possible that nucle - examine the degree of silencing defects . ocytoplasmic Hsp70 is dispensable for RNAi-dependent We noted that hsp90-A4 and mas5∆ cells formed heterochromatin assembly in S. pombe. In such cases, much “brighter” colonies and grew healthier than clr4∆ Mas5 may be acting in an Hsp70-independent manner: or dcr1∆ cells on the adenine-limiting (Fig. 1a) and ade- DnaJ domain-independent functions of Hsp40 proteins nine-lacking (Additional file 1: Fig. S6A) plates, respec- have been proposed in many studies, as reviewed in ref- tively. The colony brightness phenotype of hsp90-A4 and erences [46, 47]. mas5∆ mutants in the ade6-DN/N background was char- Despite several attempts to detect physical interac- acteristic of this class of mutants, permitting them to be tions between the S. pombe heat-shock molecular chap- readily distinguished from canonical silencing mutants erones and Ago1 using co-immunoprecipitation from such as clr4∆ . While other bright colony-forming cell extracts, we were unable to detect a positive signal silencing mutants have been isolated in similar forward stronger than background level (data not shown). This genetic screens, all of those mutants exhibited alterations suggests that the assumed physical interaction is tran- in RNA polymerase II-driven transcription [9, 11, 36]. sient, or that the involvement of these molecular chap- Although the mechanism that causes this brightness is erones in the formation of the siRNA-containing effector not yet clear, studying the defect caused by these mutants complexes is indirect at the molecular level. As the S. may lead to an understanding of a yet-unknown regula- pombe RNAi pathway forms a self-reinforcing loop that tory layer of epigenetic silencing. In this regard, colony is required for and is coupled to the assembly of the colors of heterochromatin mutants that have been stud- pericentromeric heterochromatin, a defect in any step ied in the ade6-m210 background [19, 48, 49, 51] might in the loop may result in essentially the same result: loss be worth being examined in the ade6-DN/N background of siRNA generation . u Th s, we cannot rule out the to visually classify the mutations. possibility that the formation of the effector complex is inhibited in vivo because the mutations in Hsp90 and Mas5 inhibit other steps in the RNAi pathway. Notably, Conclusions the decrease in Tas3 protein levels observed in mas5∆ Based on the results presented in this study, we propose cells, (Fig. 4e and Additional file 1: Fig. S10) may elimi- that molecular chaperones Hsp90 and Mas5 are required nate siRNA generation. Thus, Mas5 may contribute to for RNAi-dependent heterochromatin assembly in S. RNAi-dependent heterochromatin formation by main- pombe. Although the underlying molecular mechanism taining the amount of functional Tas3. Note that Mas5 remains to be elucidated, mutations in the genes encod- has been affinity-captured by Stc1 , suggesting a con - ing these chaperones greatly decreased the levels of peri- tribution of Mas5 in RNAi-dependent heterochromatin centromeric siRNA and H3K9me2 in vivo. Our results formation through this interaction. The hsp90-A4 and suggest that inhibition of the counterparts of these Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 11 of 15 chaperones in other species may have similar destructive in the immunoprecipitation and western blotting sec- effects on chromatin regulation. tion. For each biological sample, 4 × 10 cells (two tubes for one biological sample) were used for the analysis. The Methods cell extracts from the two tubes were combined, and 440 Genetic manipulations The S. pombe strains and primers µL of IP buffer (50 mM HEPES–KOH, pH 7.5, 140 mM for genetic manipulations used in this study are listed in NaCl, 1 mM EDTA, 1% Triton X-100, and 0.1% Na- Additional file 2: Tables S1 and S2, respectively. General