O-GlcNAcylation of the intellectual disability protein DDX3X exerts proteostatic cell cycle control

  1. Conor W. Mitchell
  2. Huijie Yuan
  3. Marie Sønderstrup-Jensen
  4. Florence Authier
  5. Alfonso Manuel D’Alessio
  6. Andrew T. Ferenbach
  7. Daan M.F. van Aalten

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

O-GlcNAcylation is an evolutionary conserved post-translational modification implicated in neurodevelopment. Missense variants of O-GlcNAc transferase (OGT) are causal for the intellectual disability syndrome OGT Congenital Disorder of Glycosylation (OGT-CDG). The observation of microcephaly in OGT-CDG patients suggests that dysregulation of the cell cycle and aberrant neurogenesis may contribute to disease aetiology. Here, we identify Ser584 O-GlcNAcylation of DDX3X, a known intellectual disability and microcephaly associated protein, as a key regulator of G1/S-phase transition, inhibiting proteasome-dependent degradation of DDX3X. DDX3X levels are reduced in a mouse model of OGT-CDG, alongside the DDX3X-target gene and synaptogenic regulator cyclin E1. These data reveal how a single DDX3X O-GlcNAc site exerts control of the cell cycle and highlights dysregulation of DDX3X-dependent translation, and concomitant impairments in cortical neurogenesis, as a possible pathway disrupted in OGT-CDG.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Reviewer 1:

    Although HEK cells are effective for studying molecular mechanisms and post-translational modifications through siRNA and variant overexpression manipulations, they lack functional relevance in a neuronal context. Consequently, the connection between molecular findings and observed phenotypes in mice is tenuous. It is suggested that the authors attempt to replicate these results (Figures 4 and 5) using a neuronal differentiation model employing ESCs or iPSCs.

    We have previously attempted to generate DDX3XSer584Ala knock-in ESCs via CRISPR-Cas9 because, as the reviewer points out, this would facilitate investigating the role of DDX3X O-GlcNAcylation in a neuronal differentiation model. However, clones derived after puromycin selection stop proliferating and perish during clonal outgrowth - we will include a statement to this effect in the revised manuscript. A similar phenomenon has been reported previously in Neuro2a cells by Lennox et al. (2020), who reported that installation of DDX3X patient variants is potentially toxic in certain cell lines. Therefore, the HEK293 KD/overexpression approach, also used for the study of clinically relevant DDX3X variants in other studies, while sub-optimal, is the best possible currently accessible model.

    While employing variant overexpression following siRNA-mediated reduction of the endogenous protein is a direct method to illustrate the effects of mutated DDX3X variants, the authors stress a connection between this regulatory mechanism and neurodevelopmental defects. Therefore, it would be justifiable for the authors to create cell lines by editing the endogenous DDX3X gene and demonstrate the effects of O-GlcNAc, disruption of DDX3X target levels, and cell cycle regulation. Combining these approaches (from points 1 and 2), the authors could generate iPSC/ESC lines containing the DDX3X mutations and examine their effects within a neuronal differentiation context. Such an approach would significantly enhance the impact of this study.

    As mentioned above for point 1, we have previously tried to edit the endogenous DDX3X gene with a Ser584Ala point mutation for this purpose. However, after trying this approach in both ESCs and HEK293T cells, we consistently observed stalled proliferation and cell death during clonal outgrowth. Therefore, as desirable as this experiment is, we are limited to siRNA-mediated reduction of DDX3X and rescue via over-expression, as also extensively used in other studies.

    Given the points raised by reviewers 1 and 3 about the appropriateness of model system and the links drawn between DDX3X O-GlcNAcylation and neurodevelopmental defects, we will revise the manuscript to highlight the correlative nature of this link. In addition we will add further data from OGT-CDG mouse models that strengthens this possible link (see response to point 4 by reviewer 1).

    The finding of diminished DDX3X levels in OGT mutant mice and the consequent reduction in O-GlcNAc represents a pivotal connection to the observed neurodevelopmental defects in OGT-CDG. However, this aspect of the research remains somewhat unclear, as it has not been definitively demonstrated that O-GlcNAc levels of DDX3X in OGT mutant mice are indeed decreased. Without this confirmation, the causal relationship between OGT malfunction, O-GlcNAc, and reduced DDX3X levels cannot be firmly established. There is a possibility of indirect effects, and merely observing correlation does not suffice to draw the robust conclusions presented in this paper. To address the uncertainty surrounding Figure 6D, attributed to the antibody's declared lack of specificity, the authors should conduct additional experiments.

