Tatton-Brown-Rahman Syndrome-associated DNMT3A mutations de-repress cortical interneuron differentiation to disrupt neuronal network function

  1. Gareth Chapman
  2. Julianna J Determan
  3. John R Edwards
  4. Faiza Batool
  5. James E Huettner
  6. Ramachandran Prakasam
  7. Sydney R Crump
  8. Travis E Law
  9. Haley Jetter
  10. Harrison W Gabel
  11. Kristen L Kroll

  • Curated by eLife

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    eLife Assessment

    This is an important study that develops multiple human iPSC-based models to study the consequences of DNMT3A mutations in Tatton-Brown-Rahman Syndrome. Convincing evidence shows dysregulation of GABAergic interneuron development and function, and the authors identify some of the key signaling mechanisms underlying these changes. This study will be of interest for understanding the functions of DNMT3A in brain development and the causes of neurological dysfunction in Tatton-Brown-Rahman Syndrome.

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Abstract

Pathogenic mutations in DNMT3A cause Tatton-Brown-Rahman Syndrome (TBRS), a disorder characterized by somatic overgrowth of multiple tissues including the brain and intellectual disability (OGID). Here, we investigated TBRS etiology using new human pluripotent stem cell models, modeling varying levels of TBRS-associated loss of DNMT3A function. We identified lineage-specific overgrowth in TBRS ventral forebrain medial ganglionic eminence (MGE)-like progenitors, due in part to increased signaling through the PIK3/AKT/mTOR pathway that could be modulated to ameliorate this phenotype. By contrast, reduced DNA methylation during MGE-like progenitor differentiation into GABAergic interneurons caused premature expression of neuronal and synaptic genes, triggering precocious neuronal maturation. As a result, TBRS GABAergic neurons exhibited sufficient hyperactivity to alter the development and structure of neuronal networks, likely contributing to the intellectual disability and autism spectrum disorder common to TBRS patients. Together, this work elucidates new roles for DNMT3A-mediated gene repression in human cortical development, identifying critical requirements for regulating GABAergic neuron production and neuronal network function. These findings also provide evidence for interrelated pathogenic mechanisms underlying TBRS and other OGIDs, including PIK3CA-related overgrowth syndrome and Weaver Syndrome, providing a foundation and rationale for future studies to identify common paradigms to treat these related disorders.

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  1. Version published to 10.7554/elife.111056.1 on eLife
  2. Version published to 10.7554/elife.111056 on eLife
  3. eLife

    eLife Assessment

    This is an important study that develops multiple human iPSC-based models to study the consequences of DNMT3A mutations in Tatton-Brown-Rahman Syndrome. Convincing evidence shows dysregulation of GABAergic interneuron development and function, and the authors identify some of the key signaling mechanisms underlying these changes. This study will be of interest for understanding the functions of DNMT3A in brain development and the causes of neurological dysfunction in Tatton-Brown-Rahman Syndrome.

  4. eLife

    Reviewer #1 (Public review):

    Summary:

    This is an important study that describes the consequences of the DNMT3A mutation in human neuronal development for the first time. The selective impact of DNMT3A function on GABAergic interneurons is interesting and an important feature of future therapeutics. The claims made in that manuscript are supported by strong evidence for the most part. And the data are of high quality in general and presented well.

    Strengths:

    The strengths of the work include: Characterization of multiple DNMT3A loss-of-function alleles, including two misense variants, R882H, P904L, and a deletion allele. The missense mutation lines both include an ideal control with the same genetic background. The CRISPRi-mediated DNMT3A knockdown has also been included. The study identifies the mTOR-PI3K pathway as a factor of overgrowth issues found in the mutant organoid. In bulk mRNA sequencing and whole-genome bisulfite sequencing, identify hypomethylated genomic regions associated with gene expression repression. Again, this is more pronounced in the ventral organoid compared to the dorsal organoid. In addition, the extensive electrophysiological characterizations with a high-density microelectrode array support the more mature status of mutant interneurons.

