Author response:
In this manuscript, we have addressed one of the possible modes of recruitment of Swi6 to the putative heterochromatin loci.
Our investigation was guided by earlier work showing ability of HP1 a to bind to a class of RNAs and the role of this binding in recruitment of HP1a to heterochromatin loci in mouse cells (Muchardt et al). While there has been no clarity about the mechanism of Swi6 recruitment given the multiple pathways being involved, the issue is compounded by the overall lack of understanding as to how Swi6 recruitment occurs only at the repeat regions. At the same time, various observations suggested a causal role of RNAi in Swi6 recruitment.
Thus, guided by the work of Muchardt et al we developed a heuristic approach to explore a possibly direct link between Swi6 and heterochromatin through RNAi pathway. Interestingly, we found that the lysine triplet found in the hinge domain in HP1, which influences its recruitment to heterochromatin in mouse cells, is also present in the hinge domain of Swi6, although we were cautious, keeping in mind the findings of Keller et al showing another role of Swi6 in binding to RNAs and channeling them to the exosome pathway.
Accordingly, we envisaged that a mode of recruitment of Swi6 through binding to siRNAs to cognate sites in the dg-dh repeats shared among mating type, centromere and telomere loci could explain specific recruitment as well as inheritance following DNA replication. In accordance we framed the main questions as follows: i) Whether Swi6 binds specifically and with high affinity to the siRNAs and the cognate siRNA-DNA hybrids and whether the Swi63K-3A mutant is defective in this binding, ii) whether this lack of binding of Swi63K-3A affects its localization to heterochromatin, iii) whether the this specificity is validated by binding of Swi6 but not Swi63K-3A to siRNAs and siRNA-DNA hybrids in vivo and iv) whether the binding mode was qualitatively and quantitatively different from that of Cen100 RNA or random RNAs, like GFP RNA.
We think that our data provides answers to these lines of inquiry to support a model wherein the Swi6-siRNA mediated recruitment can explain a cis-controlled nucleation of heterochromatin at the cognate sites in the genome. We have also partially addressed the points raised by the study by Keller et al by invoking a dynamic balance between different modes of binding of Swi6 to different classes of RNA to exercise heterochromatin formation by Swi6 under normal conditions and RNA degradation under other conditions.
While we aver about our hypothesis, we do acknowledge the need for more detailed investigation both to buttress our hypothesis and address the dynamics of siRNA binding and recruitment of Swi6 and how Swi6 functions fit in the context of other components of heterochromatin assembly, like the HDACs and Clr4 on one hand and exosome pathway on the other. Our future studies will attempt to address these issues.
Public Reviews:
Reviewer #1 (Public review):
Summary:
This manuscript explores the RNA binding activities of the fission yeast Swi6 (HP1) protein and proposes a new role for Swi6 in RNAi-mediated heterochromatin establishment. The authors claim that Swi6 has a specific and high affinity for short interfering RNAs (siRNAs) and recruits the Clr4 (Suv39h) H3K9 methyltransferases to siRNA-DNA hybrids to initiate heterochromatin formation. These claims are not in any way supported by the incomplete and preliminary RNA binding or the in vivo experiments that the authors present. The proposed model also lacks any mechanistic basis as it remains unclear (and unexplored) how Swi6 might bind to specific small RNA sequences or RNA-DNA hybrids. Work by several other groups in the field has led to a model in which siRNAs produced by the RNAi pathway load onto the Ago1-containing RITS complex, which then binds to nascent transcripts at pericentromeric DNA repeats and recruits Clr4 to initiate heterochromatin formation. Swi6 facilitates this process by promoting the recruitment of the RNA-dependent RNA polymerase leading to siRNA amplification.
Weaknesses:
(1) a) The claims that Swi6 binds to specific small RNAs or to RNA-DNA hybrids are not supported by the evidence that the authors present. Their experiments do not rule out non-specific charged-based interactions.
We disagree. We have used synthetic siRNAs of 20-22 nt length to do EMSA assay, as mentioned in the manuscript. Further, we have sequenced the small RNAs obtained after RIP experiments to validate the enrichment of siRNA in Swi6 bound fraction as compared to the mutant Swi6-bound fraction. These results are internally consistent regardless of the mode of binding. In any case the binding occurs primarily through the chromodomain although it is influenced by the hinge domain (see below).