    The aim of this study is not to confirm whether there is a causal link between OGT catalytic deficiency and DDX3X, but rather to report the function of DDX3X O-GlcNAcylation and propose a possible link between OGT catalytic deficiency, DDX3X loss of activity, and neurodevelopmental defects. However, we do agree that further investigation is required to determine whether there is a correlative link between OGT catalytic deficiency and DDX3X levels (and O-GlcNAcylation) in the mouse brain. Towards this end, we will repeat the immunoprecipitation of DDX3X from mouse brain lysate of wild type and OGT-CDG mice and blot for O-GlcNAc using different pan-specific O-GlcNAc antibodies/ far western techniques (CTD110.6, GST-CpOGAD298N). We will also incorporate an internal negative control (competition with free GlcNAc) to verify the specificity of the O-GlcNAc signals.

    The study places significant emphasis on this phenotype and seeks to elucidate it, at least partially, through the O-GlcNAcylation of DDX3X. However, a precise description or depiction of this phenotype is absent. Understanding the phenotype of the OGT-CDG mice necessitates consulting existing literature. The authors ought to contemplate providing brain sections with relevant staining to (i) showcase the microcephaly phenotype and (ii) bolster their assertion regarding the dysregulated cell cycle by utilising appropriate marker stainings for the progenitor cells during embryonic development.

    We have recently published a manuscript reporting a mouse model of OGT-CDG (OGTC921Y). OGTC921Y mice display microcephaly as determined by brain weight and skull length. An additional mouse model of the N648Y OGT-CDG variant also displays microcephaly (Authier et al., 2024 reports the C921Y mouse; the N648Y mice line is on BioRxiv: https://doi.org/10.1101/2023.08.23.554427 and subject to peer review elsewhere). As part of the revisions for this manuscript, we will report a micro CT-based analysis of reduced skull/brain volume that supports the microcephaly phenotype. O-GlcNAc, OGT, OGA, DDX3X and cyclin E1 western blots for three brain regions from these mouse models will also be provided. Furthermore, we will include NeuN staining of cortical brain sections to establish whether cortical density is reduced in OGTN648Y mouse brains. The proposed staining of progenitor cells during embryonic corticogenesis is a very good suggestion for future investigation, but is a time-consuming experiment (est. 1 year) that falls beyond the scope of this study and would delay sharing of our current findings.

    The proposed staining of progenitor cells during embryonic corticogenesis is a time consuming experiment (est. 1 year) that falls beyond the scope of this study.

    Reviewer 2:

    Figure 6 D - text last paragraph on page 16, and in supplement where you use RL2 - You need to do the control to show that RL2 staining goes away in the presence of free GlcNAc or when you galactosylate the protein. This control would indicate that you are detecting the sugar, not the protein backbone.

    We agree with the reviewer that an internal negative control will help validate the specificity of the RL2 signal. We will repeat the immunoprecipitation/ O-GlcNAc western blot with a free GlcNAc control.

    • Reviewer 3:*

    Lack of Appropriate Model System: Although the authors initially address the role of O-GlcNAcylation (OGT/OGA) and DDX3X in cerebral development, given that mutations in these enzymes are causative for neurological malformations and disabilities, the experiments addressing the consequences of DDX3X or OGT silencing are predominantly performed in HEK293T cells rather than a nervous system model. This limits the relevance of the findings.

    As discussed above (see Reviewer 1, points 1 and 2), we have previously tried to generate ESC knock-ins of the DDX3XSer584Alain ESCs to establish a neuronal differentiation model. However, clones perish during outgrowth, and thus these experiments are not possible. We have however performed preliminary biochemical analysis of DDX3X levels in the brains of OGT-CDG mice. It is important to emphasise that whether there is a causal link between OGT catalytic deficiency, DDX3X and cerebral development, lies beyond the remit of this manuscript. This manuscript aims to highlight DDX3X loss of activity as a candidate conveyor of neurodevelopmental defects in the mouse brain.

