Author response:
The following is the authors’ response to the original reviews.
Reviewer #1(Public review):
Summary:
In this study, the authors aim to understand how Rhino, a chromatin protein essential for small RNA production in fruit flies, is initially recruited to specific regions of the genome. They propose that asymmetric arginine methylation of histones, particularly mediated by the enzyme DART4, plays a key role in defining the first genomic sites of Rhino localization. Using a combination of inducible expression systems, chromatin immunoprecipitation, and genetic knockdowns, the authors identify a new class of Rhinobound loci, termed DART4 clusters, that may represent nascent or transitional piRNA clusters.
Strengths:
One of the main strengths of this work lies in its comprehensive use of genomic data to reveal a correlation between ADMA histones and Rhino enrichment at the border of known piRNA clusters. The use of both cultured cells and ovaries adds robustness to this observation. The knockdown of DART4 supports a role for H3R17me2a in shaping Rhino binding at a subset of genomic regions.
Weaknesses:
However, Rhino binding at, and piRNA production from, canonical piRNA clusters appears largely unaffected by DART4 depletion, and spreading of Rhino from ADMArich boundaries was not directly demonstrated. Therefore, while the correlation is clearly documented, further investigation would be needed to determine the functional requirement of these histone marks in piRNA cluster specification.
The study identify piRNA cluster-like regions called DART4 clusters. While the model proposes that DART4 clusters represent evolutionary precursors of mature piRNA clusters, the functional output of these clusters remains limited. Additional experiments could help clarify whether low-level piRNA production from these loci is sufficient to guide Piwi-dependent silencing.
In summary, the authors present a well-executed study that raises intriguing hypotheses about the early chromatin context of piRNA cluster formation. The work will be of interest to researchers studying genome regulation, small RNA pathways, and the chromatin mechanisms of transposon control. It provides useful resources and new candidate loci for follow-up studies, while also highlighting the need for further functional validation to fully support the proposed model.
We sincerely thank Reviewer #1 for the thoughtful and constructive summary of our work. We appreciate the reviewer’s recognition that our study provides a comprehensive analysis of the relationship between ADMA-histones and Rhino localization, and that it raises intriguing hypotheses about the early chromatin context of piRNA cluster formation.
We fully agree with the reviewer that our data primarily demonstrate correlation between ADMA-histones and Rhino localization, rather than direct causation. In response, we have carefully revised the text throughout the manuscript to avoid overstatements implying causality (details provided below).
We also acknowledge the reviewer’s important point that the functional requirement of ADMA-histones for piRNA clusters specification remains to be further established. We have now added the discussion about our experimental limitations (page 18).
Overall, we have revised the manuscript to present our findings more cautiously and transparently, emphasizing that our data reveal a correlation between ADMA-histone marks and the initial localization of Rhino, rather than proving a direct mechanistic requirement. We thank the reviewer again for highlighting these important distinctions.
Reviewer #2 (Public review):
This study seeks to understand how the Rhino factor knows how to localize to specific transposon loci and to specific piRNA clusters to direct the correct formation of specialized heterochromatin that promotes piRNA biogenesis in the fly germline. In particular, these dual-strand piRNA clusters with names like 42AB, 38C, 80F, and 102F generate the bulk of ovarian piRNAs in the nurse cells of the fly ovary, but the evolutionary significance of these dual-strand piRNA clusters remains mysterious since triple null mutants of these dual-strand piRNA clusters still allows fly ovaries to develop and remain fertile. Nevertheless, mutants of Rhino and its interactors Deadlock, Cutoff, Kipferl and Moonshiner, etc, causes more piRNA loss beyond these dual-strand clusters and exhibit the phenotype of major female infertility, so the impact of proper assembly of Rhino, the RDC, Kipferl etc onto proper piRNA chromatin is an important and interesting biological question that is not fully understood.
