C. elegans molting requires rhythmic accumulation of the Grainyhead/ LSF transcription factor GRH ‐1

  1. Milou W M Meeuse
  2. Yannick P Hauser
  3. Smita Nahar
  4. A Alexander T Smith
  5. Kathrin Braun
  6. Chiara Azzi
  7. Markus Rempfler
  8. Helge Großhans

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Abstract

C. elegans develops through four larval stages that are rhythmically terminated by molts, that is, the synthesis and shedding of a cuticular exoskeleton. Each larval cycle involves rhythmic accumulation of thousands of transcripts, which we show here relies on rhythmic transcription. To uncover the responsible gene regulatory networks (GRNs), we screened for transcription factors that promote progression through the larval stages and identified GRH‐1, BLMP‐1, NHR‐23, NHR‐25, MYRF‐1, and BED‐3. We further characterize GRH‐1, a Grainyhead/LSF transcription factor, whose orthologues in other animals are key epithelial cell‐fate regulators. We find that GRH‐1 depletion extends molt durations, impairs cuticle integrity and shedding, and causes larval death. GRH‐1 is required for, and accumulates prior to, each molt, and preferentially binds to the promoters of genes expressed during this time window. Binding to the promoters of additional genes identified in our screen furthermore suggests that we have identified components of a core molting‐clock GRN. Since the mammalian orthologues of GRH‐1, BLMP‐1 and NHR‐23, have been implicated in rhythmic homeostatic skin regeneration in mouse, the mechanisms underlying rhythmic C. elegans molting may apply beyond nematodes.

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  1. Version published to 10.15252/embj.2022111895
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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The manuscript describes how a rhythmically active transcription factor is important for molting cycles. The first part of the manuscript focuses on oscillating genes, and the authors nicely show a rhythmic transcription of these genes. Indeed, using RNAPolII Chip-Seq experiments, they show a rhythmic recruitment of the RNAPolII to the promoters of the oscillatory genes they have previously described. They then demonstrate that GFPs driven by the promoter of these oscillating genes, and inserted as a single copy, can very accurately recapitulate the rhythmic transcription of oscillating genes. It is interesting to see the weak impact of introns and 3'UTR on the rhythmic expression. In the second part of the manuscript, the authors perform an RNAi screen, looking for oscillating transcription factors (TF) important for molting. The goal of this approach is to identify core oscillators that could control molting cycles. Focusing their screen on oscillating TF allows them to exclude TF that would be required for embryonic or larval viability unrelated to molting. Among the 6 candidates they have identified, 5 have already been linked to molting. They focus their study on grh-1, which has never before been described during larval development. They characterize the molting phenotype of grh-1 defective worms using the AID degron system. They monitor the molting cycles using a luciferase assay in a liquid culture where transgenic worms for luciferase are grown in the presence of bacteria supplemented with luciferin. Using this approach (which is quantitative and allows high-throughput analysis), they show that grh-1 is required for each molt in a dose-dependent manner. The GRH-1 protein oscillates and peaks shortly before each molt entry, reinforcing the idea that GRH-1 is an important core TF for molting. The authors finally show that the oscillating activity of GRH-1 is crucial for the molting.

    Major comments:

    Overall, the data are clear and convincing, and the results are quantified with care. The first part of the manuscript represents a significant amount of work with the RNAPolII Chip-Seq, the different single-copy integrants and RT-PCRs. Then, the authors provide a quantitative assessment of the molting process by combining their grh-1-AID construct with the luciferase system. This strengthens the quality of the manuscript.

    My substantial suggestion is that the authors could consider extending the scope of the study in two alternative ways:

    • One question that immediately springs to mind is: what are the targets of grh-1? By applying their luciferase assay to further possible downstream targets of grh-1, they could attempt to phenocopy the grh-1 molting defect, and then look if the oscillating expression of these targets is eliminated in grh-1 defective animals. The binding site of grh-1 is apparently known (Venkatesan, 2003), so is it possible to reduce the potential target list among the oscillating genes using a bioinformatics approach? This requires a substantial amount of work (2 to 3 months) but it would help tell a more complete story.
    • It is striking that myrf-1 and nhr-23 RNAi display the same molting defects as grh-1 RNAi as show in figure 2B. Have the authors considered testing the genetic interactions of grh-1 with these two other candidates? Do they belong to the same GRN? Does grh-1 depletion impact the expression of the nhr-23 and myrf-1, or vice versa? Do they have the same target genes (Chip-seq data for nhr-23 are available)? This, again, would significantly strengthen the paper and would make the second part more complete. This alternative piece of work would require less experiments than the first suggestion but would be also of great interest. These two points are only suggestions as it represents a significant amount of work, and the paper could very well be published in its current form.