    Weaknesses:

    Although a strong study overall, some weaknesses are noted. These include:

    (1) The lack of validation data for the generated iPSCs and hESCs, such as the chromosomal contents, ploidy, and pluripotency states.

    (2) Other weaknesses relate to data interpretation and insufficient discussion of related matters, as detailed in the recommendations to the authors.

    (3) Also, some errors are noted and detailed in the recommendation section.

  5. eLife

    Reviewer #2 (Public review):

    Summary:

    Chapman, Determan et al. investigate how pathogenic mutations in DNMT3A, which cause Tatton-Brown-Rahman Syndrome (TBRS), disrupt human cortical developmental processes using a comprehensive panel of human pluripotent stem cell models spanning DNMT3A loss-of-function severity. The authors aim to identify the cellular and molecular mechanisms underlying TBRS-associated brain overgrowth and intellectual disability, and to test whether mechanistic convergence exists between TBRS and other overgrowth-intellectual disability disorders (OGIDs) caused by mutations in EZH2 (Weaver syndrome) or PIK3CA pathway components. Their central conclusion is that GABAergic interneuron development is selectively vulnerable to DNMT3A mutation, where reduced DNA methylation causes premature de-repression of neuronal and synaptic genes, driving precocious neuronal maturation and hyperactivity sufficient to disrupt neuronal network synchrony. This report adds to a growing literature supporting the vulnerability of GABAergic interneurons in NDDs and further provides a mechanistic view of this vulnerability, potentially convergent across OGIDs. The mechanistic claims around H3K27me3 compensation and mTOR-based therapeutic convergence, while promising, rest on more preliminary evidence and would benefit from the distinction between correlation and mechanism being made more explicit in the text. Overall, this is a compelling study with a rigorous experimental design and novel findings with a potential impact on a better understanding of the OGID pathophysiology.

    Strengths:

    (1) A major strength of this work is the breadth and rigor of the disease modeling approach. Four independent TBRS model systems are used in tandem: a patient-derived iPSC line with isogenic CRISPR-corrected control (R882H), a knock-in hESC model (P904L) with its wild-type isogenic, patient deletion iPSC lines (Del1/2), and CRISPRi knockdown models (G1/G2), collectively spanning a range of DNMT3A loss-of-function that correlates with phenotypic severity. This allelic series design substantially strengthens causal inference beyond what any single isogenic pair could provide.

    (2) The multi-omic integration across matched developmental stages provides a strong mechanistic foundation for the cellular phenotyping and provides significantly enhanced novelty. RNA-seq, whole-genome bisulfite sequencing, and H3K27me3 CUT&Tag are combined in the same cell types, and timepoints show that DNMT3A loss reduces CG methylation at neuronal and synaptic gene loci, leading to premature transcriptional activation.

    (3) The selective vulnerability of ventral (GABAergic) versus dorsal (glutamatergic) progenitors is one of the study's most important findings. This lineage specificity is consistently observed across all model systems and in both 2D and organoid formats, where ventral NPCs show increased proliferation, premature neuronal gene expression, and increased neurogenesis, while dorsal NPCs are largely unaffected at the transcriptomic and cellular level despite exhibiting comparable DNA methylation changes. This adds to a body of emerging work showing GABAergic interneuron vulnerability in NDDs where ubiquitously expressed genes such as chromatin modifiers are perturbed, and provides additional molecular insights into potential mechanisms of "resilience" of dorsal populations.

    (4) The functional characterization follows a logical progression from single-neuron electrophysiology (demonstrating GABAergic hyperactivity with increased action potential amplitude and firing rate) to network-level analysis using high-density multi-electrode arrays. The HD-MEA experimental design - pairing TBRS or control GABAergic neurons with a constant background of control iGlut neurons - cleanly isolates GABAergic dysfunction as the driver of network hypersynchrony.