This study tries to first test ectopic expression of Rhino via engineering a Dox-inducible Rhino transgene in the OSC line that only expresses the primary Piwi pathway that reflects the natural single pathway expression the follicle cells and is quite distinct from the nurse cell germline piRNA pathway that is promoted by Rhino, Moonshiner, etc. The authors present some compelling evidence that this ectopic Rhino expression in OSCs may reveal how Rhino can initiate de novo binding via ADMA histone marks, a feat that would be much more challenging to demonstrate in the germline where this epigenetic naïve state cannot be modeled since germ cell collapse would likely ensue. In the OSC, the authors have tested the knockdown of four of the 11 known Drosophila PRMTs (DARTs), and comparing to ectopic Rhino foci that they observe in HP1a knockdown (KD), they conclude DART1 and DART4 are the prime factors to study further in looking for disruption of ADMA histone marks. The authors also test KD of DART8 and CG17726 in OSCs, but in the fly, the authors only test Germ Line KD of DART4 only, they do not explain why these other DARTs are not tested in GLKD, the UAS-RNAi resources in Drosophila strain repositories should be very complete and have reagents for these knockdowns to be accessible.
The authors only characterize some particular ADMA marks of H3R17me2a as showing strong decrease after DART4 GLKD, and then they see some small subset of piRNA clusters go down in piRNA production as shown in Figure 6B and Figure 6F and Supplementary Figure 7. This small subset of DART4-dependent piRNA clusters does lose Rhino and Kipferl recruitment, which is an interesting result.
However, the biggest issue with this study is the mystery that the set of the most prominent dual-strand piRNA clusters. 42AB, 38C, 80F, and 102F, are the prime genomic loci subjected to Rhino regulation, and they do not show any change in piRNA production in the GLKD of DART4. The authors bury this surprising negative result in Supplementary Figure 5E, but this is also evident in no decrease (actually an n.s. increase) in Rhino association in Figure 5D. Since these main piRNA clusters involve the RDC, Kipferl, Moonshiner, etc, and it does not change in ADMA status and piRNA loss after DART4 GLKD, this poses a problem with the model in Figure 7C. In this study, there is only a GLKD of DART4 and no GLKD of the other DARTs in fly ovaries.
One way the authors rationalize this peculiar exception is the argument that DART4 is only acting on evolutionarily "young" piRNA clusters like the bx, CG14629, and CG31612, but the lack of any change on the majority of other piRNA clusters in Figure 6F leaves upon the unsatisfying concern that there is much functional redundancy remaining with other DARTs not being tested by GLKD in the fly that would have a bigger impact on the other main dual-strand piRNA clusters being regulated by Rhino and ADMA-histone marks.
Also, the current data does not provide convincing enough support for the model Figure 7C and the paper title of ADMA-histones being the key determinant in the fly ovary for Rhino recognition of the dual-strand piRNA clusters. Although much of this study's data is well constructed and presented, there remains a large gap that no other DARTs were tested in GLKD that would show a big loss of piRNAs from the main dual-strand piRNA clusters of 42AB, 38C, 80F, and 102F, where Rhino has prominent spreading in these regions.
As the manuscript currently stands, I do not think the authors present enough data to conclude that "ADMA-histones [As a Major new histone mark class] does play a crucial role in the initial recognition of dual-strand piRNA cluster regions by Rhino" because the data here mainly just show a small subset of evolutionarily young piRNA clusters have a strong effect from GLKD of DART4. The authors could extensively revise the study to be much more specific in the title and conclusion that they have uncovered this very unique niche of a small subset of DART4-dependent piRNA clusters, but this niche finding may dampen the impact and significance of this study since other major dual-strand piRNA clusters do not change during DART4 GLKD, and the authors do not show data GLKD of any other DARTs. The niche finding of just a small subset of DART-4-dependent piRNA clusters might make another specialized genetics forum a more appropriate venue.
We are deeply grateful to Reviewer #2 for the detailed and insightful review that carefully situates our study in the broader context of Rhino-mediated piRNA cluster regulation. We appreciate the reviewer’s recognition that our inducible Rhino expression system in OSCs provides a valuable model to explore de novo Rhino recruitment under a simplified chromatin environment.
At the same time, we agree that the current data mainly support a role for DART4 in regulating a subset of evolutionarily young piRNA clusters, and do not demonstrate a requirement for ADMA-histones at the major dual-strand piRNA clusters such as 42AB or 38C. We have therefore revised the title and main conclusions to more accurately reflect the scope of our findings.