Furthermore, we have carried out EMSA experiments using Swi6 mutants carrying all three possible double mutations of the K residues in the KKK triplet and found that there was no difference in the binding pattern as compared to the wt Swi6: only the triple mutant “3K-3A” showed the effect. These results suggest that that the bdining is not completely dependent on the basic residues. These results will be included in the revised version.
We also have some preliminary data from SAXS study showing that the CD of wt Swi6 shows a change in its structure upon binding to the siRNA, while the “3K-3A” mutant of Swi6 has a compact, folded structure that occludes the binding site of Swi6 in the chromodomain.” We propose to mention this preliminary finding in the revised version as unpublished data.
b) Claims about different affinities of Swi6 for RNAs of different sizes are based on a comparison of KD values derived by the authors for a handful of S. pombe siRNAs with previous studies from the Buhler lab on Swi6 RNA binding. The authors need to compare binding affinities under identical conditions in their assays.
Thus, the EMSA data do suggest sequence specificity in binding of Swi6 to specific siRNA sequences (Figure S5) and implies specific residues in Swi6 being responsible for that. Thus, Identification of the residues in Swi6 involved in siRNA binding in the CD would definitely be interesting, as also the experimental confirmation of the consensus siRNA sequence. It may however be noted that as against the binding of Swi6 to siRNAs occurs through CD, that of Cen100 or GFP RNA was shown be through the hinge domain by Keller et al.
The estimation of Kd by the Buhler group was based on NMR study, which we are not in a position to perform in the near future. Nonetheless, we did carry out EMSA study using the ‘Cen100’ RNA, same as the one used by the Keller et al study. Surprisingly, in contrast with the result of EMSA in agarose gel showing binding of Swi6 to “Cen100” RNA as reported by Keller et al, we fail to observe any binding in EMSA done in acrylamide gel. (The same is true of the RevCen 100). While this raises issues of why the Keller et al chose to do EMSA in agarose gel instead of the conventional approach of using acrylamide gel, it does lend support to our claim of stronger binding of Swi6 to siRNAs. Another relevant observation of binding of Swi6 to the “RevCen” RNA precursor RNAs but a detectable binding to siRNAs denoted as VI-IX (as measured by competition experiments, that are derived from RevCen RNA; Figure S4 and S7), which are derived by Dcr1 cleavage of the ‘’RevCen’’ RNA.
We also disagree that we carried out EMSA with a small bunch of siRNAs. As indicated in Figure 1 and S1, we synthesized nearly 12 siRNAs representing the dg-dh repeats at Cen, mat and tel loci and measured their specificity of binding to Swi6 using EMSA assay by labeling the ones labelled “D”, “E” and “V” directly and those of the remaining ones by the latter’s ability to compete against the binding (Figure 1, S4). These results point to presence of a consensus sequence in siRNAs that shows highly specific and strong binding to Swi6 in the low micromolar range.
Further, our claim of binding of Swi6 and not Swi63K>3A to siRNA in vivo is validated by RIP experiments, as shown in Fig 2 and S9.
c) The regions of Swi6 that bind to siRNAs need to be identified and evidence must be provided that Swi6 binds to RNAs of a specific length, 20-22 mers, to support the claim that Swi6 binds to siRNAs. This is critical for all the subsequent experiments and claims in the study.
We have provided both in vitro data, which is va;idiated in vivo by RIP experiments, as mentioned above. However, we agree that it wpuld be very interesting to identify the residues in Swi6 chromdomain responsible for binding to siRNA. However, such an investigation is beyond the scope of the present study.
(2) a) The in vivo results do not validate Swi6 binding to specific RNAs, as stated by the authors. Swi6 pulldowns have been shown to be enriched for all heterochromatic proteins including the RITS complex. The sRNA binding observed by the authors is therefore likely to be mediated by Ago1/RITS.
