Essential function of transmembrane transcription factor MYRF in promoting transcription of miRNA lin-4 during C. elegans development
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eLife assessment
The microRNA lin-4, originally discovered in C. elegans, has a key role in controlling developmental timing across species, but how its expression is developmentally regulated is poorly understood. Here, the authors provide convincing evidence that two MYRF transcription factors are essential positive regulators of lin-4 during early C. elegans larval development. These results provide important insight into the molecular control of developmental timing that could have significant implications for understanding these processes in more complex systems.
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Abstract
Precise developmental timing control is essential for organism formation and function, but its mechanisms are unclear. In C. elegans , the microRNA lin-4 critically regulates developmental timing by post-transcriptionally downregulating the larval-stage-fate controller LIN-14. However, the mechanisms triggering the activation of lin-4 expression toward the end of the first larval stage remain unknown. We demonstrate that the transmembrane transcription factor MYRF-1 is necessary for lin-4 activation. MYRF-1 is initially localized on the cell membrane, and its increased cleavage and nuclear accumulation coincide with lin-4 expression timing. MYRF-1 regulates lin-4 expression cell-autonomously and hyperactive MYRF-1 can prematurely drive lin-4 expression in embryos and young first-stage larvae. The tandem lin-4 promoter DNA recruits MYRF-1 GFP to form visible loci in the nucleus, suggesting that MYRF-1 directly binds to the lin-4 promoter. Our findings identify a crucial link in understanding developmental timing regulation and establish MYRF-1 as a key regulator of lin-4 expression.
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Author Response
The following is the authors’ response to the previous reviews.
Thank you for your continued review and for providing insightful suggestions. Below, I share some unpublished new findings related to the MYRF ChIP, comment on the potential interplay between myrf-1 and myrf-2, and describe the modifications we've implemented to address the reviewers' comments.
(1) MYRF-1 ChIP
Our collaboration with the modERN (Model Organism Encyclopedia of Regulatory Networks) project has recently yielded MYRF ChIP data. The results demonstrate clear and consistent MYRF binding across samples, notably on the lin-4 promoter. Given the significant detail and extensive description required to adequately present these findings, we have decided it is impractical to include them in the current paper. These results will be more suitably published …
Author Response
The following is the authors’ response to the previous reviews.
Thank you for your continued review and for providing insightful suggestions. Below, I share some unpublished new findings related to the MYRF ChIP, comment on the potential interplay between myrf-1 and myrf-2, and describe the modifications we've implemented to address the reviewers' comments.
(1) MYRF-1 ChIP
Our collaboration with the modERN (Model Organism Encyclopedia of Regulatory Networks) project has recently yielded MYRF ChIP data. The results demonstrate clear and consistent MYRF binding across samples, notably on the lin-4 promoter. Given the significant detail and extensive description required to adequately present these findings, we have decided it is impractical to include them in the current paper. These results will be more suitably published in a separate ongoing study focused on MYRF's regulatory targets during larval development.
(2) Inter-regulation between myrf-1 and myrf-2
We acknowledge the interpretation that myrf-2 may act as a genetic antagonist to myrf-1, as suggested by the delayed arrest in myrf-1; myrf-2 double mutants and a trend towards increased lin-4 expression in myrf-2 mutants. Additionally, our unpublished data suggest an elevated myrf-2 expression peak in myrf-1 null mutants during the L1-L2 transition, indicating a potential mutual repressive interaction between myrf1 and myrf-2.
On the other hand, myrf-1 and myrf-2 exhibit functional redundancy in DD synaptic rewiring and lin-4 expression. A gain of function in myrf-2 promotes early DD synaptic rewiring. Furthermore, three independent co-immunoprecipitation analyses targeting myrf-1::gfp, myrf-2::gfp, and pan-1::gfp confirm a tight association between myrf-1 and myrf-2 in vivo. These findings challenge the notion of myrf-2 primarily antagonizing myrf-1, or vice versa.
We propose a model where myrf-1 and myrf-2 collaborate and are functionally redundant, with compensatory elevated expression when one paralog is absent. For instance, the loss of myrf-1 triggers upregulation of myrf-2, which, though insufficient on its own, accelerates the transcriptional program and exacerbates system deterioration, leading to accelerated death. How exactly this takes place is currently unclear. We notice the MYRF binding on both myrf-1 and myrf-2 genes in MYRF-ChIP.
Given the complexity of these interactions, we have chosen not to delve deeply into this discussion in the paper without more direct evidence, which would require detailed analysis.
(3) Revisions Addressing Reviewer Suggestions
(a) We have revised our interpretation of the mScarlet signal changes in myrf-1(ybq6) and myrf-2(ybq42) mutants to reflect a more nuanced understanding of their potential genetic relationship, as highlighted in the main text.
“The mScarlet signals exhibit a marked reduction in the putative null mutant myrf-1(ybq6) (Figure 1D, E). Intriguingly, in the putative null myrf-2(ybq42) mutants, there is a noticeable trend towards increased mScarlet signals, although this increase does not reach statistical significance (Figure 2C, D).”
(b) In response to feedback on Figure 2 and the characterization of lin-4(umn84) mutants, we've included a new series of images showing lin-4(umn84)/+ and lin-4(umn84) signals through larval stages, presented as Figure 2 Figure Supplement 2. This addition clarifies the functional status of lin-4 nulls in our study.
“Our observations revealed that mScarlet signals were not detected early L1 larvae (Figure 2C-F; Figure 2 Figure Supplement 2).”
(c) To improve the clarity of Fig 6, we've added indicator arrows in the red, green, and merge channels, enhancing the visualization of the signals.
We appreciate the opportunity to clarify these points and hope that our revisions and additional data address the concerns raised.
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eLife assessment
The microRNA lin-4, originally discovered in C. elegans, has a key role in controlling developmental timing across species, but how its expression is developmentally regulated is poorly understood. Here, the authors provide convincing evidence that two MYRF transcription factors are essential positive regulators of lin-4 during early C. elegans larval development. These results provide important insight into the molecular control of developmental timing that could have significant implications for understanding these processes in more complex systems.
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Reviewer #1 (Public Review):
In this work, the authors set out to ask whether the MYRF family of transcription factors, represented by myrf-1 and myrf-2 in C. elegans, have a role in the temporally controlled expression of the miRNA lin-4. The precisely timed onset of lin-4 expression in the late L1 stage is known to be a critical step in the developmental timing ("heterochronic") pathway, allowing worms to move from the L1 to the L2 stage of development. Despite the importance of this step of the pathway, the mechanisms that control the onset of lin-4 expression are not well understood.
