Dual Inhibition of MYRF Cleavage by Its JM Region and PAN-1 CCT Gates Developmental Timing in C. elegans
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eLife Assessment
This important study advances our understanding of developmental timing mechanisms by studying the cleavage, nuclear translocation, and oscillation of the transcription factor MYRF-1 (vertebrate MYRF) during C. elegans larval development. The evidence supporting the conclusions is solid, with elegant genome engineering experiments and state-of-the-art microscopy. The work will be of broad interest to cell and developmental biologists.
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Abstract
Post-embryonic development proceeds through discrete stages, yet the mechanisms that coordinate transitions between stages remain incompletely defined. The transmembrane transcription factor MYRF undergoes intramolecular cleavage to release a nuclear-localized fragment essential for developmental progression in C. elegans . Previously, we showed that PAN-1 as a key partner required for MYRF activity during development. PAN-1 promotes MYRF trafficking to the cell membrane—an essential step for MYRF cleavage—through interactions between their extracellular domains (Xia et al., 2021). Here, we show that MYRF-1 cleavage and nuclear translocation oscillate with larval stage transitions in C. elegans , peaking mid-to-late stage and being suppressed during molts. Using endogenous gene editing and mutant reporters, we identify an uncharacterized juxtamembrane (JM) region in MYRF-1 that self-inhibits cleavage. JM deletion triggers premature MYRF-1 nuclear entry, early lin-4 activation, growth defects, and adult lethality. We further demonstrate that the cytoplasmic tail (CCT) of the transmembrane protein PAN-1 acts as a predominant trans-inhibitor of MYRF-1 cleavage, coupling extracellular association with cytoplasmic inhibition. PAN-1 CCT deletion causes near-constitutive MYRF-1 nuclear accumulation, leading to premature lin-4 activation, accelerated M-cell division, and larval lethality. Removing these inhibitory mechanisms on MYRF-1 cleavage overrides nutrient-responsive developmental checkpoints. These findings uncover dual inhibitory mechanisms governing MYRF-1 cleavage and establish MYRF-1 cleavage as a key gatekeeper of developmental timing.
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eLife Assessment
This important study advances our understanding of developmental timing mechanisms by studying the cleavage, nuclear translocation, and oscillation of the transcription factor MYRF-1 (vertebrate MYRF) during C. elegans larval development. The evidence supporting the conclusions is solid, with elegant genome engineering experiments and state-of-the-art microscopy. The work will be of broad interest to cell and developmental biologists.
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Reviewer #1 (Public review):
The current study is a follow-up to a previously published article in eLife in 2021, demonstrating that the transcription factor MYRF-1 interacts with the transmembrane protein PAN-1, which is required for the stability and targeting of MYRF-1 to the plasma membrane. There, MYRF-1 undergoes self-catalytic cleavage of its intracellular domain and translocates to the nucleus. Here, the authors analyze the activation of MYRF-1 during the larval development of C. elegans. They nicely show that MYRF-1 cleavage and nuclear translocation oscillate with larval stage transitions. They further identify two regions in MYRF-1 and PAN-1 that negatively regulate MYRF-1 cleavage and activation, and show that relief of this negative regulation causes premature lin-4 activation and overrides nutrient-responsive developmental …
Reviewer #1 (Public review):
The current study is a follow-up to a previously published article in eLife in 2021, demonstrating that the transcription factor MYRF-1 interacts with the transmembrane protein PAN-1, which is required for the stability and targeting of MYRF-1 to the plasma membrane. There, MYRF-1 undergoes self-catalytic cleavage of its intracellular domain and translocates to the nucleus. Here, the authors analyze the activation of MYRF-1 during the larval development of C. elegans. They nicely show that MYRF-1 cleavage and nuclear translocation oscillate with larval stage transitions. They further identify two regions in MYRF-1 and PAN-1 that negatively regulate MYRF-1 cleavage and activation, and show that relief of this negative regulation causes premature lin-4 activation and overrides nutrient-responsive developmental checkpoints. The experiments are elegant and accurately support the conclusions raised. There are only minor comments and suggestions to improve the manuscript.
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Reviewer #2 (Public review):
In this study, Xu et al. investigated the regulatory mechanisms controlling intramolecular cleavage of the transmembrane transcription factor MYRF-1, an important event that controls developmental progression in C. elegans.
The authors made important advances in several aspects:
(1) Through endogenous gene editing/tagging, further supported by western blots, the authors convincingly demonstrate the novel finding that the intramolecular cleavage and nuclear translocation of MYRF-1 is not static, but temporally controlled within each developmental stage: with nuclear translocation peaking at the late stage and then declining into lethargus/molts between developmental stages (Figure 1).
(2) They demonstrate that this cleavage and nuclear translocation is controlled by external stimuli, namely starvation.
(3) …
Reviewer #2 (Public review):
In this study, Xu et al. investigated the regulatory mechanisms controlling intramolecular cleavage of the transmembrane transcription factor MYRF-1, an important event that controls developmental progression in C. elegans.
The authors made important advances in several aspects:
(1) Through endogenous gene editing/tagging, further supported by western blots, the authors convincingly demonstrate the novel finding that the intramolecular cleavage and nuclear translocation of MYRF-1 is not static, but temporally controlled within each developmental stage: with nuclear translocation peaking at the late stage and then declining into lethargus/molts between developmental stages (Figure 1).
(2) They demonstrate that this cleavage and nuclear translocation is controlled by external stimuli, namely starvation.