We disagree with the first comment. Our RIP experiments do validate the in vitro results (Fig 1, 2, S4 and S9), as argued above. The observation alluded to by the reviewer “Swi6 pulldowns have been shown to be enriched for all heterochromatic proteins including the RITS complex” is not inconsistent with our observation; it is possible that the siRNA may be released from the RITS complex and transferred to Swi6, possibly due to its higher affinity.
Thus, we would like to suggest that the role of Swi6 is likely to be coincidental or subsequent to that of Ago1/RITS (see below). We think that the binding by Swi6 to the siRNA and siRNA-DNA hybrid and could be also carried out in cis at the level of siRNA-DNA hybrids.
This point needs to be addressed in future studies.
b) Most of the binding in Figure S8C seems to be non-specific.
We would like to point out that the result in Figure S8C needs to be examined together with the Figure S8B, which shows RNA bound by Swi6 but not Swi63K-3A to hybridize with dg, dh and dh-k probes.
c) In Figure S8D, the authors' data shows that Swi6 deletion does not derepress the rev dh transcript while dcr1 delete cells do, which is consistent with previous reports but does not relate to the authors' conclusions.
The purpose of results shown in Figure S8D is just to compare the results of Swi6 with that of Swi63K-3A.
d) Previous results have shown that swi6 delete cells have 20-fold fewer dg and dh siRNAs than swi6+ cells due to decreased RNA-dependent RNA polymerase complex recruitment and reduced siRNA amplification.
This result is consistent with our results invoking a role of Swi6 in binding to, protecting and recruiting siRNAs to homologous sites.
To find if the overall production of siRNA is compromised in swi6 3K->3A mutant, we i) calculated the RIP-Seq read counts for swi6 3K->3A , swi6+ and vector control in 200 bp genomic bins , ii) divided the Swi6 3K->3A and swi6+ signals by that of control, iii) removed the background using the criteria of signal value < 25% of max signal, and iv) counted the total reads (in excess to control) in all peak regions in both samples. This revealed a total count of 10878 and 8994 respectively for Swi6 3K->3A and swi6+ samples, possibly implying that the overall siRNA production is not compromised in the Swi6 3K->3A mutant.
(3) a) The RIP-seq data are difficult to interpret as presented. The size distribution of bound small RNAs, and where they map along the genome should be shown as for example presented in previous Ago1 sRNA-seq experiments.
Please see the response to 2(d).
b) It is also unclear whether the defects in sRNA binding observed by the authors represent direct sRNA binding to Swi6 or co-precipitation of Ago1-bound sRNAs.
The correspondence between our in vivo and in vitro results suggests that the binding to Swi6 would be direct. We do not observe a complete correspondence between the Swi6- and Ago-bound siRNAs. We think Swi6 binding may be coincident with or following RITS complex formation.
This point will be discussed in the Revision.
The authors should also sequence total sRNAs to test whether Swi6-3A affects sRNA synthesis, as is the case in swi6 delete cells.
Please see response to 2(d) above.
(4) The authors examine the effects of Swi6-3A mutant by overexpression from the strong nmt1 promoter. Heterochromatin formation is sensitive to the dosage of Swi6. These experiments should be performed by introducing the 3A mutations at the endogenous Swi6 locus and effects on Swi6 protein levels should be tested.
Although we agree, we think that the heterochromatin formation is occurring in presence of nmt1-driven Swi6 but not Swi63K>3A, as indicated by the phenotype and Swi6 enrichment at otr1R::ade6, imr1::ura4 and his3-telo (Figure 3) and mating type (Fig. S10). Furthermore, the both GFP-Swi6 and GFPSwi63K>3A are expressed at similar level (Fig. S8A).
(5) The authors' data indicate an impairment of silencing in Swi6-3A mutant cells but whether this is due to a general lower affinity for nucleosomes, DNA, RNA, or as claimed by the authors, siRNAs is unclear. These experiments are consistent with previous findings suggesting an important role for basic residues in the HP1 hinge region in gene silencing but do not reveal how the hinge region enhances silencing.
Our study aims to correlate the binding of Swi6 but not Swi63K-3A to siRNA with its localization to heterochromatin. A similar difference in binding of Swi6 but not Swi63K-3A to siRNA-DNA hybrid, together with sensitivity of silencing and Swi6 localization to heterochromatin to RNaseH support the above correlations as being causally connected.