We agree with the reviewer that functional redundancy among DARTs may explain why major dual-strand piRNA clusters are unaffected by DART4 GLKD. Indeed, we have tried DART1 GLKD in the germline, which shows collapse of Rhino foci in OSCs.For DART1 GLKD, two approaches were possible:
(1) Crossing the BDSC UAS-RNAi line (ID: 36891) with nos-GAL4.
(2) Crossing the VDRC UAS-RNAi line (ID: 110391) with nos-GAL4 and UAS-Dcr2.
The first approach was not feasible because the UAS-RNAi line always arrived as dead on arrival (DOA) and could not be maintained in our laboratory. The second approach did not yield effective and stable knockdown (as follows).
DART8 and CG17726 did not alter Rhino foci in OSC knockdown experiments; therefore, we did not attempt germline knockdown (GLKD) of these DARTs in the ovary. We agree with the reviewer’s opinion that there are piRNA source loci where Rhino localization depends on DART1, and that simultaneous depletion of multiple DARTs may indeed reveal additional positive results because ADMA-histones such as H3R8me2a may be completely eliminated by the knockdown of multiple DARTs. At the same time, we note that many evolutionarily conserved piRNA clusters show a loss of ADMA accumulation compared with evolutionarily young piRNA clusters, with levels that are comparable to the background input in ChIP-seq reads. Therefore, conserved clusters such as 42AB and 38C may no longer be regulated by ADMA. Even if multiple DARTs function redundantly to regulate ADMA, it may be difficult to disrupt Rhino localization at such conserved piRNA clusters by depletion of DARTs. While disruption of Rhino localization at conserved clusters like 42AB and 38C may be challenging, we cannot exclude the possibility that DART depletion affects Rhino binding at less conserved piRNA clusters, where ADMA modification remains detectable. We added clarifications in the Discussion to acknowledge the potential redundancy with other DARTs and to note that further knockdown experiments in the germline will be necessary to test this model comprehensively (page 18).
We appreciate the reviewer’s critical feedback, which has helped us refine the message and strengthen the interpretative balance of the paper.
Reviewer #1 (Recommendations for the authors):
In multiple places, the link between ADMA histones and Rhino recruitment is presented in terms that imply causality. Please revise these statements to reflect that, in most cases, the evidence supports correlation rather than direct functional necessity. Similarly, statements suggesting that ADMA histones promote Rhino spreading should be revised unless supported by direct evidence.
We sincerely thank the reviewer for the insightful comments. We recognize that these suggestions are crucial for improving the manuscript, and we have revised it accordingly to address the concerns. The specific revisions we made are detailed below.
(1) Page 1, line 14: The original sentence “in establishing the sites” was changed to “may establish the potential sites.”
(2) Page 4, lines 11-12: The original sentence “genomic regions where Rhino binds at the ends and propagates in the areas in a DART4-dependent manner, but not stably anchored” was changed to “genomic regions that have ADMA-histones at their ends and exhibit broad Rhino spreading across their internal regions in a DART4dependent manner”
(3) Page4, lines 12-15: The original sentence “Kipferl is present at the regions but not sufficient to stabilize Rhino-genomic binding after Rhino propagates.” was changed to “In contrast to authentic piRNA clusters, Kipferl was lost together with Rhino upon DART4 depletion in these regions, suggesting that Kipferl by itself is not sufficient to stabilize Rhino binding; rather, their localization depends on DART4.”
(4) Page4, lines17-18: The original sentence “are considered to be primitive clusters” was changed to “might be nascent dual-strand piRNA source loci”.
(5) Page 8, line 7: The original sentence “Involvement of ADMA-histones in the genomic localization of Rhino was implicated.” was changed to “Correlation of ADMA-histones in the genomic localization of Rhino was implicated.”
(6) Page 8, lines 19-21: The original sentence “These results suggest that ADMAhistones, together with H3K9me3, contribute significantly and specifically to the recruitment of Rhino to the ends of dual-strand clusters in OSCs.” was changed to “These results raise the possibility that ADMA-histones, together with H3K9me3, may contribute specifically to the recruitment of Rhino to the ends of dual-strand clusters in OSCs.”