(3) They reveal modes of regulation of the intramolecular cleavage that is mildly regulated by MYRF-1's own JM domain as well as the CCT tail of interacting partner PAN-1.
The conclusions of this paper are mostly well supported by data, but some aspects of the manuscript and conclusions should be clarified and extended to strengthen its findings.
(1) The authors concluded that the intramolecular cleavage and nuclear localization of MYRF-1 were similarly temporally-regulated in all tissue types. However, the data/image presented was limited to specific regions/cell types that were inconsistently chosen across developmental windows. For example, for the cleavage/nuclear translocation across L1 into lethargus (Figures 1B, E, F, G), the heads of the worm were shown to comprise mostly neurons and muscles. While across the rest of the larval stages, only mid-body pictures were shown, comprising mostly hypodermal and some intestinal cells. A complete coverage of all tissues across all time points would better support the author's conclusion that this temporal regulation occurs similarly in all tissue types. Additionally, the authors should clearly indicate which tissue/cell-types were used in the quantifications, as these were not done for several figure panels (including but not limited to Figure 1I and J).
(2) Related to point 1 above, this inconsistency in tissue assessment was also true for downstream experiments (Figures 2-6; e.g., starvation, JM, and CCT regulation, etc.). Broad tissue specific assessment for all downstream experiments would greatly enhance the strength and relevance of the findings. Judging by the current data presented (Figures 3, 5, 6), it seems to suggest that there are tissue/cell-type differences in the regulation of MYRF-1 nuclear translocation.
(3) Developmental progression was superficially and inconsistently assessed across the study. Developmental progression was mainly assessed by hypodermal (V-lineage) division patterns and worm length in this study. Several glaring omissions that should have been examined were the lengths of larval stages/lethargus and molting defects, as well as gonad development, to help identify which developmental landmarks were affected vs. not.
(4) The phosphorylation within MYRF-1's JM domain was insufficiently investigated. There were two serine phosphorylation sites that were discovered through mass spectrometry experiments, however the authors only investigated one of the serine (S623) residues without any justifications for the choice. Additional investigation of the other residues, as well as both together, would strengthen the relevance of these phosphorylation events to cleavage and nuclear translocation, especially considering the minimal effect observed with only mutating the one residue.
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Reviewer #3 (Public review):
Summary:
In this paper, the authors identified dual inhibitory mechanisms, an intrinsic juxtamembrane (JM) region and an extrinsic cytoplasmic tail (CCT) domain in the binding protein PAN-1, that suppress MYRF-1 cleavage in C. elegans. The authors showed that MYRF-1 cleavage oscillates across larval stages, peaking in mid-to-late phases and being suppressed during molts. This oscillatory pattern is consistent with MYRF-1's role in promoting transitions of larval stages, particularly in late-L1 involving lin-4 activation and DD neuron remodeling.
Strengths:
This work generated several knock-in strains of fluorescent tags and mutations in the endogenous myrf-1 and pan-1gene loci, which will provide resources for future identification and characterization of the underlying molecular mechanisms regulating MYRF-1 …
Reviewer #3 (Public review):
Summary:
In this paper, the authors identified dual inhibitory mechanisms, an intrinsic juxtamembrane (JM) region and an extrinsic cytoplasmic tail (CCT) domain in the binding protein PAN-1, that suppress MYRF-1 cleavage in C. elegans. The authors showed that MYRF-1 cleavage oscillates across larval stages, peaking in mid-to-late phases and being suppressed during molts. This oscillatory pattern is consistent with MYRF-1's role in promoting transitions of larval stages, particularly in late-L1 involving lin-4 activation and DD neuron remodeling.
Strengths:
This work generated several knock-in strains of fluorescent tags and mutations in the endogenous myrf-1 and pan-1gene loci, which will provide resources for future identification and characterization of the underlying molecular mechanisms regulating MYRF-1 cleavage inhibition.
The results presented in the paper are solid enough to support the paper's main conclusions.
This study is valuable for establishing MYRF-1 cleavage as a key gatekeeper of the C. elegans developmental timing. Findings from C. elegans MYRF-1 may provide insight into the regulation and function of mammalian MYRF.
Weaknesses:
The following points should be discussed to further support the authors' model that MYRF-1 cleavage is a key gatekeeper of developmental timing.
(1) Recent findings by Helge Großhans and Jordan Ward groups showed that KIN-20 (CK1δ) and LIN-42 (PERIOD) are required for proper molt timing in C. elegans, and that loss of LIN-42 binding or of the phosphorylated LIN-42 tail impairs nuclear accumulation of KIN-20, resulting in arrhythmic molts (EMBO J. 44, 6368-6396, 2025). In this paper, the authors concluded that PAN-1 promotes MYRF trafficking to the cell membrane, where MYRF-1 cleavage and nuclear translocation occur, and that oscillates with developmental molting cycles in C. elegans. It is unclear whether MYRF-1 and KIN-20 interact in the nucleus and, if so, how this interaction controls developmental timing.
(2) Separately, it was previously shown that the let-7 primary transcript (pri-let-7) exhibits oscillating, pulse-like expression that peaks during each larval stage, rather than a steady increase, and directly correlates with developmental molting cycles. It is unclear whether the nuclear-localized MYRF-1 fragment regulates the oscillatory primary let-7 expression during larval transition (McCulloch and Rougvie, 2014; Van Wynsberghe et al., 2011).
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