    About the luciferase assay for grh-1, nhr-23 and myrf-1 RNAi, the authors observe "an apparent arrest in development or death following atypical molts". What do they mean by "atypical molt" at this stage of the paper? Indeed, for these candidates, the luminescence traces are highly perturbed after the second molt (for grh-1 and nrh-23 RNAi) or the third molt (for myrf-1), but these abnormal traces seem to reflect an arrest in development or larval lethality rather than an atypical molting. Can the authors clarify this point?

    In the part on the phenotypic analysis of GRH-1 depleted animals, the authors conclude the paragraph with "GRH-1 is required for viability at least in part through its role in proper cuticle formation". This role in proper cuticle formation refers to the cuticle break in the head region as observed in time lapse. It would be useful to have a visual test of the cuticle permeability using an Hoechst staining.

    The authors show GFP::GRH-1 pictures at different stages to describe a rhythmic protein accumulation (see also my minor comment on GFP picture quality). From the perspective of whether all tissues are oscillating, it would be interesting to see if all the cells they mention in the text are showing the same rhythmic fluorescence.

    In relation to the previous comment, which tissue is responsible for the defects observed by the degradation of GRH-1? Is it possible to use a tissue-specific depletion of AID-tagged GRH-1 using Seam-cell specific, rectal cell specific, vulval precursors specific promoters, etc...?

    In the last part of the results, the authors show that molting requires oscillatory GRH-1 activity by depleting GRH-1 at variable times in L2. It would be interesting to know what happens if a stable (non-oscillating) amount of GRH-1 protein is maintained over time in the worms (using a non-oscillating promoter).

    Minor comments:

    In figure 5 B, C, D, it seems that right before entering the M2, M3 and M4 respectively, there is a peak of luminescence (a thin bright line) and a strong luminescent signal is detected at the molt exit. Can the authors comment on that?

    If I understand correctly, for the GRH-1 GFP CRISPR reporter (Fig S7, S8 and S9), the authors have imaged single worms in microchambers on a spinning disk microscope. I fail to see why they used such a sophisticated approach to describe the expression pattern of GRH-1. This imaging setup is ideal for timelapse. However, in the context of which cell express GRH-1, the resolution is not good enough to fully assess cell identity. Indeed, the GFP images are a bit blurry, and it is difficult to make out the difference between real GFP fluorescence and gut autofluorescence. It would be helpful to have better quality pictures with a more regular setup, i.e., a 2% agarose pad mounted on a regular microscope or confocal. For non-specialists of the C. elegans anatomy, small insets for each category of cells mentioned (seam cells, vulval precursors etc.) would be appreciated.

    It would be easier to assess the GRH-1 expression decrease in adults if the pictures were shown in parallel with the larval pictures, with the same brightness/contrast correction (if any). Make insets to compare the same cells between different stages.

    How can the authors quantify the duration of molts 3 and 4 in fig S4 when these molts are not seen in the luciferase assay in fig 2B? Can the authors clarify this point?

    Writing/clarity: For non-specialists, mention why the authors used a PEST sequence in their constructs and explain what the eft-3 promoter is (they mention it in the Luciferase assay, but it is not clear enough).

    In Fig S4, make clearer that EV = MOCK.

    In the methods, the authors refer to the we146 mutant strain, but they neither use it nor mention it in the body of the text. Producing such a mutant strain is great and it should be mentioned in the results, with an explanation as to why they are dying. Otherwise, it should be removed from methods.

    In the Methods, the genotype of the strains is misleading. For example HW1372 : EG6699; xeSi... is not the regular way to write a C. elegans genotype. It should be written as: HW1360 xeSi131[F58H1.2p::GFP::H2B::Pest::unc-54 3', unc-119+] II; unc-119(ed3) III as the strain used to generate the MosSci insertion is described in the paragraph on Transgenic reporter strain generation.

    For the GFP CRISPR strain, the authors write either GFP::GRH-1 in Fig S7, S8 and S9, grh-1::gfp::3xflag in the methods or GRH-1-GFP fusion in the results. The authors should homogenize the way they write this reporter strain. Whether it is an Nterminal or a Cterminal fusion will determine how they should label it.