Overall, the paper provides convincing evidence that MYRF factors have a key role in promoting lin-4 expression in young larvae. Using state-of-the-art techniques (knock-in reporters and conditional alleles), the authors show that MYRF factors are …
Reviewer #1 (Public Review):
In this work, the authors set out to ask whether the MYRF family of transcription factors, represented by myrf-1 and myrf-2 in C. elegans, have a role in the temporally controlled expression of the miRNA lin-4. The precisely timed onset of lin-4 expression in the late L1 stage is known to be a critical step in the developmental timing ("heterochronic") pathway, allowing worms to move from the L1 to the L2 stage of development. Despite the importance of this step of the pathway, the mechanisms that control the onset of lin-4 expression are not well understood.
Overall, the paper provides convincing evidence that MYRF factors have a key role in promoting lin-4 expression in young larvae. Using state-of-the-art techniques (knock-in reporters and conditional alleles), the authors show that MYRF factors are essential for lin-4 activation and act cell-autonomously. Results using some unusual gain-of-function alleles are supported by consistent results using other approaches. The authors also provide evidence supporting the idea that MYRF factors activate lin-4 by directly activating its promoter. Because these results are indirect test of this, further experiments will be necessary to conclusively determine whether lin-4 is indeed a direct target of MYRF factors. myrf-1 and myrf-2 likely function redundantly to activate lin-4; potential complex interactions between these two genes will be an interesting area for future work.
Overall, the paper's results are convincing. The important findings on miRNA regulation and the control of developmental timing will make this work of interest to a broad range of developmental biologists.
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Reviewer #2 (Public Review):
Summary:
In this manuscript, the authors examine how temporal expression of the lin-4 microRNA is transcriptionally regulated.
Comments on revised version:
In the revised manuscript, the authors have suitably addressed my original concerns.
Aims achieved: The aims of the work are now achieved.
Impact: This study shows that a single transcription factor (MYRF-1) is important for the regulation of multiple microRNAs that are expressed early in development to control developmental timing.
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Author Response
The following is the authors’ response to the original reviews.
We would like to extend our sincere thanks to the editors and reviewers for their time and effort in reviewing our manuscript and offering insightful feedback. We have now completed the revisions, and the following is a summary of the key changes made.
(1) Analysis of myrf-1 and myrf-2 Mutations
A major concern raised was the characterization of the myrf-1(ju1121 G274R) mutation as a loss-of-function and myrf-1(syb1313, 1-700, gfp) as a gain-of-function mutation used in our study. These analyses have been previously detailed in our published papers (Meng, Dev Cell. 2017; Xia, Elife. 2021). In the revised manuscript, we have included a thorough explanation of this information in the introduction and added diagrams (Figure 1D, E) to illustrate the mutants …
Author Response
The following is the authors’ response to the original reviews.
We would like to extend our sincere thanks to the editors and reviewers for their time and effort in reviewing our manuscript and offering insightful feedback. We have now completed the revisions, and the following is a summary of the key changes made.
(1) Analysis of myrf-1 and myrf-2 Mutations
A major concern raised was the characterization of the myrf-1(ju1121 G274R) mutation as a loss-of-function and myrf-1(syb1313, 1-700, gfp) as a gain-of-function mutation used in our study. These analyses have been previously detailed in our published papers (Meng, Dev Cell. 2017; Xia, Elife. 2021). In the revised manuscript, we have included a thorough explanation of this information in the introduction and added diagrams (Figure 1D, E) to illustrate the mutants used in this study. A more detailed description is also available in the provisional letter I sent following the receipt of the decision letter.
We have incorporated new analyses of the endogenous lin-4 expression reporter in the myrf-1(ybq6, indel null), myrf-2(ybq42, indel null), and myrf-1(ybq6); myrf-2(ybq42) double mutants (Figure 2C). The results demonstrate complete inactivation of lin-4 expression in the double mutants. The data suggest that myrf-1 predominantly drives lin4 expression, while myrf-2 plays a minor role. This aligns with their roles in synaptic rewiring and is consistent with the observed lack of lin-4 expression in myrf-1(ju1121).
Furthermore, we have included analyses using pan-1(gk142) deletion mutants. PAN-1 is critical for MYRF trafficking to the cell membrane (Xia, Elife, 2021). In the absence of PAN-1, MYRF is trapped in the ER and subsequently degraded. The pan-1 mutants exhibit impaired synaptic rewiring, similar to myrf-1; myrf-2 double mutants, but somehow show larval arrest significantly later than myrf-1 mutants. Notably, lin-4 expression is not activated in pan-1 mutants. (references on the larval arrest phenotypes in pan-1 mutants: Gao G, Dev Biol. 2012 PMID: 22342905; Gissendanner CR, BMC Dev Biol. 2013. PMID: 23682709.)
Overall, these findings provide substantial evidence that MYRF is crucial for activating lin-4 during larval development.
(2) Regarding the Use of maIs134 as a lin-4 Expression Reporter
In response to the concerns raised about the use of the 2.4 kb Plin-4-gfp reporter (maIs134) as an indicator of lin-4 transcription, as detailed in the provisional letter, there is no evidence suggesting that maIs134 is an unsuitable reporter for lin-4 transcription. Recently, Kinney et al. (Dev Cell 2023, PMID: 37643611) showed that the pulse control element (PCE), located approximately 2.8 kb upstream, is not essential for lin-4 expression. Their findings also imply that a 2.4 kb region, encompassing what is referred to as the "short" regulatory region in their paper, contains essential elements required for driving the expression of lin-4. Nevertheless, I acknowledge that using an endogenously tagged reporter would be more ideal. It's important to note that we employed the endogenous expression reporter in our analyses of the myrf-1; myrf-2 double mutants, pan-1 mutants, and in the gain-of-function analysis of myrf-1. The outcomes from these studies corroborate our principal conclusions, reinforcing the validity of using maIs134 in our research context.
(3) Direct Binding of MYRF to the lin-4 Promoter
The technical challenges of MYRF ChIP (Chromatin Immunoprecipitation) have proven to be significant. Consequently, we have decided not to postpone the manuscript revision while awaiting additional results. We have included a section titled 'Limitations of the Study' to acknowledge our current lack of direct evidence for MYRF-1 binding to the endogenous lin-4 promoter. If it aligns better with eLife's format policy, we are open to relocating this paragraph to the discussion section.