(7) Page 10, lines 11-13: The original sentence “These results suggest that DART1 and DART4 are involved in Rhino recruitment at distinct genomic sites through the decreases in ADMA-histones in each of their KD conditions (H4R3me2a and H3R17me2a, respectively).” was changed to ”These results suggest that DART1 and DART4 could contribute to Rhino recruitment at distinct genomic sites through the decreases in ADMA-histones in each of their KD conditions (H4R3me2a and H3R17me2a, respectively).”
(8) Page 13, line 2: The original sentence “Genomic regions where Rhino spreads in a DART4-dependent manner, but not stably anchored, produce some piRNAs“ was changed to “Genomic regions where Rhino binds broadly in a DART4-dependent manner, but not stably anchored, produce some piRNAs”
(9) Page 13, lines 21-22: The original sentence “These results support the hypothesis that ADMA-histones are involved in the genomic binding of Rhino both before and after Rhino spreading, resulting in stable genome binding.” was changed to “These results raise the possibility that a subset of Rhino localized to genomic regions correlating with ADMA-histones may serve as origins of spreading.”
(10) Page 16, lines 6-8: The original sentence “In this study, we took advantage of cultured OSCs for our analysis and found that chromatin marks (i.e., ADMA-histones) play a crucial role in the loading of Rhino onto the genome.” was changed to “In this study, we took advantage of cultured OSCs for our analysis and found that chromatin marks (i.e., bivalent nucleosomes containing H3K9me3 and ADMA-histones) appear to contribute to the initial loading of Rhino onto the genome.”
(11) Page16, line 12: The original sentence “We propose that the process of piRNA cluster formation begins with the initial loading of Rhino onto bivalent nucleosomes containing H3K9me3 and ADMA-histones (Fig. 7C). In OSCs, the absence of Kipferl and other necessary factors means that Rhino loading into the genome does not proceed to the next step.” was removed.
Major points
(1) Clarify the limited colocalization between Rhino and H3K9me3 in OSCs. The observation that FLAG-Rhino foci show minimal overlap with H3K9me3 in OSCs appears inconsistent with the proposed model by the authors in the discussion, in which Rhino is initially recruited to bivalent nucleosomes bearing both H3K9me3 and ADMA marks. This discrepancy should be addressed.
We thank the reviewer’s insightful comments. Indeed, ChIP-seq shows that Rhino partially overlaps with H3K9me3 (Fig. 1F), but immunofluorescence did not reveal any detectable overlap (Fig. 1A). We interpret this discrepancy as arising from the fact that immunofluorescence primarily visualizes H3K9me3 foci that are localized as broad domains in the genome, such as those at centromeres, pericentromeres, or telomeres (named chromocenters), whereas the sharp and interspersed H3K9me3 signals along chromosome arms are difficult to detect by immunofluorescence. We now have these explanations in the revised text (page 6).
(2) Please indicate whether the FLAG-Rhino used in OSCs has been tested for functionality in vivo-for example, by rescuing Rhino mutant phenotypes. This is particularly relevant given that no spreading is observed with this construct.
We thank the reviewer for raising this important point. We have not directly tested the functionality of FLAG-Rhino construct used in OSCs in living Drosophila fly; i.e., it has not been used to rescue Rhino mutant phenotypes in flies. We acknowledge that FLAGRhino has not previously been expressed in OSCs, and that its localization pattern in OSCs differs from that observed in ovaries, where Rhino is endogenously expressed. However, several lines of evidence suggest that the addition of the N-terminal FLAG tag is unlikely to compromise Rhino function
(1) In previous studies, N-terminally tagged Rhino (e.g., 3xFLAG-V5-Precision-GFPRhino) was expressed in a living Drosophila ovary and was shown to localize properly to piRNA clusters, indicating that the tag does not prevent Rhino from binding its genomic targets (Baumgartner et al., 2022; eLife. Fig. 3 supplement 1G).
(2) In Drosophila S2 cells, FLAG-tagged tandem Rhino chromodomains construct was shown to bind H3K9me3/H3K27me3 bivalent chromatin, demonstrating that the FLAG tag does not impair this fundamental chromatin interaction (Akkouche et al., 2025; Nat Struct Mol Biol. Fig. 4b).
(3) GFP-tagged Rhino has been demonstrated to rescue the transposon derepression phenotype of Rhino mutant flies, further supporting that the addition of tags does not abolish its in vivo function. (Parhad et al., 2017; Dev Cell. Fig.1D).