In terms of mechanism of binding, we need to clarify that the primary mode of binding is through the CD and not the hinge domain, although the hinge domain does influence this binding. This result is different from those of Keller et al.
We have some structural data based on preliminary SAXS experiment supporting binding of siRNA to the CD and influence of the hinge domain on this binding. However, this line of investigation need to be extended and will be subject of future investigations.
(6) RNase H1 overexpression may affect Swi6 localization and silencing indirectly as it would lead to a general reduction in R loops and RNA-DNA hybrids across the genome. RNaseH1 OE may also release chromatin-bound RNAs that act as scaffolds for siRNA-Ag1/RITS complexes that recruit Clr4 and ultimately Swi6.
These are formal possibilities. However, the correlation between swi6 binding to siRNA-DNA hybrid and delocalization upon RNase H1 treatment argues for a more direct link.
(7) Examples of inaccurate presentation of the literature.
a) The authors state that "RNA binding by the murine HP1 through its hinge domains is required for heterochromatin assembly (Muchardt et al, 2002). The cited reference provides no evidence that HP1 RNA binding is required for heterochromatin assembly. Only the hinge region of bacterially produced HP1 contributes to its localization to DAPI-stained heterochromatic regions in fixed NIH 3T3 cells.
Noted. Statement will be corrected.
b) "... This scenario is consistent with the loss of heterochromatin recruitment of Swi6 as well as siRNA generation in rnai mutants (Volpe et al, 2002)." Volpe et al. did not examine changes in siRNA levels in swi6 mutant cells. In fact, no siRNA analysis of any kind was reported in Volpe et al., 2002.
Correct. We only say that Swi6 recruitment is reduced in rnai mutants and correlate it with ability of SWi6 to bind to siRNA generated by RNAi and subsequently to siRNA-DNA hybrid.
Reviewer #2 (Public review):
The aim of this study is to investigate the role of Swi6 binding to RNA in heterochromatin assembly in fission yeast. Using in vitro protein-RNA binding assays (EMSA) they showed that Swi6/HP1 binds centromere-derived siRNA (identified by Reinhardt and Bartel in 2002) via the chromodomain and hinge domains. They demonstrate that this binding is regulated by a lysine triplet in the conserved region of the Swi6 hinge domain and that wild-type Swi6 favours binding to DNA-RNA hybrids and siRNA, which then facilitates, rather than competes with, binding to H3K9me2 and to a lesser extent H3K9me3.
However, the majority of the experiments are carried out in swi6 null cells overexpressing wild-type Swi6 or Swi63K-3A mutant from a very strong promoter (nmt1). Both swi6 null cells and overexpression of Swi6 are well known to exhibit phenotypes, some of which interfere with heterochromatin assembly. This is not made clear in the text.
We think that the argument is not valid as we show that swi6 but not Swi63K-3A could restore silencing at imr1::ura4, otr1::ade6 and his3-telo (Fig 3) and mating type (Fig. S10), when transformed into a swi6D strain.
Whilst the RNA binding experiments show that Swi6 can indeed bind RNA and that binding is decreased by Swi63K-3A mutation in vitro (confusingly, they only much later in the text explained that these 3 bands represent differential binding and that II is likely an isotherm). The gels showing these data are of poor quality and it is unclear which bands are used to calculate the Kd.
We disagree with the comment about the quality of EMSA data. We think it is of similar quality or better than that of Keller et al, except in some cases, like Fig 1D, a shorter exposure shown to distinguish the slowest shifted band has caused the remaining bands to look fainter.
RNA-seq data shows that overall fewer siRNAs are produced from regions of heterochromatin in the Swi63K-3A mutant so it is unsurprising that analysis of siRNA-associated motifs also shows lower enrichment (or indeed that they share some similarities, given that they originate from repeat regions).
Please see response to comment 2(d) of the first reviewer above.
It is not clear which bands are being alluded to. However, we‘ll rectify any gaps in information in the revision.