(4) Specific Issues
We have provided responses to each specific question following the respective inquiries (see below).
Reviewer #1 (Public Review):
In this work, the authors set out to ask whether the MYRF family of transcription factors, represented by myrf-1 and myrf-2 in C. elegans, have a role in the temporally controlled expression of the miRNA lin-4. The precisely timed onset of lin-4 expression in the late L1 stage is known to be a critical step in the developmental timing ("heterochronic") pathway, allowing worms to move from the L1 to the L2 stage of development. Despite the importance of this step of the pathway, the mechanisms that control the onset of lin-4 expression are not well understood.
Overall, the paper provides convincing evidence that MYRF factors have a role in the regulation of lin-4 expression. However, some of the details of this role remain speculative, and some of the authors' conclusions are not fully supported by the studies shown. These limitations arise from three concerns. First, the authors rely heavily on a transcriptional reporter (maIs134) that is known not to contain all of the regulatory elements relevant for lin-4 expression. Second, the authors use mutant alleles with unusual properties that have not been completely characterized, making a definitive interpretation of the results difficult. Third, some conclusions are drawn from circumstantial or indirect evidence that does not use field-standard methods.
The authors convincingly demonstrate that the cytoplasmic-to-nuclear translocation of MYRF-1 coincides with the activation of lin-4 expression, making MYRF-1 a good candidate for mediating this activation. However, the evidence that MYRF-1 is required for the activation of lin-4 is somewhat incomplete. The authors provide convincing evidence that lin-4 activation fails in animals carrying the unusual mutation myrf1(ju1121), which the authors describe as disrupting both myrf-1 and myrf-2 activity. The concern here is that it is difficult to rule out that ju1121 is not also disrupting the activity of other factors, and it does not disentangle the roles of myrf-1 and myrf-2. Partially alleviating this issue, they also find that expression from the maIs134 reporter is disrupted in putative myrf-1 null alleles, but making inferences from maIs134 about the regulation of endogenous lin-4 is problematic. Helpfully, an endogenous Crisprgenerated lin-4 reporter allele is used in some studies, but only using the ju1121 allele. Together, these findings provide solid evidence that MYRF factors probably do have a role in lin-4 activation, but the exact roles of myrf-1 and myrf-2 remain unclear because of limitations of the unusual ju1121 allele and the use of the maIs134 reporter. The creative use of a conditional myrf-1 alleles (floxed and using the AID system) partially overcomes these concerns, providing strong evidence that myrf-1 acts cellautonomously to regulate lin-4, though again, these key experiments are only carried out with the maIs134 transgene.
A second important question asked by the authors is whether MYRF activity is sufficient to activate lin-4 expression. The authors provide evidence that supports this idea, but this support is somewhat incomplete, because the authors rely partially on the maIs104 array and, more importantly, on mutant alleles of MYRF-1 that they propose are constitutively active but are not completely characterized here.
The authors also approach the question of whether MYRF-1 regulates lin-4 via direct interaction with its promoter. The evidence presented here is consistent with this idea, but it relies on indirect evidence involving genetic interactions between myrf-1 and the presence of multiple copies of the lin-4 promoter, as well as the detection of nuclear foci of MYRF-1::GFP in the presence of multiple copies of the lin-4 promoter. This is not the field-standard approach for testing this kind of hypothesis, and the positive control presented (using the TetR/TetO interaction) is unconvincing. Thus, the evidence here is consistent with the authors' hypothesis, but the studies shown are incomplete and do not represent a rigorous test of this possibility.
Finally, the authors ask whether MYRF factors have a role in the regulation of other miRNAs. The evidence provided (RNAseq experiments, validated by several reporter transgenes) solidly supports this idea, with the provision that it is not completely clear that ju1121 is disrupting only the activity of myrf-1 and myrf-2.
Reviewer #2 (Public Review):
In this manuscript, the authors attempt to examine how the temporal expression of the lin-4 microRNA is transcriptionally regulated. However, the experimental support for some claims is incomplete. The authors repeatedly use the ju1121(G247R) mutation of myrf-1, but more information is required to evaluate their claim that this mutation "abolishes its DNA binding capability but also negatively interferes with its close paralogue MYRF-2". Additionally, in the lin-4 scarlet endogenous transcriptional reporter, the lin-4 sequence is removed. Since lin-4 has been reported to autoregulate, it seems possible that the removal of lin-4 coding sequence could influence reporter expression. Further, concrete evidence for direct lin-4 regulation by MYRF-1 is lacking, as the approaches used are indirect and not standard in the field. Overall, while the aims of the work are mostly achieved, data regarding the direct regulation of lin-4 by MYRF-1 and placing the work into the context of previous related reports is lacking. Because of its very specific focus, this paper reports useful findings on how a single transcription factor family might control the expression of a microRNA.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
(1) p.4 Authors should be cautious about this statement: "Once produced, lin-4 can selfenhance its own transcription by directly interacting with cis-elements in the promoter region[26, 27]." As reference 27 shows, this autoregulation is apparently an artifact of the reporter transgene; lin-4 does not appear to have the same role at the endogenous locus.
The discussion of “auto regulation” has been removed from the introduction.
(2) p.4 please provide a reference: "It is worth noting that external food signals are insufficient to drive lin-4 expression as lin-4 doesn't promptly turn on when animals encounter food."
This statement is derived from a combination of our unpublished observations and personal deductions. Within the sequence of events occurring during L1 development, the initiation of lin-4 expression happens relatively late. Therefore, the original sentence has been revised to “Given that lin-4 expression initiates in late L1, it is reasonable to deduce that merely providing food is inadequate to induce lin-4 expression.”
(3) Please provide more detail about myrf-1(syb1468) and myrf-1(syb1491) - are they likely null alleles? Are the phenotypes recessive? Please show the specific locations of deletions.
Please also refer to our response for the main issues raised. The two alleles under discussion are documented in Xia et al. (eLife, 2021). Both of these alleles are recessive and functionally equivalent to null mutations. Interestingly, all the myrf-1 alleles we have analyzed show recessive characteristics in various phenotypic aspects, including growth, synaptic rewiring, and M-cell division. The precise location of these genetic alterations is visually represented in Figure 1D and E.
(4) Fig 2C: are these animals heterozygous for the lin-4 Crispr reporter? If not, this is a lin-4 null. If lin-4 is required for the maintenance of its own expression, this result might be misleading about the role of myrf-1.