Therefore, we interpret the partial localization of FLAG-Rhino in OSCs as reflecting the specific chromatin environment and regulatory context of OSCs rather than functional impairment due to the FLAG tag.
(3) Given the low levels of piRNA production and the absence of measurable effects on transposon expression or fertility upon DART4 knockdown, the rationale for classifying these regions as piRNA clusters should be clearly stated. Additional experiments could help clarify whether low-level piRNA production from these loci is sufficient to guide Piwidependent silencing. The authors should also consider and discuss the possibility that some of these differences may reflect background-specific genomic variation rather than DART4-dependent regulation per see.
We thank the reviewer for the insightful comments. As noted, DART4 knockdown did not measurably affect transposon expression or fertility. piRNAs generated from DART4associated clusters associate with Piwi but are insufficient for target repression. Although loss of DART4 largely eliminated piRNAs from these clusters, the cluster-derived transcripts themselves were unchanged. To clarify this point, we now refer to these regions as DART4-dependent piRNA-source loci (DART4 piSLs) in the revised text. We also acknowledge that some observed differences may reflect strain-specific genomic variation and have added this caveat on page 16.
(4) The authors should describe the genomic context of DART4 clusters in more detail. Specifically, it would be helpful to indicate whether these regions overlap with known transposable elements, gene bodies, or intergenic regions, and to report the typical size range of the clusters. Are any of the piRNAs produced from these clusters predicted to target known transcripts?
We thank the reviewer’s insightful comments. The overlap of DART4 piSL with transposable elements, gene bodies, and intergenic regions is shown in the right panel of Supplementary Fig. 6E (denoted as “Rhino reduced regions in DART4 GLKD” in the figure). The typical size range of these clusters is presented in Supplementary Fig. 6G. The annotation of piRNA reads derived from these piSL is shown in the right panel of Supplementary Fig. 6F, indicating that most of them appear to target host genes. The specific genes and transposons matched by the piRNAs produced from DART4 piSL are listed in Supplementary Table 8.
(5) While correlations between Rhino and ADMA histone marks (especially H3R8me2a,H3R17me2a, H4R3me2a) are robust, many ADMA-enriched regions do not recruit Rhino. Please discuss this observation and consider the possible involvement of additional factors.
We thank the reviewer’s insightful comments. As pointed out, not all ADMA-enriched regions recruit Rhino; rather, Rhino is recruited only at sites where ADMAs overlap with H3K9me3. Furthermore, the combination of H3K9me3 and ADMAs alone does not fully account for the specificity of Rhino recruitment, suggesting the involvement of additional co-factors (for example, other ADMA marks such as H3R42me2a, or chromatininteracting proteins). In addition, since histone modifications—including arginine methylation—have the possibility that they are secondary consequences of modifications on other proteins rather than primary regulatory events, it is possible that DART1/4 contribute to Rhino recruitment not only through histone methylation but also via arginine methylation of non-histone chromatin-interacting factors. However, methylation of HP1a does not appear to be involved (Supplementary Fig. 3G). We have added new sentences about these points in the Discussion section (page 18).
(6) The manuscript states that Kipferl is present at DART4 clusters but does not stabilize Rhino binding. Please specify which experimental results support this conclusion and explain.
We apologize for the lack of clarity regarding Kipferl data. Supplementary Fig. 7A and 7B show that Kipferl localizes at major DART4 piSL. This Kipferl localization is lost together with Rhino upon DART4 GLKD, indicating that Rhino localization at DART4 piSL depends on DART4 rather than on Kipferl. From these results, we infer that, unlike at authentic piRNA clusters, Kipferl may not be sufficient to stabilize the association of Rhino with the genome at DART4 piSL. We have added this interpretation on page 14.
Minor points
(1) Figure 1D: Please specify which piRNA clusters are included in the metaplot - all clusters, or only the major producers?
We thank the reviewer for the question. The metaplot was not generated from a predefined list of “all” piRNA clusters or only the “major producers.” Instead, it was constructed from Rhino ChIP–seq peaks (“Rhino domains”) that are ≥1.5 kb in length.These Rhino domains mainly correspond to the subregions within major dual-strand clusters (e.g., 42AB, 38C) as well as additional clusters such as 80F, 102F, and eyeless, among others. We have provided the full list of domains and their corresponding piRNA clusters (with genomic coordinates) in Supplementary Table 9 and added the additional explanation in Fig. 1d legend.