deoxycholate) containing protease inhibitors (P08215, yeast manipulation methods and culture conditions were Sigma-Aldrich) was added to obtain 880 µL of whole-cell as documented elsewhere [36, 52]. For N-terminal tag- extract for each biological sample. An aliquot (200 µL) of ging of Ago1, p3FLAGago1N-natMX4 or p3MYCago1N- secondary antibody-conjugated magnetic beads was pre- natMX4 was integrated into the ago1 locus. To construct pared as described in the immunoprecipitation and west- these plasmids, 3xFLAG or 3xMyc (respectively) epitope- ern blotting section. After centrifuging the pooled cell encoding sequences, including an NdeI linker, were extract at 20,000×g at 4 °C for 15 min, an aliquot (850 µL) inserted between the promoter (extending from nt -259 of the resulting supernatant was incubated with the mag- to the ORF start codon) and the sequences encoding netic beads for 2 h at 4 °C. Beads were washed twice with the N-terminus (from the second codon to nt 733 of the 500 µL per wash of IP buffer containing protease inhibi - ORF) of the ago1 gene in the natMX gene plasmid. The tors and resuspended in 500 µL of IP buffer containing respective plasmids were digested with XhoI, purified by protease inhibitors. Of the 500 µL of immunoprecipitate, ethanol precipitation, and introduced into host strains one 100 µL aliquot was stored for evaluation by western using a yeast transformation kit for S. pombe (Wako Pure blotting. For the remaining 400 µL, the supernatant was Chemical Industries) according to the manufacturer’s removed and the beads were suspended in 250 µL of AE instructions. clonNAT-resistant clones were selected on buffer (50 mM sodium acetate pH 5.2, 10 mM EDTA). YES plates containing 100 mg/L clonNAT (Werner Bio- After mixing with 250 µL of citrate-saturated phenol, Agents). All of the tagged sequences were subjected to samples were frozen at − 80 °C and thawed at 65 °C. After DNA sequencing to confirm that no additional mutation centrifugation at 20,000×g at 25 °C for 5 min, the aqueous had been introduced during construction. layer was mixed with 250 µL of phenol/chloroform/isoa- Chromatin immunoprecipitation Chromatin immuno- myl alcohol (25:24:1). After centrifugation at 20,000×g at precipitation and subsequent qPCR were performed as 25 °C for 5 min, the resulting aqueous layer was mixed described elsewhere . The primary antibody used for with 25 µL of 3 M sodium acetate, pH 5.2, 625 µL of etha- immunoprecipitation of H3K9me2, the secondary anti- nol, and 2 µL of Ethachinmate (312-01791, Nippon Gene) body-conjugated magnetic beads, and the primers used and centrifuged at 20,000×g at 4 °C for 20 min. The pellet for qPCR were as described in the previous study . was rinsed with 500 µL of 80% ethanol and resuspended Cells growing logarithmically in YES medium at 30 °C in 20 µL of DEPC-treated water. An aliquot (10 µL) of the were used for the analyses. Cell density in each culture RNA sample was used for the northern analysis. was measured with a particle counter (CDA-500, Sys- Immunoprecipitation and western blotting Cells mex) according to the manufacturer’s instructions. To (2 × 10 ) growing logarithmically in YES at 30 °C were avoid inaccurate measurements caused by cell floccula - washed with 10 mL of distilled water and with 1 mL of tion, cells were diluted and briefly sonicated by directly IP buffer. Cell pellets were resuspended in 220 µL of IP immersing the cuvettes in an ultrasonic cleaner bath buffer containing protease inhibitor cocktail. Cells were (Branson 5510) prior to counting. disrupted in a Multi-Beads Shocker (MB400U, Yasui RNA preparation, RT-qPCR, and northern blotting Kikai). IP buffer (400 µL) containing protease inhibi - Methods for total RNA extraction, RT-qPCR, and north- tors was added to the cell extract, and the mixture was ern blotting were as documented