The lin-4 gene is located at Chr II: -0.86, and myrf-1 is positioned at Chr II: +2.98. Both of these alleles are balanced by mIn1. As a result, homozygotes for myrf-1 are also homozygotes for umn84. Regarding the role of lin-4 microRNA in its own transcription, research from Frank Slack’s lab has concluded that lin-4 microRNA does not affect the transcription of the lin-4 gene.
(5) p.8: please provide evidence/citation: "however, this experiment used a short promoter of dpy-7, which is not activated in seam cells..."
The dpy-7 promoter has been extensively utilized for transgene experiments in both Andrew Chisholm’s lab and our own. For reference, the original publication has been cited (PMID: 9121480).
(6) Fig 4B. Do the hypodermal knockout animals arrest at L1/L2?
This specific dual allele does not display arrest at the L1/L2 stages. A comprehensive description of the phenotype related to myrf-1LoxP(ybq98); Pdpy-7-Cre(tmIs1028) has been incorporated into the main text, and corresponding new data have been integrated into Figure 4.
(7) p. 11-13. I suggest that the authors consider making the section "MYRF-1 interacts with lin-4 promoter directly" much more succinct. The unsuccessful gelshift experiments can be explained in 1-2 sentences. The backstory about the weak Daf-C phenotype of the floxed allele is likely to confuse readers who are not experts in the field.
We have omitted the description of the gel shift experiments. However, we chose to retain the explanation of the Daf-C phenotype, simplifying the narrative for clarity. The Daf-C phenotype, which we have thoroughly analyzed, is considered significant. Our current research is exploring how nutrients facilitate the cleavage of myrf-1 on the cell membrane."
(8) Fig. 6G. I see no obvious change in the localization of TetR-RFP with or without the presence of TetO DNA, even though the authors use this as a positive control and claim that it validates the use of this approach to study MYRF-1/lin-4p interaction. What tissue is being imaged here? Hypodermis?
The intensity of fluorescent foci varies across transgenic F1 individuals. To assist with visualization, white arrows have been included in Figure 6G. These arrows highlight the formation of puncta in TetR::tagRFP(ybqSi233) due to the presence of a 7xTetO sequence-containing DNA array (indicated by white arrows), while simultaneously showing the lack of aggregation in GFP::MYRF-1. MYRF-1 is expressed across a broad range of tissues, and our analysis did not focus on any specific tissue type. The nuclei shown in the images are derived from a variety of tissues, including the intestine, epidermis, neurons, and the somatic cells of the reproductive system, as inferred from their morphology.
(9) Fig. 7A. Please consider using a different color scheme for the wt vs mutant data. These colors are too similar to those used for the expression-level heatmap. (Also, it's unclear how the fold-change data are normalized - i.e, fold-change compared to what?)
The color scheme in the clustering heatmap has been revised for enhanced contrast. This heatmap does not simply display raw read counts (TPM) or log2 values, though log2 transformation is part of the math process. If displayed directly, variables with low values can overshadow those with high values in the color representation. Instead, the read count data have undergone a series of transformations, including rlog transformation, size factor normalization, and gene-wise scaling, which leads to a more visually informative display of expression changes. Initially, we utilized a web-based tool (https://www.bioinformatics.com.cn/en) for creating the heatmap. However, due to the lack of detailed documentation on this site, we opted to reanalyze the data using functions from the DESeq2 package in R. This reanalysis enabled us to update the graph along with a revised figure legend, aiming to enhance clarity and comprehension.
(10) p. 14: "Remarkably, 6 out of the 7 up-regulated microRNAs are clustered on one phylogenetic branch" - does this mean upregulated in the mutant compared to WT?
The sentence has been revised to “Notably, 6 of the 7 microRNAs showing increased expression in myrf-1(ju1121) compared to wild type are clustered on a single phylogenetic branch,..”
(11) Fig. 7C: Authors might comment on which tissues show expression of these miRNAs.
A sentence has been added: “The reporter for mir-48 is primarily detected in the pharynx, mir-73 is present in both the pharynx and seam cells, whereas mir-230 is detected in seam cells.”
(12) p. 16: "Our report includes the partial dauer-constitutive phenotype caused by the interaction between the lin- 4 promoter DNA and MYRF-1." - consider rewording this; according to the author's model, it's not the interaction per se that causes the Daf-C phenotype, but rather the sequestration of MYRF-1 (or -2?) by excess lin-4p.
The sentence has been revised to “Our observations suggest that the tandem array of lin-4 promoter DNA may sequester a certain amount of MYRF protein. This sequestration could limit the availability of MYRF, potentially leading to a partial dauerconstitutive phenotype.”
Reviewer #2 (Recommendations For The Authors):
(1) The use of L1 (and not even defining what L1 means) in the abstract is very C. elegans-field specific. Make the writing more accessible to a general audience
This sentence has been revised.
(2) Instead of writing in the context of upregulation and downregulation - I advise using activation/induction and repression instead. e.g. MYFR-1 is necessary for lin-14 induction in late stage L1.
The wording of “upregulation” and “downregulation” has been changed.
(3) We find that lin-4 transcription reporter fails to be upregulated in myrf-1(ju1121) at any viable stages that can be analyzed - should this just say 'fails to be expressed'?
“at any viable stages that can be analyzed” has been removed.
(4) The section starting with this sentence is strange as in the previous section the authors showed that MYRF-1 expressed in muscle or epidermis IS sufficient to drive lin4 expression - 'The next question was whether MYRF-1 is sufficient to drive the upregulation of lin-4.'
The sentence has been updated to reflect our research focus: “Given that both the induction of lin-4 and the cleavage of MYRF at the cell membrane happen within a specific time window, we investigated whether a gain of function in MYRF-1 alone is adequate to modify the onset timing of lin-4.”
(5) This sentence needs modifying "A series of MYRF-1 variants were expressed in HEK cells by transfection, and cell lysis was tested for their binding with 498 bp DNA of the lin-4 promoter." This sentence suggests that cell lysis tests the binding of the protein to DNA which is obviously incorrect.
We have chosen to omit the description of these experiments from our text due to their inconclusive results.
(6) Typographical changes/suggestions to aid clarity:
Introduction: lin-4 and lin-14 are the two that have been studied in details - change to lin4 and lin-14 are the two that have been studied in detail
Results: Write NAA solution in full the first time it is mentioned.
Remove ', a collagen,' when describing the dpy-7 promoter. The authors don't describe what myo-3 encodes so keep this consistent.
Page 14 'itself was upregulated in the mutants.' be more specific. Which mutants?
All four identified places have been appropriately corrected or revised.