    Weaknesses:

    (1) The concomitant induction of proliferation and differentiation in TBRS V-NPCs is conceptually striking, since these are generally considered antagonistic developmental programs. The authors partially address this tension by noting that DNMT3A LOF alone is insufficient to initiate neuronal differentiation, i.e., V-NPCs upregulate neuronal and synaptic genes while retaining progenitor identity, implying that transcriptomic priming and commitment to differentiation are decoupled. However, the relationship between the proliferative phenotype and the epigenetic priming phenotype remains mechanistically unresolved. The manuscript documents mTOR pathway upregulation at the protein level and identifies shared DEGs that include proliferative regulators, but it does not establish whether mTOR-driven proliferation and mCG-loss-driven neuronal gene de-repression/enhanced differentiation are causally linked or represent two independent consequences of DNMT3A LOF.

    (2) Relatedly, the rapamycin rescue experiment is a valuable proof-of-concept for the PIK3/AKT/mTOR convergence but is limited to a single dose in a single model (882) with a single readout (Ki67+ proliferation). Given the prominence of mTOR pathway convergence in the manuscript as a potential shared therapeutic avenue across OGIDs, the data supporting this claim are somewhat preliminary. It remains unknown whether mTOR inhibition rescues downstream phenotypes (neurogenesis, gene expression, neuronal maturation) or whether less severe TBRS models respond similarly. This might also help tackle the first comment above. e.g., if mTOR inhibition rescued proliferation but not the transcriptomic priming, that would support two independent mechanisms.

    (3) The claim that H3K27me3 compensates for mCG loss is an important mechanistic point, but the current data do not distinguish between active compensation, in which EZH2 is recruited in response to methylation loss, and functional redundancy, in which H3K27me3 is independently established and becomes the dominant repressive mark once DNA methylation is reduced. The EZH2 knockdown/inhibition experiments show that H3K27me3 is sufficient to maintain repression at hypo-DMR sites, but they do not establish that H3K27me3 gain is itself a response to methylation loss. Because H3K27me3 profiling was performed only in the severe 882 model, it is also unclear whether H3K27me3 gain scales with DNMT3A LOF severity, as a compensatory model would predict. Finally, the EZH2 overexpression rescue is performed in V-NPCs, whereas the compensation model is developed primarily in D-NPCs, making it difficult to assess whether the same mechanism operates in the lineage where it was originally inferred.

    (4) The narrative framing of dorsal neuron development as unaffected by DNMT3A LOF is somewhat at odds with the data presented. The 882 D-NPCs show substantial DNA methylation changes, and TBRS D-INs exhibit what the authors describe as "substantive transcriptomic differences" involving persistent expression of pluripotency and progenitor genes, which seems to be a distinct but potentially significant phenotype. The impact of DNMT3A loss between ventral and dorsal lineages might be more accurately framed as divergent in nature rather than specific to a certain population.

    (5) SST stainings are not entirely convincing. They appear mostly nuclear, and some instances localized to rosettes in organoids, whereas the protein is largely confined to processes and is expected to be found outside progenitor-rich zones like rosettes.

  6. eLife

    Reviewer #3 (Public review):

    Summary:

    In this manuscript, the authors investigated TBRS etiology by using new human pluripotent stem cell models, modeling varying levels of TBRS-associated loss of DNMT3A function. They identified increased lineage-specific proliferation of precursors in TBRS ventral MGE-like progenitors, which they propose was related to increased signaling through the PIK3/AKT/mTOR pathway. Furthermore, they show that reduced DNA methylation during MGE-like progenitor differentiation into GABAergic interneurons can cause a premature expression of neuronal and synaptic genes, triggering precocious neuronal maturation. In conclusion, they propose that TBRS-derived GABAergic neurons exhibit hyperactivity that can alters the development and structure of neuronal networks.