    Inconclusive Cell Cycle Analysis: The authors' analysis regarding cell cycle characterization is not sufficiently conclusive. First, they need to accurately define the link between DDX3X and cyclin E1. The study they refer to (Lai et al., 2010) is rather superficial, and requires a more in-depth analysis, in order to appreciate the existing link between the two given molecules. Indeed, the current experiments do not clarify whether cells are stuck in G1 due to cyclin E1 downregulation or if cyclin E1 is downregulated because cells are blocked in G1. A suggested approach would be to perform rescue experiments with cyclin E1 overexpression, by using Quantitative Image-Based Cytometry (QIBC) or flow cytometry (EdU incorporation + Hoecsht staining) to monitor cell cycle changes and define the interplay between these molecules mechanistically. However, this alone cannot exclude the presence of alternative substrates of DDX3X regulation influencing cell cycle phase transition. A more holistic approach, such as an interactome analysis through mass spectrometry, may be helpful. Additionally, mild mitotic stress often results in cell cycle arrest in the subsequent G0/G1 phase, which can resemble the G1/S transition impairment described by the authors. Consequently, the statement "Here we identify Ser584 O-GlcNAcylation of DDX3X (...) as a key regulator of G1/S transition" is not well-supported (to do so, the authors should define the temporal pattern of such post-translational modification). It would also be interesting to determine whether the reduction in cell viability is due to a simple slowdown of the cell cycle or apoptotic induction.

    We agree that performing cyclin E1 over-expression could provide mechanistic insights into the link between DDX3X, cyclin E1, and the cell cycle. We will therefore repeat the cell cycle analysis by flow cytometry shown in Fig. 5B with the addition of cyclin E1 over-expression in cells co-transfected with siRNA against DDX3X and siRNA-resistant DDX3XSer584Ala, to investigate whether cyclin E1 rescues the observed accumulation of cells in G1.

    We acknowledge the reviewer's point that quiescence (G0 entry) and G1/S stalling can provide similar cell cycle profiles to that observed in Fig. 5B. We will therefore re-write the necessary sections of the manuscript to emphasise that we cannot be certain whether the observed cell cycle defects resulting from loss of DDX3X Ser584 O-GlcNAcylation stem from G1/S phase stalling or mitotic stress followed by quiescence.

    Regarding the possibility that our observed reductions in the number of viable cells stems from stalled cell cycle progress or apoptosis, we will knock-down DDX3X and blot for cleaved caspase-3 as a marker for apoptosis to investigate the latter.

    The proposed interactome analysis of DDX3X is a tangential experiment. Given that DDX3X regulates cell cycle progression through its RNA helicase and transcriptional co-regulator functions, interactome analysis would not provide direct (or indeed, useful) readouts of how loss of DDX3X Ser584 O-GlcNAcylation affects cell cycle progression (i.e. it is possible there are significant effects on DDX3X activity without affecting the interactome).

    Weak Correlative Link: While the link between OGT/OGA and the cell cycle is well-established (seereviewSaunders et al., 2023) due to the multitude of targets subjected to this post-translational regulation, the correlative link between DDX3X mutations and cell cycle effects is further weakened by the fact that cyclin E1 knockout mice are indistinguishable from their wild-type littermates.

    DDX3X controls the transcription and translation of hundreds of genes, not just cyclin E1. For example, DDX3X controls Klf4 transcription which, in certain cell lines, regulates S-phase entry (Canizarro et al., FEBS Lett. 2018). Thus, it is possible for one candidate conveyor of the observed cell cycle defects to not produce significant phenotypes in a KO mouse. In the interests of emphasising this point, we will add a discussion point regarding the multiple pathways through which loss of DDX3X O-GlcNAcylation may affect the cell cycle in the discussion.

    Throughout the text, the authors refer to figures out of alphanumerical order, making the reading experience extremely difficult. To enhance readability, it is essential to present figures in a logical, sequential manner. Additionally, for further comments I would suggest to implement the text with line numbers.

    We will correct the text to ensure all figures are referenced and presented in a sequential manner, and line numbers will be added.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In the manuscript, Mitchell et al. explore the interplay between O-GlcNAcylation and the cell cycle, with a particular focus on how DDX3X, an RNA helicase of the DEAD box family, influences the G1/S phase transition. The authors begin with a bioinformatics strategy to identify patterns of correlation between cell cycle regulators and the enzymes OGT and OGA in a dataset of temporal transcriptomics of the human prefrontal cortex. They then narrow their analysis to DDX3X, due to its strong potential correlation with the cell cycle and its candidacy as a substrate for O-GlcNAcylation. Subsequently, the authors biochemically investigate how O-GlcNAcylation regulates DDX3X. Finally, given that DDX3X has been previously shown to regulate cyclin E1, the authors assess the effect of DDX3X depletion on the cell cycle in vitro.