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eLife assessment
The microRNA lin-4, originally discovered in C. elegans, has a key role in developmental timing across species, but how its expression is developmentally controlled is poorly understood. Here, the authors provide convincing evidence that two MYRF transcription factors are essential positive regulators of lin-4 during early C. elegans larval development. These results provide important insight into the molecular nature of developmental timing that could have significant implications for understanding these processes in more complex systems.
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Reviewer #1 (Public Review):
In this work, the authors set out to ask whether the MYRF family of transcription factors, represented by myrf-1 and myrf-2 in C. elegans, have a role in the temporally controlled expression of the miRNA lin-4. The precisely timed onset of lin-4 expression in the late L1 stage is known to be a critical step in the developmental timing ("heterochronic") pathway, allowing worms to move from the L1 to the L2 stage of development. Despite the importance of this step of the pathway, the mechanisms that control the onset of lin-4 expression are not well understood.
Overall, the paper provides convincing evidence that MYRF factors have a role in the regulation of lin-4 expression. Using state-of-the-art techniques (knock-in reporters and conditional alleles), the authors show that MYRF factors are essential for …
Reviewer #1 (Public Review):
In this work, the authors set out to ask whether the MYRF family of transcription factors, represented by myrf-1 and myrf-2 in C. elegans, have a role in the temporally controlled expression of the miRNA lin-4. The precisely timed onset of lin-4 expression in the late L1 stage is known to be a critical step in the developmental timing ("heterochronic") pathway, allowing worms to move from the L1 to the L2 stage of development. Despite the importance of this step of the pathway, the mechanisms that control the onset of lin-4 expression are not well understood.
Overall, the paper provides convincing evidence that MYRF factors have a role in the regulation of lin-4 expression. Using state-of-the-art techniques (knock-in reporters and conditional alleles), the authors show that MYRF factors are essential for lin-4 activation and act cell-autonomously. While there are some minor concerns about the use of unusual gain-of-function alleles, these are mitigated by consistent results using other approaches. The authors also provide evidence that MYRF factors activate lin-4 by directly activating its promoter. While their results are certainly consistent with this possibility, they rely on indirect measurements and are therefore not definitive. Further experiments will be necessary to determine whether this model is accurate.
Some details about the relative roles of the two C. elegans MYRF factors, myrf-1 and myrf-2, remain unclear. myrf-1 clearly seems to play the more important role lin-4 activation and the regulation of developmentally timed processes. However, there are numerous hints that myrf-2 may act in the opposite direction, either by inhibiting myrf-1 itself or its ability to activate its targets. Further work will be necessary to understand the genetic and mechanistic relationships between these two genes.
Overall, the findings in this paper are convincing, and the results will be of interest to a wide range of developmental biologists.
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Reviewer #2 (Public Review):
Summary:
In this manuscript, the authors examine how temporal expression of the lin-4 microRNA is transcriptionally regulated.In the revised manuscript, the authors have suitably addressed my original concerns.
Aims achieved: The aims of the work are now achieved.
Impact: This study shows that a single transcription factor (MYRF-1) is important for the regulation of multiple microRNAs that are expressed early in development to control developmental timing.
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Author Response
We express our gratitude to the editors for acknowledging the significance of our findings and facilitating the review process. We would also like to thank the reviewers for dedicating their time to thoroughly read the manuscript and provide valuable insights.
During the revision process, we will address the raised issues and concerns, confident that our revisions will enhance the clarity and strength of the paper.
In response to the reviewers' feedback, we acknowledge that some of the relevant information was previously presented in our published papers (Meng, Dev Cell. 2017; Xia, Elife. 2021). However, we recognize that in the current version of the manuscript, we may not have expounded on these details as clearly as needed. We will rectify this shortcoming in the revised version to provide a more comprehensive account …
Author Response
We express our gratitude to the editors for acknowledging the significance of our findings and facilitating the review process. We would also like to thank the reviewers for dedicating their time to thoroughly read the manuscript and provide valuable insights.
During the revision process, we will address the raised issues and concerns, confident that our revisions will enhance the clarity and strength of the paper.
In response to the reviewers' feedback, we acknowledge that some of the relevant information was previously presented in our published papers (Meng, Dev Cell. 2017; Xia, Elife. 2021). However, we recognize that in the current version of the manuscript, we may not have expounded on these details as clearly as needed. We will rectify this shortcoming in the revised version to provide a more comprehensive account of our research.
We also explain our perspective on why the discovery of MYRF controlling lin-4 upregulation is crucial in addressing unanswered key questions in developmental biology.
The Loss of Function Characteristics of myrf-1(ju1121 G274R)
We would like to present the evidence supporting the characteristics of myrf-1(ju1121) as a loss-of-function mutation affecting both myrf-1 and myrf-2. In our initial paper (Meng, Dev Cell. 2017), the nature of this mutation was a significant focus of our research.
Our investigation involved analyzing multiple alleles (tm, ok, gk alleles from CGC, and indel alleles made in-house) of myrf-1 and myrf-2, as well as their double mutants. Here is a summary of our current understanding based on these analyses:
myrf-1 single loss-of-function (l.f.) mutants exhibit penetrant arrest at the end of L1 or early L2 stages. However, they only display very mild deficiency in DD synpatic remodeling at 21 hours, primarily caused by a delay.
myrf-2 single l.f. mutants behave similarly to the wild type, exhibiting no significant developmental abnormalities, including synpatic remodeling.
myrf-1 and myrf-2 double l.f. mutants exhibit penetrant arrest during L2, occurring approximately half a stage later than in myrf-1 single mutants.
Remarkably, myrf-1 and myrf-2 double l.f. mutants exhibit severe blockage in synaptic remodeling, indicating that both genes act collaboratively to drive this essential process (Meng, Figure 5).
The myrf-1(ju1121 G274R) mutation exhibits severe synaptic remodeling blockage and arrest during L2, closely resembling myrf-1 myrf-2 double mutants (Meng, Figure 1 and 2).
Therefore, despite myrf-1's more significant role in development based on the arrest phenotype, synaptic remodeling requires the combined function of myrf-1 and myrf-2. This redundancy is further supported by the analysis of the new set of specific myrf-1 mutants (Xia, Figure 6).
Both myrf-1 and myrf-2 are broadly expressed (Meng, Figure 3 and S5), and they undergo developmentally regulated cell-membrane to nucleus translocation (Xia, Figure 4 and Supplement 1). Overexpressing N-MYRF-1 and full-length MYRF-2 in DD neurons leads to precocious synaptic remodeling (Meng, Figure 4 and 5). Interestingly, overexpressing full-length myrf-1 does not have the same effect, indicating potential regulatory differences between these two factors.