The experiments are seemingly linked yet fail to substantiate their overall conclusions. For instance, the authors show that the Swi63K-3A mutant displays reduced siRNA binding in vitro (Figure 1D) and that H3K9me2 levels at heterochromatin loci are reduced in vivo (Figure 3C-D). They conclude that Swi6 siRNA binding is important for Swi6 heterochromatin localization, whilst it remains entirely possible that heterochromatin integrity is impaired by the Swi63K-3A mutation and hence fewer siRNAs are produced and available to bind. Their interpretation of the data is really confusing.
Our argument is that the lack of binding by Swi63K>3A to siRNA can explain the loss of recruitment to heterochromatin loci and thus affect the integrity of heterochroamtin; the recruitment of Swi6 can occur possibly by binding initially to siRNA and thereafter as siRNA-DNA hybrid. However, the overall level of siRNAs is not affected, as in 2(D) above. This interpretation is supported by results of ChIP assay and confocal experiments, as also by the effect of RNaseH1 in the recruitment of Swi6.
The authors go on to show that Swi63K-3A cells have impaired silencing at all regions tested and the mutant protein itself has less association with regions of heterochromatin. They perform DNA-RNA hybrid IPs and show that Swi63K-3A cells which also overexpress RNAseH/rnh1 have reduced levels of dh DNA-RNA hybrids than wild-type Swi6 cells. They interpret this to mean that Swi6 binds and protects DNA-RNA hybrids, presumably to facilitate binding to H3K9me2. The final piece of data is an EMSA assay showing that "high-affinity binding of Swi6 to a dg-dh specific RNA/DNA hybrid facilitates the binding to Me2-K9-H3 rather than competing against it." This EMSA gel shown is of very poor quality, and this casts doubt on their overall conclusion.
We do agree with the reviewer about the quality of EMSA (Fig. 5B). However, as may be noticed in the EMSA for siRNA-DNA hybrid binding (Fig 4A), the bands of Swi6-bound siRNA-DNA hybrid are extremely retarded. Hence the EMSA for subsequent binding by H3-K9-Me peptides required a longer electrophoretic run, which led to reduction in the sharpness of the bands. Nevertheless, the data does indicate binding efficiency in the order H3K9-Me2> H3-K9-Me3 > H3-K9-Me0. Having said that, we plan to repeat the EMSA or address the question by other methods, like SPR.
Unfortunately, the manuscript is generally poorly written and difficult to comprehend. The experimental setups and interpretations of the data are not fully explained, or, are explained in the wrong order leading to a lack of clarity. An example of this is the reasoning behind the use of the cid14 mutant which is not explained until the discussion of Figure 5C, but it is utilised at the outset in Figure 5A.
We tend to agree somewhat and will attempt to submit a revised version with greater clarity, as also the explanation of experiment with cid14D strain.
Another example of this lack of clarity/confusion is that the abstract states "Here we provide evidence in support of RNAi-independent recruitment of Swi6". Yet it then states "We show that...Swi6/HP1 displays a hierarchy of increasing binding affinity through its chromodomain to the siRNAs corresponding to specific dg-dh repeats, and even stronger binding to the cognate siRNA-DNA hybrids than to the siRNA precursors or general RNAs." RNAi is required to produce siRNAs, so their message is very unclear. Moreover, an entire section is titled "Heterochromatin recruitment of Swi6-HP1 depends on siRNA generation" so what is the author's message?
The reviewer has correctly pointed out the error. Indeed, our results actually indicate an RNAi-dependent rather than independent mode of recruitment. Rather, we would like to suggest an H3-K9-Me2-indpendnet recruitment of Swi6. We will rectify this error in our revised manuscript.
The data presented, whilst sound in some parts is generally overinterpreted and does not fully support the author's confusing conclusions. The authors essentially characterise an overexpressed Swi6 mutant protein with a few other experiments on the side, that do not entirely support their conclusions. They make the point several times that the KD for their binding experiments is far higher than that previously reported (Keller et al Mol Cell 2012) but unfortunately the data provided here are of an inferior quality and thus their conclusions are neither fully supported nor convincing.
We have used the method of Heffler et al (2012) to compute the Kd from EMSA data.