elsewhere [11, 36, 44]. then centrifuged at 20,000×g at 4 °C for 15 min to obtain For the northern blotting of pericentromeric siRNA, we the supernatant as input extract. An aliquot (200 µL) of used oligonucleotide DNA probes that are complemen- M-280 anti-mouse sheep antibody-conjugated magnetic tary to the sequenced siRNAs named “A” to “L” [44, 53]. beads (112-02, Thermo Fisher Scientific) was washed The siRNA named “D” (5′-UGG AUU AAG GAG AAG twice with IP buffer, incubated with 3 µL of mouse CGG UA-3′)  was omitted for northern detection, monoclonal anti-FLAG antibody (M2, Sigma-Aldrich) because the probe complementary to this sequence tends at 4 °C for 2 h, and washed three times with IP buffer. to detect unwanted background RNA. For northern blot- Beads were resuspended in 500 µL of the input extract, ting of RNA in FLAG-Ago1 immunoprecipitates, RNA incubated at 4 °C for 2 h, washed three times with 500 was prepared as follows. Extracts of cells growing loga- µL per wash of IP buffer containing protease inhibitors, rithmically in YES at 30 °C were obtained as described and resuspended in 50 µL of SDS sample buffer. Western Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 12 of 15 blotting and detection of epitope-tagged proteins were 20 mM of dimethyl pimelimidate dihydrochloride performed as described previously . Mouse monoclo- (D8388, Sigma-Aldrich), rotated for 30 min at room tem- nal anti-Myc antibody 9E10 (1:2000), anti-G196 ascites perature, and washed twice with 10 mL per wash of 0.2 M (1:2000), anti-α-tubulin antibody DM1A (1:2000), and ethanolamine, pH 8.0. Beads then were resuspended in anti-FLAG antibody M2 (1:2000) were used as primary 10 mL of 0.2 M ethanolamine, pH 8.0; rotated for 2 h at antibodies. HRP-conjugated goat anti-mouse antibody room temperature; washed twice with 10 mL per wash (1:5000, Rockland Immunochemicals) was used as sec- of phosphate-buffered saline (PBS); washed once with ondary antibody. 10 mL of 100 mM glycine–HCl, pH 2.5; washed twice Isolation of hsp90-A4 silencing mutant In a genetic with 0.2 M Tris–HCl, pH 8.0; washed twice with 10 mL screen described previously , six mutants that exhib- per wash of IP buffer containing 0.05% sodium azide; and ited a defect in pericentromeric silencing were isolated; finally suspended in 500 µL of IP buffer containing 0.05% characterization of three of these mutants was reported sodium azide. The resulting antibody-conjugated Sepha - elsewhere [11, 36]. For the present work, we character- rose beads were stored at 4 °C. ized one of the remaining three mutants, A4, which was Extracts of cells growing logarithmically in YES at named after its original mutant pool designation (“A”). 30 °C were obtained as described in the immunoprecipi- Sequence analysis of the whole genome of an A4 strain tation and western blotting section. For each biological was performed as described previously . Five mis- sample, 1 × 10 cells (five tubes for one biological sample) sense mutations were found in the protein-coding genes were used for the analysis. Cell extracts from two tubes of the A4 genome. Genetic analysis of the A4 strain were combined and 1.3 mL of IP buffer containing pro - revealed that a mutation in the hsp90 gene, causing the tease inhibitors was added to obtain 2.4 mL of whole- R33C mutation, could not be genetically separated from cell extract for each biological sample. Insoluble debris the A4 phenotype: all of the tested A4 progeny with the was removed by centrifuging the extract three times at silencing phenotype possessed the mutation (n = 9). 