    Major comments:

    Despite the authors' interesting identification of a novel substrate for O-GlcNAcylation, most conclusions drawn from the study are correlative. The manuscript suffers from three major drawbacks:

    1. Lack of Appropriate Model System: Although the authors initially address the role of O-GlcNAcylation (OGT/OGA) and DDX3X in cerebral development, given that mutations in these enzymes are causative for neurological malformations and disabilities, the experiments addressing the consequences of DDX3X or OGT silencing are predominantly performed in HEK293T cells rather than a nervous system model. This limits the relevance of the findings.
    2. Inconclusive Cell Cycle Analysis: The authors' analysis regarding cell cycle characterization is not sufficiently conclusive. First, they need to accurately define the link between DDX3X and cyclin E1. The study they refer to (Lai et al., 2010) is rather superficial, and requires a more in-depth analysis, in order to appreciate the existing link between the two given molecules. Indeed, the current experiments do not clarify whether cells are stuck in G1 due to cyclin E1 downregulation or if cyclin E1 is downregulated because cells are blocked in G1. A suggested approach would be to perform rescue experiments with cyclin E1 overexpression, by using Quantitative Image-Based Cytometry (QIBC) or flow cytometry (EdU incorporation + Hoecsht staining) to monitor cell cycle changes and define the interplay between these molecules mechanistically. However, this alone cannot exclude the presence of alternative substrates of DDX3X regulation influencing cell cycle phase transition. A more holistic approach, such as an interactome analysis through mass spectrometry, may be helpful. Additionally, mild mitotic stress often results in cell cycle arrest in the subsequent G0/G1 phase, which can resemble the G1/S transition impairment described by the authors. Consequently, the statement "Here we identify Ser584 O-GlcNAcylation of DDX3X (...) as a key regulator of G1/S transition" is not well-supported (to do so, the authors should define the temporal pattern of such post-translational modification). It would also be interesting to determine whether the reduction in cell viability is due to a simple slowdown of the cell cycle or apoptotic induction.
    3. Weak Correlative Link: While the link between OGT/OGA and the cell cycle is well-established (see review Saunders et al., 2023) due to the multitude of targets subjected to this post-translational regulation, the correlative link between DDX3X mutations and cell cycle effects is further weakened by the fact that cyclin E1 knockout mice are indistinguishable from their wild-type littermates.

    Minor comments:

    Throughout the text, the authors refer to figures out of alphanumerical order, making the reading experience extremely difficult. To enhance readability, it is essential to present figures in a logical, sequential manner. Additionally, for further comments I would suggest to implement the text with line numbers.

    Significance

    Overall, the major novelty of the manuscript is the interesting link between DDX3X, its O-GlcNAcylation, and cell cycle regulation. However, this section requires the most intense revision to ensure robustness and clarity of the findings.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Missense mutations in OGT are causative in X-linked intellectual disability disorders, termed OGT-CDG. This paper identifies O-GlcNAcylation (OGN) at Ser584 of DDX3X, a known intellectual disability and microcephaly associated protein, as a regulator of G1/S transition, inhibiting the proteasome degradation of DDX3X. This OGN site controls the degradation of DDX3X. Lack of OGN at Ser 584 results in more rapid degradation of the protein, which affects the cell cycle. The study shows that dysregulation of DDX3X-dependent translation and concomitant impairments in cortical neurogenesis as a pathway disrupted in OGT-CDG. Overall, a well written paper. The data support the author's conclusions.

    Figure 6 D - text last paragraph on page 16, and in supplement where you use RL2 - You need to do the control to show that RL2 staining goes away in the presence of free GlcNAc or when you galactosylate the protein. This control would indicate that you are detecting the sugar, not the protein backbone.

    Significance

    This well prepared paper is highly significant. 1) It provides mechanistic insights into a cause of human intellectual disability; 2) It helps elucidate the role of O-GlcNAcylation in nutrient regulation of the cell cycle. 3) It provides more data on the roles of DDX3X.