The myrf-1(ju1121 G274R) mutation is located in the N-terminal region of the Ig-fold type DNA-binding domain, specifically within the loop between a and b Ig-fold strands. This site is conserved across all metazoan MYRFs (Meng, Figure 1D and 6A). The mutant myrf-1(G274R) loses its DNA binding ability, as demonstrated by a gel mobility shift assay using the counterpart residue mutation in mammalian MYRF (Meng, Figure 6B).
MYRF-1(ju1121 G274R) mutant interfering with normal MYRF’s function has been supported by molecular genetics experiments (Meng, Figure 6C-E) and biochemical analysis. In essence, the MYRF-1(G274R) mutant does not impact MYRF trimerization or MYRF-1-MYRF-2 interaction, but blocks DNA binding. Substantial evidence has confirmed the physical binding of MYRF-1 and MYRF-2 both in vitro and in vivo (Meng, Figure 5G and S6; Xia, Figure 1A). Importantly, MYRF- 1(ju1121 G274R) is still able to bind to MYRF-2, as supported by coIP analysis (Meng, Figure S7), indicating that the G274R mutation does not disrupt the MYRF-1-MYRF-2 interaction. This observation is consistent with the characteristics of the MYRF structure (PMID: 28160598; PMID: 34345217). The critical interface of the MYRF trimer is located in the alpha-helix upstream of the ICE domain, the beta sheets of the ICE, and the beta-helix of the bridge region between ICE and DBD. Therefore, since MYRF-1(ju1121 G274R) is not situated in this critical interface of the MYRF trimer, it is unlikely that the mutation affects MYRF trimerization.
With all available evidence, we propose a reasonable model where myrf-1(ju1121) has two effects: rendering myrf-1 defective in DNA binding and negatively interfering with MYRF-2 by forming a non-functional trimer consisting of monomer MYRF-1(ju1121) and wild-type MYRF-2.
Regarding the potential neomorphic function of myrf-1(ju1121), the myrf-1(ju1121)/+ individuals appear superficially wild type and show no defects in synaptic remodeling. Furthermore, we have generated a myrf-1 minigene array that results in a complete rescue of the developmental phenotype in myrf-1(ju1121) (Meng, Figure 3A-D). Notably, the transgene is expected to be low copy numbered, as it was generated by injecting at a very low concentration of 0.1 ng/μl. The complete rescue of the phenotype strongly suggests that any potential aberrant effects caused by myrf-1(ju1121) mutants are minimal.
In summary, myrf-1(ju1121) behaves similarly to myrf-1 myrf-2 double mutants, and we utilized this allele for the convenience of analysis.
Due to the essential role of MYRF-controlled processes in larval development and the lack of detectable phenotypic effects in myrf-2 single loss-of-function mutants, it is evident that myrf-2 plays a minor role in these developmental events. Considering that development regulation rarely follows a simple linear or accumulative fashion, deciphering the relative contributions of each myrf-1 and myrf-2 in specific developmental events may not be straightforward. Consequently, our primary focus remains on investigating the functions of myrf-1.
Nevertheless, we concur that providing a clear description of the impact of myrf-1 and myrf-2 single mutants on lin-4 expression is crucial. We are actively conducting ongoing analyses, and the new findings will be incorporated in the revised version of our manuscript.
Characterizing myrf-1(syb1313, 1-700) as a Hyperactive Allele of myrf-1
The cleavage and release of N-MYRF are developmentally regulated and occur in late L1. We have substantial evidence supporting the interaction between the non-cytoplasmic region of MYRF and another transmembrane protein, PAN-1, which is crucial for delivering MYRF onto the cell membrane (Xia, Figure 1, 7, 8, 10, 11 and 13). The myrf-1(syb1313, 1-700) mutant lacks the non-cytoplasmic region of MYRF, which is the interaction site for PAN-1. Initial analyses revealed that in the mutants, MYRF-1(syb1313) remains in the cytoplasmic, ER-like structure, resulting in larval arrest during L2 (Xia, Figure 8).
However, a more careful analysis unveiled that a small amount of N-MYRF is processed and enters the nucleus, but this process is not dependent on the normal developmental timing and may take place during early-mid L1. Consequently, this leads to precocious yet discordant DD synaptic remodeling and M-cell lineage division (Xia, Figure 6 and 9). Considering the precocious development, the low quantity of nuclear N-MYRF, and the overall larval arrest phenotype observed in the mutants, we conclude that myrf-1(syb1313) represents an inconsistent, weak hyperactive form of MYRF-1. Moreover, the hyperactive function may be context-dependent, for instance, presence of myrf-1(syb1313) may be sufficient for certain needs in neurons but insufficient for epidermis. Our ongoing research to identify the downstream targets of MYRF also supports this notion.
Given that the myrf-1(syb1313) mutant has been thoroughly characterized and published, it is the most suitable option for use in our current investigations on lin-4 expression.
Furthermore, we employed the MYRF-1(delete 601-650) deletion mutant construct, which is a significantly more effective hyperactive MYRF-1 mutant when overexpressed. This reagent stems from our ongoing study, which is dedicated to identifying the self-inhibitory mechanisms of MYRF cleavage. The extensive volume of data that led to this discovery makes it impractical to include in the current manuscript. However, we are eager to share the substantial effects of MYRF-1(delete 601-650) mutants in activating lin-4 expression, which strengthens the role of MYRF in regulating lin-4. We will take care to revise this section to provide clearer references.
The lin-4p::nls::mScarlet(umn84) knock-in reporter is loss-of-function for lin-4; however, lin-4 mature microRNA does not affect lin-4 expression.
Indeed, the lin-4 knock-in reporter umn84 removes lin-4 coding sequence. As a result, the homozygous reporter strain is also lin-4 null mutants. Since both lin-4 and myrf-1 are located on Chr II and are less than 4 m.u. apart, the constructed strain is myrf-1 lin-4(umn84) / mIn1 (balanced by mIn1). Consequently, the myrf-1 homozygous animal is also lin-4 reporter homozygous.
Regarding the endogenous function of the "auto-regulating element," we are aware of the follow-up paper by Frank Slack's group, in which they concluded that the previously reported sequence is dispensable for lin-4 expression, and the loss of lin-4 does not affect the expression of its primary transcript (PMID: 29324872). To avoid confusion, we will remove or revise the introductory sentences as necessary to accurately reflect this information.