(2) Supplemental Figure 5E is referred to as 5D in the main text.
We corrected the figure citations on pages 11-12: the reference to Supplementary Fig. 5E has been changed to 5D, and the reference to Supplementary Fig. 5F has been changed to 5E.
(3) Supplemental Figure 7C: The color legend does not match the pie chart, which may confuse readers.
We thank the reviewer for the helpful comment. We are afraid we were not entirely sure what specific aspect of the legend was confusing, but to avoid any possible misunderstanding, we revised Supplemental Fig. 7C so that the color boxes in the legend now exactly match the corresponding colors in the pie chart. We hope this modification improves clarity.
(4) Since the manuscript focuses on the roles of DART1 and DART4, including their expression profiles in OSCs and ovaries would help contextualize the observed phenotypes. Please consider adding this information if available.
We thank the reviewer for the suggestion. We have now included a scatter plot comparing RNA-seq expression in OSCs and ovaries (Supplementary Fig. 3H). In these datasets, DART1 is strongly expressed in both tissues, whereas DART4 shows no detectable reads. Notably, ref. 28 reports strong expression of both DART1 and DART4 in ovaries by western blot and northern blot. In our own qPCR analysis in OSCs, DART4 expression is about 3% of DART1, which, although low, may still be sufficient for functional roles such as modification of H3R17me2a (Fig. 3C, Supplementary Fig. 3F and 3I). We have added these new data and additional explanation in the revised manuscript (page 11).
(5) Several of the genome browser snapshots, particularly scale and genome coordinates, are difficult to read.
We apologize for the difficulty in reading several of the genome browser snapshots in the original submission. We have re-generated the relevant figures using IGV, which provides clearer visualization of scale and genome coordinates. The previous images have been replaced with the improved versions in the revised manuscript.
Reviewer #2 (Recommendations for the authors):
(1) The authors need to elaborate on what this sentence means, as it is very unclear what they are describing about Rhino residency: "The results show that Rhino in OSCs tends to reside in the genome where Rhino binds locally in the ovary (Fig. 1C)."
We apologize for the lack of clarity in the original sentence. The text has been revised as follows:
”Rhino expressed in OSCs bound predominantly to genomic sites exhibiting sharp and interspersed Rhino localization patterns in the ovary, while showing little localization within broad Rhino domains, including major piRNA clusters.”
In addition, to clarify the behavior of Rhino at broad domains, we have added the phrase “the terminal regions of broad domains, such as major piRNA clusters” to the subsequent sentence.
(2) The red correlation line is very confusing in Figure 5F. What sort of line does this mean in this scatter plot?
We apologize for the lack of clarity regarding the red line in Fig. 5F. The red line represents the least-squares linear regression fit to the data points, calculated using the lm() function in R, and was added with abline() to illustrate the correlation between ctrl GLKD and DART4 GLKD values. In the revised figure, we have clarified this in the legend by specifying that it is a regression line.
(3) There is no confirmation of the successful knockdown of the various DARTs in the OSCs.
We thank the reviewer for the comment. The knockdown efficiency of the various DARTs in OSCs was confirmed by RT–qPCR. The data are now shown in Supplementary Fig. 3J.
(4) What is the purpose of an unnumbered "Method Figure" in the supplementary data file? Why not just give it a number and mention it properly in the text?
We thank the reviewer for the suggestion. We have now assigned a number to the previously unnumbered "Method Figure" and have included it as Supplementary Fig. 9.
The figure is now properly cited in the Methods section.
(5) For Figure 5A, those fly strain numbers in the labels are better reserved in the Methods, and a more appropriate label is to describe the GAL4 driver and the UAS-RNAi construct by their conventional names.
We thank the reviewer for the suggestion. The labels in Fig. 5A have been updated to use the conventional names of the GAL4 drivers and UAS-RNAi constructs. Specifically, they now read Ctrl GLKD (nos-GAL4 > UAS-emp) and DART4 GLKD (nos-GAL4 > UASDART4). The original fly strain numbers are listed in the Methods section.