    The paper is of significant general interest.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    O-GlcNAcylation is a post-translational modification and plays a crucial role in neurodevelopment. Variants of O-GlcNAc transferase (OGT) are linked to OGT Congenital Disorder of Glycosylation (OGT-CDG), a syndrome causing intellectual disability. Microcephaly in OGT-CDG patients suggests the involvement of cell cycle dysregulation and abnormal neurogenesis. Mitchell et al. identify Ser584 O-GlcNAcylation of DDX3X, a protein associated with intellectual disability and microcephaly, as a regulator of G1/S-phase transition. They show that this PTM of DDX3X stabilises and prevents the targeting of DDX3X to the proteasome and therefore its degradation. Reduced DDX3X levels in an OGT-CDG mouse model and decreased expression of DDX3X-target gene cyclin E1 suggest impaired cell cycle control and cortical neurogenesis as pathways affected in OGT-CDG.

    This study introduces DDX3X as a novel target for O-GlcNAcylation, which enhances its stability and ensures proper regulation of the cell cycle, particularly through its target cyclin E1. While the findings regarding DDX3X O-GlcNAcylation and the identification of the modified residue are compelling, addressing several issues regarding the link between O-GlcNAc of DDX3X-cell cycle regulation and neurodevelopmental impact would enhance the study's robustness.

    Major points requiring attention:

    1. Although HEK cells are effective for studying molecular mechanisms and post-translational modifications through siRNA and variant overexpression manipulations, they lack functional relevance in a neuronal context. Consequently, the connection between molecular findings and observed phenotypes in mice is tenuous. It is suggested that the authors attempt to replicate these results (Figures 4 and 5) using a neuronal differentiation model employing ESCs or iPSCs.
    2. While employing variant overexpression following siRNA-mediated reduction of the endogenous protein is a direct method to illustrate the effects of mutated DDX3X variants, the authors stress a connection between this regulatory mechanism and neurodevelopmental defects. Therefore, it would be justifiable for the authors to create cell lines by editing the endogenous DDX3X gene and demonstrate the effects of O-GlcNAc, disruption of DDX3X target levels, and cell cycle regulation. Combining these approaches (from points 1 and 2), the authors could generate iPSC/ESC lines containing the DDX3X mutations and examine their effects within a neuronal differentiation context. Such an approach would significantly enhance the impact of this study.
    3. The finding of diminished DDX3X levels in OGT mutant mice and the consequent reduction in O-GlcNAc represents a pivotal connection to the observed neurodevelopmental defects in OGT-CDG. However, this aspect of the research remains somewhat unclear, as it has not been definitively demonstrated that O-GlcNAc levels of DDX3X in OGT mutant mice are indeed decreased. Without this confirmation, the causal relationship between OGT malfunction, O-GlcNAc, and reduced DDX3X levels cannot be firmly established. There is a possibility of indirect effects, and merely observing correlation does not suffice to draw the robust conclusions presented in this paper. To address the uncertainty surrounding Figure 6D, attributed to the antibody's declared lack of specificity, the authors should conduct additional experiments.
    4. The study places significant emphasis on this phenotype and seeks to elucidate it, at least partially, through the O-GlcNAcylation of DDX3X. However, a precise description or depiction of this phenotype is absent. Understanding the phenotype of the OGT-CDG mice necessitates consulting existing literature. The authors ought to contemplate providing brain sections with relevant staining to (i) showcase the microcephaly phenotype and (ii) bolster their assertion regarding the dysregulated cell cycle by utilising appropriate marker stainings for the progenitor cells during embryonic development.

    Minor issue:

    Consider replacing in cellulo with in vitro

    Significance

    The study provides an in-depth biochemical analysis of the O-GlcNAcylation of DDX3X, identifying it as a novel target. However, limitations stem from the absence of a robust causal connection between OGT-DDX3X and neurodevelopmental effects, as well as the utilization of HEK cells rather than a neuronal model. Moreover, the study would gain from exploring endogenous proteins instead of solely relying on siRNA and OE methods to investigate the cellular and functional impacts of DDX3X O-GlcNAcylation. Overall, the study provides a mechanistic advance regarding O-GlcNAc PTM and the targets of OGT. This work could be of interest to audiences interested in neurodevelopment, as well as PTM of non-histone proteins.

  5. Version published to 10.1101/2024.02.28.582457v1 on bioRxiv