    Enlarge the font size for Fig S8 and 9 for the scale bar.

    Significance

    The author's prior analysis (Meeuse, et al 2020) showed that mRNA oscillations are coupled with developmental processes, including molting. The present paper extends that finding by showing that oscillating transcript levels are directly linked to a rhythmic recruitment of the RNAPolII on their promoter. Then Meeuse and her colleagues use the molting as a model system to access the importance of oscillating TF for rhythmic processes. Through an RNAi screen, they have identified 6 candidates involved in the molting process. One of the candidates, grh-1, is characterized further. They combine a quantitative-based analysis (luciferase assay) with a time and dose-controlled degradation of GRH-1 to clearly describe the impact of grh-1 depletion on molting. This time and dose control is very smart and key to their study. Overall, the paper adds some interesting piece of information to the field of rhythmic control of molting cycles, as it shows that oscillating transcription factors provide a developmental clock in this process. But this notion is not completely new, as it has been shown in other developmental processes like the circadian clock. Moreover, how molting cycles are controlled by GRH-1 remains to be elucidated.

    My field of expertise is GNRs studies, the genetic of C. elegans, embryonic and larval development in C.elegans, timelapse and confocal imaging. I do not have expertise in Chip-Seq analysis.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this paper, Meeuse et al. analyze the determinants of rhythmical molting in C. elegans using a combination of RNA-polymerase ChIP-seq, RNA-seq, RNAi and imaging coupled to targeted degradation. The main finding is the discovery of the importance for the molting process of GRH-1, a transcription factor homolog to Vertebrate Grainyhead, as well as the identification of 5 additional transcription factors for which molting is defective.

    Major comments:

    The experiments are generally well performed, and the results convincingly demonstrate the function of GRH-1 in molting.

    The coupled RNA-seq and ChIP-seq experiment (Fig. 1A/B) was done only once.

    One of the claims (p3) is that rhythmic transcription of oscillating genes is driven by rhythmic RNA pol II occupancy. If this is suggested visually by Figure 1A/B, I think this claim merits further statistical analysis. How is ChIP-seq correlated with RNA-seq globally and at the individual gene level? How good is the correlation? Can the authors provide a supplementary table with this correlation at the gene level? In the same paragraph, a couple examples of genes for which RNA polII ChIP does not correlate with RNA-seq would be helpful to the reader (they are currently cited as "instances where oscillating mRNA levels were not accompanied by rhythmic RNAPII promoter binding"). Please provide gene names.

    The oscillatory transcription of several promoters is tested using GFP fusions coupled with q-RT PCR. It is not clear to me whether these experiments were repeated. Additionally, the authors state that each qPCR was repeated (only once), hence both data points should be shown in the graphs on Fig. 1C and S2.

    The authors perform then RNAi knock-down of 92 transcription factors involved in molting using bioluminescence and identify 6 genes involved in molts, three of which have been characterized previously. They focus on one of the other three, grh-1, as its orthologs are involved in epidermal biology in other organisms. Targeted degradation of GRH-1 using the auxin degron confirms the function of GRH-1 in each molt, in an auxin-concentration dependent manner. GFP tagging of GRH-1 shows an accumulation prior to each molt, suggesting the transcription factor is necessary for the onset of molting. As the GRH-1 target site is known (PMID 12888489), the paper would be greatly strengthened if the authors could loop back to the RNA-seq/ChIP-seq dataset and highlight which cycling genes have indeed a GRH-1 binding site in their promoter sequence and whether this correlates with one specific phase of the molting cycle.

    Similar to the ChIP/RNA-seq experiments, it is not clear to me whether the different bioluminescence experiments were performed once or twice.

    As stated above, the results are very convincing and the conclusions quite clear. Additionally, all wet lab experiments are described in very many details. However, I feel that the description of the data analysis is too succinct to allow reproducing the experiments, even using the GEO data. I would expect the authors to provide a github public link with the scripts to perform the RNA-seq and ChIP-seq analyses, the Matlab scripts to analyze bioluminescence experiments (Fig. 2,5,7) and the CNN used to analyze single worm molting, even if the method is to be described elsewhere.

    Minor comments:

    The authors should provide descriptive statistics for their high-throughput sequencing (read number, mapping statistics etc...).