20,000×g at 4 °C for 15 min. An aliquot (30 µL) of the Reintroduction of the mutation into the wild-type anti-G196 antibody-conjugated Sepharose beads (pre- genome via a selective marker (kanMX::hsp90-A4) as pared as described above) were washed with 1 mL of IP described previously  yielded the same phenotype buffer. The beads were suspended in a 1-mL aliquot of the as that observed in the original A4 mutant (Additional soluble extract and incubated for 3 h at 4 °C under rota- file 1: Fig. S12). Therefore, we named the mutant allele tion. After removal of the supernatant, the beads were hsp90-A4. suspended in another 1-mL aliquot of the soluble extract Identification of Mas5 as a silencing factor In order to and incubated for 3 h at 4 °C under rotation. The beads identify candidate proteins that might act as silencing were washed four times with IP buffer containing pro - factors, we screened for proteins that interacted with tease inhibitors and finally suspended in 10 mL of sample either RNA polymerase II or Spt6, an RNA polymer- buffer. The precipitates were separated on a 12% SDS- ase II-associated histone chaperone, both of which are PAGE gel, silver-stained (Additional file 1: Fig. S13), and involved in heterochromatic silencing [36, 54, 55]. G196- subjected to mass spectrometric analysis as described tagged protein complexes were immunoprecipitated previously . Proteins identified with over 95% proba - from four strains : a wild-type strain not expressing bility were assigned using the scaffold3 software ver. 3.5.1 a G196-tagged protein (HKM-1100); a wild-type strain (Proteome Software, Inc.). Isolation of transcription- expressing C-terminally G196-tagged Spt6 from its own related proteins in an Spt6- or Rpb3-dependent manner promoter (HKM-2064); a iws1∆ strain expressing the was confirmed by successful, selective co-immunoprecip - G196-tagged Spt6 (HKM-2066); and a wild-type strain itation (Additional file 2: Table S3). Non-essential genes expressing C-terminally G196-tagged Rpb3, an RNA pol- corresponding to ten of these proteins were (individually) ymerase II subunit, from its own promoter (HKM-2061). genetically deleted and evaluated for contribution to the For preparing anti-G196 antibody-conjugated Sepha- silencing of the ade6 marker gene inserted in the pericen- rose beads, 500 µL of Protein G-Sepharose (4 Fast Flow, tromere by observing the colony color of deletants grow- GE Healthcare) was washed three times with 10 mL per ing on YES medium that contained limiting amounts of wash of 20 mM Tris–HCl, pH 8.0. The beads were resus - adenine (Additional file 2: Table S4). In this screen, Mas5 pended in 10 mL of 20 mM Tris–HCl, pH 8.0, mixed with was identified as a potential silencing regulator. 2 mL of anti-G196 murine ascites, and incubated over- Drawing of phylogenetic trees and of schematic repre- night under rotation at 4 °C. After removal of the super- sentations of homologous proteins Amino acid sequences natant, the beads were washed three times with 10 mL and Gene3D domain information for Hsp40 and Hsp70 per wash of borate buffer (0.2 M sodium borate, pH 9.0). proteins were obtained using the BioMart tool of Ensem- The beads were resuspended in borate buffer containing ble (Release 35). The dataset for the S. pombe proteins Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 13 of 15 was ASM294v2; S. cerevisiae, R64-1-1; and D. mela- presented as means + SD (n = 3). (C) Strand‑specific RT ‑ qPCR for the ade6 + + nogaster, BDGP6. Phylogenetic trees in Additional and ura4 genes located in the endogenous loci (ade6 and ura4 ) and in + + the pericentromere (otr1R::ade6 and imr1L::ura4 ). Values are normalized file 1: Figs. S1 and S3 were drawn with the web software to that of the sense strand of ribosomal 28S RNA, and are presented as Phylogeny.fr . Order of the proteins was manually means + SD (n = 3). **P < 0.01 (Student’s t‑test). Figure S7. Hsp90 and changed on the web site. Colors for protein names were Mas5 are dispensable for the silencing at the mating‑type locus. Strand‑ specific RT ‑ qPCR for the cenH transcript from the mating‑type locus. Val‑ changed (using the vector