Additionally, besides analyzing the expression of the knock-in reporter of lin-4 (umn84), we also conducted a thorough analysis of mature microRNA expression using targeted qPCR and genomic analysis via microRNA sequencing. Both sets of results indicate severely defective upregulation of lin-4 mature microRNA in myrf-1(ju1121).
No evidence indicates that the 2.4 kb reporter of Plin-4-gfp (maIs134) is an inappropriate reporter for lin-4 transcription.
maIs134 is originated from the Ambros lab, and to date, there is no single evidence demonstrating that maIs134 cannot be regarded as a reliable transcription reporter for lin-4 expression. The Stec et al. (Curr Biol 2021. PMID: 33357451) paper suggests that the PCE or CEA site (at ~ -2.8 kb) outside the 2.4 kb region confers enhancing effects for lin-4 transcription, but no other published paper has studied lin-4 transcription and cited this finding.
While the Stec et al. paper provides elaborate mechanistic descriptions, the basic characterization of the importance of CE-A and blmp-1 to lin-4 expression is lacking. Deletion of CE-A in the lin-4 promoter reporter using an Ex array transgene resulted in highly variable reporter expression (Stec, Figure 4D). Notably, two high expression data points indicated that a transgene reporter without CE-A can be highly expressed, suggesting that CE-A is unnecessary for lin-4 transcription. Only when both CE-A and CE-D (within 2.4 kb) were deleted, the reporter expression was significantly decreased. Moreover, deletion of CE-C (proximal region) alone caused severe loss of reporter activity, supporting that proximal CE-C is the essential element, while CE-A is not.
It is important to note that the effect of CE-A on lin-4 expression has not been analyzed using stable transgenes or genetic deletions in the endogenous lin-4 region. Furthermore, there is no data on how blmp-1 mutants affect the expression of the wild-type lin-4 promoter reporter, CEA deletion reporter, or lin-4 mature microRNA, despite the paper’s main claim that blmp-1 boosts lin-4 expression. While CE-A can confer an enhancing effect in epidermal expression when fused to the gst-5 promoter, there is no data showing that CE-A is sufficient to drive lin-4 transcription by itself.
In summary, there is currently insufficient evidence to establish whether CE-A is necessary or sufficient for regulating lin-4 expression. In fact, the data presented in Stec et al. (Curr Biol 2021) suggest that CE-A is unnecessary for lin-4 expression. As such, I do not see any reason to consider the 2.4 kb reporter in maIs134 as inappropriate for analyzing lin-4 transcription. Furthermore, our presented data using the knock-in reporter of lin-4 (umn84) demonstrated that its regulation by myrf is essentially consistent with the observations drawn from the maIs134 analysis.
The Significance of the Finding: MYRF Regulating lin-4 Upregulation
We are grateful that the editors find our results valuable for those interested in lin-4 expression. However, we acknowledge that the editors may not share the same enthusiasm as we do, seeing this as a landmark discovery in understanding postembryonic development, a fundamental question in the field of developmental biology.
Importance of Understanding lin-4 Upregulation in Development
The foundation of developmental biology has been built on the principles derived from studying embryonic development in model organisms like Drosophila, exemplified by the Nobel laureates Lewis, Nusslein-Volhard, and Wieschaus. These principles explain what occurs during embryonic development, including patern formation, morphogenesis, and differentiation. However, these existing principles do not fully explain the phenomena of postembryonic development, including growth. For instance, during C. elegans development in L1, it remains unclear what controls the initiation of P cell division. If we may exclude dividing cells from the discussion, numerous stage-specific changes occur in non-dividing cells, including neurons. The extensive, systematic expression studies of transcription factors in C. elegans have failed to provide evidence that such developmental progression is driven by sequential activation of transcriptional cascades, as commonly observed during embryonic differentiation. A different approach to ask a similar question is to inquire how developmental timing is controlled, e.g., "why does it take a boy 12 years to reach adolescence?" This perspective highlights the need to identify potential unidentified checkpoints that control postembryonic stages (An example of insightful review: The Systemic Control of Growth. Cold Spring Harb Perspect Biol. 2015. PMID:
The upregulation of lin-4 represents a system’s checkpoint during postembryonic development. Deciphering the mechanism controlling lin-4 expression is instrumental in understanding the principles of postembryonic development, even extending to adult development, including life span control.
Importance of the Finding: MYRF's Control of lin-4 Upregulation
To date, no other essential, positive regulator of lin-4 transcription has been identified, although several negative regulators have been reported. A landmark paper by Victor Ambros identified FLYWCH as a repressor of lin-4 expression during embryogenesis (PMID: 18794349). FLYWCH mutants fail to progress to normal hatched larvae, implying that FLYWCH is crucial. The paper indeed suggested that FLYWCH has additional functions beyond suppressing lin-4, although these functions have not been thoroughly characterized. The significance of the FLYWCH finding lies in the elaborate control during the transition from embryo to larval development, where lin- 4 is actively suppressed. This control may ensure the robustness of subsequent lin-4 activation. The process during the embryo-to-larvae transition, as well as the counterpart process in mammalian development perinatally, remains poorly understood.
Another negative regulator of lin-4 is lin-42, as reported in three papers in 2014 (PMID: 25319259; PMID: 24699545; PMID: 25032706). Lin-42 negatively regulates lin-4 expression, despite the main focus of the papers being lin-42's repression of let-7. However, the precise mechanisms by which this repression is achieved are not fully understood.
Amy Pasquinelli's lab conducted a genome-wide screen to identify factors responsible for driving lin-4 upregulation but did not identify a critical factor that promotes lin-4 transcription (PMID: 20937268).
In the recent paper by Stec et al. (Curr Biol 2021. PMID: 33357451), they reported blmp-1's role in enhancing lin-4 expression. However, the significance of blmp-1 in regulating lin-4 remains vaguely described, despite a large amount of data describing elaborate epigenetic controls. The paper did not provide data on how endogenous lin-4 expression is affected in blmp-1 mutants, nor did it demonstrate how full-length reporter expression is affected in blmp-1 mutants. The only relevant data appears to be on the CE-A-gst-5 promoter reporter in blmp-1 mutants. As a result, it remains unclear how blmp-1 affects lin-4 transcription.
In summary, no single factor has been identified, the loss of which leads to significant deficiencies in lin-4 upregulation. MYRF is the first and a critical factor identified in this context. This finding represents a significant advancement in our understanding of lin-4 regulation and its crucial role in development.
