Essential Function of Membrane-Bound Transcription Factor MYRF in Promoting Transcription of miRNA lin-4 during C. elegans Development

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.

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.

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:

1. 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.

2. myrf-2 single l.f. mutants behave similarly to the wild type, exhibiting no significant developmental abnormalities, including synpatic remodeling.

3. 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.

4. 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).

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.