design software Graphic ver. ues are normalized to that of the sense strand of ribosomal 28S RNA, and 3.0.1 (Autodesk, Inc.)) by processing the scalable vector are presented as means + SD (n = 6). **P < 0.01 (Student’s t‑test). Figure graphics images that were downloaded from the Phylog- S8. Reduction of Ago1 protein level in hsp90-A4 and mas5∆ cells. Two‑fold serially diluted whole‑ cell extracts were separated by SDS‑PAGE followed eny.fr web site. Schematic representations of proteins in by western blotting and Coomassie brilliant blue staining. The membrane Additional file 1: Figs. S1–S3 were drawn with the generic for detecting FLAG‑Ago1 with an antibody against FLAG epitope was rep ‑ graphic functions “plot” and “polygon” of the R statistical robed with an antibody against α‑tubulin. Figure S9. mRNA expression levels of arb1 and tas3 genes. Strand‑specific RT ‑ qPCR for the arb1 and environment. Localization signals and transmembrane tas3 genes. Values are normalized to that of the sense strand of ribosomal helices were predicted with the web software TargetP, ver. 28S RNA, and are presented as means + SD (n = 6). *P < 0.05, **P < 0.01 1.1 , and TMHMM, ver. 2.0 , respectively. (Student’s t‑test). Figure S10. Protein expression level of Tas3. Detection of proteins in whole‑ cell extracts by western blotting. The membrane for detecting Tas3‑Myc with an antibody against Myc epitope was reprobed Additional files with an antibody against α‑tubulin to confirm that equal amounts of samples were loaded. For the mas5 deletion, four independently con‑ structed clones were tested. Figure S11. Colony colors of otr1R::ade6 Additional file 1: Figure S1. Fission yeast Hsp70 proteins and their strains are dependent on the ade6 alleles in the endogenous ade6 locus. homologs. (A) Phylogenic tree of Hsp70 proteins. Scale‑bar unit indicates Cells were streaked on normal YES plates (YES) and YES containing limited the number of amino acid substitutions per site. Names of proteins are amount of adenine (Low adenine), and incubated at 30°C for four days. associated with two‑letter abbreviations and color ‑ coded to indicate the + Strains with otr1R::ade6 in the ade6-DN/N genetic background formed species: “sp” for Schizosaccharomyces pombe (red), “sc” for Saccharomyces darker colonies. Note that the colony colors of canonical heterochromatin cerevisiae (black), and “dm” for Drosophila melanogaster (blue). (B) Domain mutants (i.e. clr4∆) in the ade6-m210 background were faint pink, and structure of Hsp70 proteins. Protein names are depicted as in (A) and + are not as white as those of strains with the native ade6 allele in the their amino acid lengths are shown. Protein domains are drawn as boxes endogenous ade6 locus. Figure S12. Reintroduction of the R33C muta‑ according to the CATH‑ Gene3D classification. The N‑terminal ATPase tion phenocopied the original A4 isolate. (A) Schematic diagram showing domains (ATPase) are composed of three internal domains that belong to the introduction of the mutation (R33C) found in the original A4 isolate. the CATH superfamilies 3.30.420.40, 22.214.171.124, and 3.90.640.10. Substrate‑ (B) Cells were streaked on normal YES plates (YES) and YES containing binding domains (SB) belong to the 126.96.36.199 superfamily. The C‑terminal limited amount of adenine (Low adenine), and incubated at 30°C for four lid domains (Lid) belong to the 1.20.1270.10 superfamily. Predicted days. In HKM‑1565 and HKM ‑1618, the G418‑resistant kanMX cassette was localization signals for mitochondria ( TargetP‑M) and for the endoplasmic located upstream of the hsp90 promoter. Both strains were generated by reticulum or beyond ( TargetP‑S) are shown as shaded boxes. Locations introducing kanMX‑ containing DNA fragments with or without the R33C of the proteins in the cell are indicated. For clarity, D. melanogaster Hsp70 