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eLife assessment
This useful study investigates the roles of C. elegans MYRF transcription factors myrf- and myrf-2 in the temporally controlled activation of the miRNA lin-4, a key step in larval developmental timing. While some of the findings are solid, other evidence is incomplete because of concerns about the technical approaches. This study provides information that will be useful to those interested in the regulation of lin-4 expression in C. elegans.
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Reviewer #1 (Public Review):
In this work, the authors set out to ask whether the MYRF family of transcription factors, represented by myrf-1 and myrf-2 in C. elegans, have a role in the temporally controlled expression of the miRNA lin-4. The precisely timed onset of lin-4 expression in the late L1 stage is known to be a critical step in the developmental timing ("heterochronic") pathway, allowing worms to move from the L1 to the L2 stage of development. Despite the importance of this step of the pathway, the mechanisms that control the onset of lin-4 expression are not well understood.
Overall, the paper provides convincing evidence that MYRF factors have a role in the regulation of lin-4 expression. However, some of the details of this role remain speculative, and some of the authors' conclusions are not fully supported by the …
Reviewer #1 (Public Review):
In this work, the authors set out to ask whether the MYRF family of transcription factors, represented by myrf-1 and myrf-2 in C. elegans, have a role in the temporally controlled expression of the miRNA lin-4. The precisely timed onset of lin-4 expression in the late L1 stage is known to be a critical step in the developmental timing ("heterochronic") pathway, allowing worms to move from the L1 to the L2 stage of development. Despite the importance of this step of the pathway, the mechanisms that control the onset of lin-4 expression are not well understood.
Overall, the paper provides convincing evidence that MYRF factors have a role in the regulation of lin-4 expression. However, some of the details of this role remain speculative, and some of the authors' conclusions are not fully supported by the studies shown. These limitations arise from three concerns. First, the authors rely heavily on a transcriptional reporter (maIs134) that is known not to contain all of the regulatory elements relevant for lin-4 expression. Second, the authors use mutant alleles with unusual properties that have not been completely characterized, making a definitive interpretation of the results difficult. Third, some conclusions are drawn from circumstantial or indirect evidence that does not use field-standard methods.
The authors convincingly demonstrate that the cytoplasmic-to-nuclear translocation of MYRF-1 coincides with the activation of lin-4 expression, making MYRF-1 a good candidate for mediating this activation. However, the evidence that MYRF-1 is required for the activation of lin-4 is somewhat incomplete. The authors provide convincing evidence that lin-4 activation fails in animals carrying the unusual mutation myrf-1(ju1121), which the authors describe as disrupting both myrf-1 and myrf-2 activity. The concern here is that it is difficult to rule out that ju1121 is not also disrupting the activity of other factors, and it does not disentangle the roles of myrf-1 and myrf-2. Partially alleviating this issue, they also find that expression from the maIs134 reporter is disrupted in putative myrf-1 null alleles, but making inferences from maIs134 about the regulation of endogenous lin-4 is problematic. Helpfully, an endogenous Crispr-generated lin-4 reporter allele is used in some studies, but only using the ju1121 allele. Together, these findings provide solid evidence that MYRF factors probably do have a role in lin-4 activation, but the exact roles of myrf-1 and myrf-2 remain unclear because of limitations of the unusual ju1121 allele and the use of the maIs134 reporter. The creative use of a conditional myrf-1 alleles (floxed and using the AID system) partially overcomes these concerns, providing strong evidence that myrf-1 acts cell-autonomously to regulate lin-4, though again, these key experiments are only carried out with the maIs134 transgene.
A second important question asked by the authors is whether MYRF activity is sufficient to activate lin-4 expression. The authors provide evidence that supports this idea, but this support is somewhat incomplete, because the authors rely partially on the maIs104 array and, more importantly, on mutant alleles of MYRF-1 that they propose are constitutively active but are not completely characterized here.
The authors also approach the question of whether MYRF-1 regulates lin-4 via direct interaction with its promoter. The evidence presented here is consistent with this idea, but it relies on indirect evidence involving genetic interactions between myrf-1 and the presence of multiple copies of the lin-4 promoter, as well as the detection of nuclear foci of MYRF-1::GFP in the presence of multiple copies of the lin-4 promoter. This is not the field-standard approach for testing this kind of hypothesis, and the positive control presented (using the TetR/TetO interaction) is unconvincing. Thus, the evidence here is consistent with the authors' hypothesis, but the studies shown are incomplete and do not represent a rigorous test of this possibility.
Finally, the authors ask whether MYRF factors have a role in the regulation of other miRNAs. The evidence provided (RNAseq experiments, validated by several reporter transgenes) solidly supports this idea, with the provision that it is not completely clear that ju1121 is disrupting only the activity of myrf-1 and myrf-2.
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Reviewer #2 (Public Review):
In this manuscript, the authors attempt to examine how the temporal expression of the lin-4 microRNA is transcriptionally regulated. However, the experimental support for some claims is incomplete. The authors repeatedly use the ju1121(G247R) mutation of myrf-1, but more information is required to evaluate their claim that this mutation "abolishes its DNA binding capability but also negatively interferes with its close paralogue MYRF-2". Additionally, in the lin-4 scarlet endogenous transcriptional reporter, the lin-4 sequence is removed. Since lin-4 has been reported to autoregulate, it seems possible that the removal of lin-4 coding sequence could influence reporter expression. Further, concrete evidence for direct lin-4 regulation by MYRF-1 is lacking, as the approaches used are indirect and not standard …
Reviewer #2 (Public Review):
In this manuscript, the authors attempt to examine how the temporal expression of the lin-4 microRNA is transcriptionally regulated. However, the experimental support for some claims is incomplete. The authors repeatedly use the ju1121(G247R) mutation of myrf-1, but more information is required to evaluate their claim that this mutation "abolishes its DNA binding capability but also negatively interferes with its close paralogue MYRF-2". Additionally, in the lin-4 scarlet endogenous transcriptional reporter, the lin-4 sequence is removed. Since lin-4 has been reported to autoregulate, it seems possible that the removal of lin-4 coding sequence could influence reporter expression. Further, concrete evidence for direct lin-4 regulation by MYRF-1 is lacking, as the approaches used are indirect and not standard in the field. Overall, while the aims of the work are mostly achieved, data regarding the direct regulation of lin-4 by MYRF-1 and placing the work into the context of previous related reports is lacking. Because of its very specific focus, this paper reports useful findings on how a single transcription factor family might control the expression of a microRNA.
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