    In figure 1C, it would be helpful to the reader to write peak phase and amplitude of the tested genes on the graph.

    In the same figures, for gene F58H1.2, for which the correlation between the reporter and the endogenous gene is not perfect, the authors "suspect [...] that the reporter may lack relevant promoter or intronic enhancer elements". An alternative explanation is that post-transcriptional regulation occurs for this mRNA, a hypothesis which should be added to the text.

    Significance

    As stated above, this manuscript highlights convincingly that GRH-1 is involved in the molting cycle in the nematode C. elegans, a conserved function of the gene between evolutionary distant species for skin biology.

    Field of expertise: C. elegans, high-throughput sequencing methods, imaging.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Meeuse and colleagues describes a series of detailed and elegant experiments addressing the molecular mechanisms underlying oscillatory gene expression patterns in the nematode Caenorhabditis elegans and how these are required for molting between larval stages. By performing ChIP-seq on synchronised populations at 12 different timepoints (each separated by 1h), the authors find that RNA pol II (RNAPII) occupancy at >2000 promoters shows an oscillating behaviour that is paralleled by similar changes in mRNA abundance. To identify transcription factors (TFs) involved in generating these cycles, the authors conducted a candidate RNAi screen on 92 TFs annotated as been expressed in an oscillating manner themselves. Monitoring developmental progression of individual animals by a luminescence-based assay, the authors identify 6 TFs that caused either death (or arrest) after aberrant molts (3 TFs) or prolonged duration on molts (3 TFs). One of these, the Grainyhead/LSF transcription factor GRH-1 is then investigated in further details. Temporal control of GRH-1 depletion is achieved by TIR1/auxin-mediated protein degradation. This revealed that GRH-1 is required during each larval molt. In agreement with the criteria for including grh-1 among the candidate RNAi clones, GRH-1 protein levels are shown to peak immediately before entry to each molt.

    I am very enthusiastic about the manuscript at several levels: conceptualisation, experimental design, quality of data and clarity of text and discussion. I have only two comments in the category of "major comments":

    1. I do not see how the experiments presented in Figure 7 can be used as argument to conclude that "Molting requires oscillatory GRH-1 activity" (title of last Results section and also reflected in the title of the manuscript). I think the experiment nicely shows that there is a timepoint beyond which removal of GRH-1 no longer interferes with the upcoming molt but that doesn't imply that GRH-1 necessarily needs to oscillate. Stronger evidence could be provided by inducible, non-oscillating GRH-1 expression.
    2. After reading the manuscript, one is left with the obvious question: which of the many oscillating genes are direct targets of the oscillating TF GRH-1? Experiments to answer this are not strictly needed for the current manuscript, which stands perfectly on its own, but it would make a significant increase in the overall understanding of oscillating genes and GRH-1 in particular.

    Minor comments:

    The sampling used to generate the data represented in Figure 1 correspond to from 22 hours until 33 hours of post-embryonic development at 25{degree sign}C. It would be useful to indicate how these time points relate to larval development L1-L4.

    The authors state that co-oscillation of RNAPII and mRNA is not observed for all genes. Although this might indeed be due to technical limitations are suggested by the authors, it would be relevant to provide more information (e.g. number or percentage of genes).

    Are the images of GRH-1::GFP expression in larvae (Fig S8) and adults (Fig S9) acquired with identical settings? I assume so, but it is not obvious, particularly because the images are in separate figures.

    Have the authors examined if GRH-1 activity is not only controlled by oscillating transcription, but also post-translationally (e.g. phosphorylation, nuclear import, etc.) as is the case for many TFs. The authors could potentially "overlay" the GRH-1::GFP data with transcriptional data (available at least for 22-33h) to see if the shapes coincide.

    Significance

    Oscillating genes are found in many biological contexts and are fascinating examples of tightly controlled gene expression. The Großhans laboratory has previously identified close to 4,000 oscillating transcripts and is a leader in this field. The current manuscript incorporates a variety of sophisticated techniques that together enable the authors to identify six genes that are required for rhythmic molting in C. elegans. The protein most deeply studied in the manuscript, GRH-1, is homologous to Grainyhead, which is involved in ECM remodeling and other cyclic processes. The findings in this manuscript are therefore of potential relevance across a broad evolutionary scale.

  6. Version published to 10.1101/2022.03.01.482504v2 on bioRxiv
  7. Version published to 10.1101/2022.03.01.482504v1 on bioRxiv