mutation into the genome of the wild‑type strain HKM ‑1100. Figure proteins other than Hsc70‑4 are omitted. Figure S2. Hsp40 family proteins S13. Silver staining of immunoprecipitated proteins that were analyzed in fission yeast. Domain structure of all 26 fission yeast Hsp40 proteins by mass spectrometry. Immunoprecipitates from indicated cells were are shown. Protein names and amino acid lengths are indicated. Protein separated by SDS‑PAGE and silver ‑stained (see Methods). Each lane in the domains are drawn as boxes according to the CATH‑ Gene3D, Pfam, gel was sliced as indicated by red lines. Proteins in each gel piece were and Prosite classifications. Predicted localization signals are shown as analyzed by nano‑liquid chromatography tandem mass spectrometry. in Additional file 1: Figure S1. Predicted trans‑membrane helix regions Black arrows indicate visible protein bands that are absent or barely seen ( TMhelix) are indicated as gray boxes. The DnaJ domains belong to the in the untagged control (no‑tag). CATH superfamily 1.10.287.110. Type‑I Hsp40 proteins are characterized by Additional file 2: Table S1. Strains used in this study. Table S2. Primers C‑terminal (purple) and central (pink) domains that belong to the CATH used in this study. Table S3. Transcription related factors detected in superfamilies 2.60.260.20 and 188.8.131.52, respectively. The type‑II Hsp40 massspectrometry analysis. Table S4. List of candidate genes for silencing protein Psi1 also contains the C‑terminal domain but lacks the central factors suggested by massspectrometry. domain. The other proteins are classified as the type ‑III Hsp40 proteins. Figure S3. Fission yeast type‑I and ‑II Hsp40 proteins and their homologs. (A) Phylogenic tree of Hsp40 proteins. Scale‑bar unit indicates the number of amino acid substitutions per site. Names of proteins are depicted as in Additional file 1: Figure S1. (B) Domain structure of Hsp40 proteins. Protein names are depicted as in (A) and amino acid lengths are shown. Protein Abbreviations domains and predicted localization signals are drawn as in Additional ARC : Argonaute small interfering RNA chaperone; ER: endoplasmic reticulum; file 1: Figure S2. Locations of proteins in the cell are indicated. For clarity, 5‑FOA: 5‑fluoroorotic acid; Hsp: heat ‑shock molecular chaperone protein; D. melanogaster Hsp40 proteins other than Droj2 are omitted. Figure S4. + + Hsp40: heat‑shock protein 40; Hsp70: heat ‑shock protein 70; Hsp90: heat ‑ Schematic representation of marker integration sites. The ade6 and ura4 shock protein 90; H3K9me: methylation of histone H3 at Lys‑9; RNAi: RNA marker genes were located in the SphI site of otr1R (otr1R(SphI)::ade6 ) interference; RITS: RNA‑induced transcriptional silencing; siRNA: small interfer ‑ and in the NcoI site of imr1L (imr1L(NcoI)::ura4 ), respectively. Figure S5. ing RNA. Hsp90 and Mas5 are dispensable for red pigment formation. Cells were spotted on normal YES plates (YES) and YES containing limited amount Authors’ contributions of adenine (Low adenine), and incubated at 30°C for six days. Figure S6. KO performed most of the experiments. KO and TI performed the northern Derepression of marker genes inserted in the pericentromere. (A) Cells blotting analyses. JN and KS performed the mass spectrometry analysis. TU were serially diluted, spotted on normal EMMS plates and EMMS lacking and YM were involved in study design and material preparation. KO and HK adenine (EMMS ‑ adenine) or uracil (EMMS ‑ uracil), and incubated at 30°C designed the study. HK was a major contributor in writing the manuscript. All for 6 days. (B) Strand‑specific RT ‑ qPCR for the ura4 marker gene. Values authors read and approved the final manuscript. are normalized to that of the sense strand of ribosomal 28S RNA, and are Okazaki et al. Epigenetics & Chromatin (2018) 11:26 Page 14 of 15 Author details 9. Kato H, Goto DB, Martienssen RA, Urano T, Furukawa K, Murakami Y. RNA Department of Biochemistry, Shimane University School of Medicine, polymerase II is required for RNAi‑ dependent heterochromatin assembly. 89‑1 Enya‑cho, Izumo, Shimane 693‑8501, Japan. Division of Cytogenetics, Science. 2005;309:467–9. National Institute of Genetics, Mishima, 1111 Yata, Mishima 411‑8540, Japan. 10. Kawakami K, Hayashi A, Nakayama J, Murakami Y. A novel RNAi protein, Proteomics Support Unit, RIKEN Center for Developmental Biology, Kobe, Dsh1, assembles RNAi machinery on chromatin to amplify heterochro‑ Hyogo 650‑0047, Japan. Division of Chromatin Regulation, National Institute matic siRNA. Genes Dev. 2012;26:1811–24. for Basic Biology, Okazaki, Aichi 444‑8585, Japan. Department of Chemistry, 11. Oya E, Kato H, Chikashige Y, Tsutsumi C, Hiraoka Y, Murakami Y. Faculty of Science, Hokkaido University, Sapporo, Hokkaido 060‑0810, Japan. Mediator directs co‑transcriptional heterochromatin assembly by RNA Present Address: KNC Laboratories Co. Ltd., Kobe, Hyogo 651‑2271, Japan. interference‑ dependent and ‑independent pathways. PLoS Genet. Present Address: Laboratory for Genome Regeneration, Institute for Quanti‑ 2013;9:e1003677. tative Biosciences, The University of Tokyo, Bunkyo‑ku, Tokyo 113‑0032, Japan. 12. Buker SM, Iida T, Buhler M, Villen J, Gygi SP, Nakayama J, Moazed D. Present Address: National Cerebral and Cardiovascular Center, Suita, Osaka Two different Argonaute complexes are required for siRNA generation 565‑8565, Japan. and heterochromatin assembly in fission yeast. Nat Struct Mol Biol. 2007;14:200–7. Acknowledgements 13. Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, Grewal SI, Moazed D. RNAi‑ We thank Dr. R. C. Allshire for providing strains and Dr. K. Kawakami for gener‑ mediated targeting of heterochromatin by the RITS complex. Science. ating yeast strains, and we would like to acknowledge the technical expertise 2004;303:672–6. of the Interdisciplinary Center for Science Research, Organization for Research 14. Ishida M, Shimojo H, Hayashi A, Kawaguchi R, Ohtani Y, Uegaki K, and Academic Information, Shimane University. Nishimura Y, Nakayama J. Intrinsic nucleic acid‑binding activity of Chp1 chromodomain is required for heterochromatic gene silencing. Mol Cell. Competing interests 2012;47:228–41. The authors declare that they have no competing interests. 15. Petrie VJ, Wuitschick JD, Givens CD, Kosinski AM, Partridge JF. RNA interfer‑ ence (RNAi)‑ dependent and RNAi‑independent association of the Chp1 Availability of data and materials chromodomain protein with distinct heterochromatic loci in fission yeast. The datasets used and analyzed during the current study are available from Mol Cell Biol. 2005;25:2331–46. the corresponding author on reasonable request. 16. Partridge JF, DeBeauchamp JL, Kosinski AM, Ulrich DL, Hadler MJ, Noffsinger VJ. Functional separation of the requirements for establish‑ Consent for publication ment and maintenance of centromeric heterochromatin. Mol Cell. Not applicable. 2007;26:593–602. 17. Sugiyama T, Cam H, Verdel A, Moazed D, Grewal SI. RNA‑ dependent RNA Ethics approval and consent to participate polymerase is an essential component of a self‑ enforcing loop coupling Not applicable. heterochromatin assembly to siRNA production. Proc Natl Acad Sci USA. 2005;102:152–7. Funding 18. Motamedi MR, Verdel A, Colmenares SU, Gerber SA, Gygi SP, Moazed D. This study was supported by JSPS KAKENHI Grant Number JP26116513 (to Two RNAi complexes, RITS and RDRC, physically interact and localize to HK), and by a Grant‑in‑Aid for JSPS Research Fellow (to KO, 13J07740). noncoding centromeric RNAs. Cell. 2004;119:789–802. 19. 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Epigenetics & Chromatin – Springer Journals
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