    Strengths:

    Overall, the data presented is convincing, from an early developmental point of view, given that the iPSC-derived 2D cultures or organoids used do not get to reach a mature state. Nonetheless, the data clearly show the effects that deleterious mutations in TBRS can cause during the period of neurogenesis, which was missing in the field.

    Weaknesses:

    (1) Li et al., 2022 (referred to in the manuscript) seems to already show the interplay between H3K27me3 and Dnmt3a discussed in this study i.e., that in the absence of DNA methylation, there is an expansion of polycomb-like repression. These data should be better acknowledged in the paragraph 'Repressive H3K27me3 compensates for severe loss of DNA methylation' (page 9), given it supports the data presented in this manuscript and suggests this as a common mechanism in the interplay between these two repressive marks, as it is well established in the literature.

    (2) The authors should acknowledge that the omics data come from a mixed population of cells.

    (3) The authors are encouraged to further discuss whether the overgrowth observed in ventral GABAergic cultures or organoids compares to the overgrowth observed in diseased patients. One expects MRIs to have been performed in patients and that these could be harnessed to discern if overgrowth occurs in the cortex or ventral regions of the brain.

  7. eLife

    Author response:

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    This is an important study that describes the consequences of the DNMT3A mutation in human neuronal development for the first time. The selective impact of DNMT3A function on GABAergic interneurons is interesting and an important feature of future therapeutics. The claims made in that manuscript are supported by strong evidence for the most part. And the data are of high quality in general and presented well.

    Strengths:

    The strengths of the work include: Characterization of multiple DNMT3A loss-of-function alleles, including two misense variants, R882H, P904L, and a deletion allele. The missense mutation lines both include an ideal control with the same genetic background. The CRISPRi-mediated DNMT3A knockdown has also been included. The study identifies the mTOR-PI3K pathway as a factor of overgrowth issues found in the mutant organoid. In bulk mRNA sequencing and whole-genome bisulfite sequencing, identify hypomethylated genomic regions associated with gene expression repression. Again, this is more pronounced in the ventral organoid compared to the dorsal organoid. In addition, the extensive electrophysiological characterizations with a high-density microelectrode array support the more mature status of mutant interneurons.

    Weaknesses:

    Although a strong study overall, some weaknesses are noted. These include:

    (1) The lack of validation data for the generated iPSCs and hESCs, such as the chromosomal contents, ploidy, and pluripotency states.

    We thank the reviewer for their constructive feedback. We previously validated our 882 models with whole genome sequencing and teratoma formation upon mouse fat pad injection, while the parental human embryonic stem cell line (WA01 hESCs) used for P904L variant knock-in was validated by our Genome Engineering Stem Cell (GESC) core upon derivation of that variant knock-in model. We have now added both karyotyping and pluripotency staining (SOX2/OCT4) for all other hPSC lines as (new) Supplementary Figure S17 and included further description in our Methods section under “hPSC Model Generation and Culture”.

    New Data: Supplemental Figure S17 (SOX2/OCT4 staining in hPSCs and karyotyping of all lines used)

    Text edits: Additional language confirming hPSC line validation will be added to the Methods section under “hPSC Model Generation and Culture” on page 18.

    (2) Other weaknesses relate to data interpretation and insufficient discussion of related matters, as detailed in the recommendations to the authors.

    We thank the reviewer for their insightful suggestions and have detailed our responses in the “recommendations to the authors” section.

    (3) Also, some errors are noted and detailed in the recommendation section.

    We thank the reviewer for catching these errors and have since corrected them, with detailed responses below.

    Reviewer #2 (Public review):

    Summary:

    Chapman, Determan et al. investigate how pathogenic mutations in DNMT3A, which cause Tatton-Brown-Rahman Syndrome (TBRS), disrupt human cortical developmental processes using a comprehensive panel of human pluripotent stem cell models spanning DNMT3A loss-of-function severity. The authors aim to identify the cellular and molecular mechanisms underlying TBRS-associated brain overgrowth and intellectual disability, and to test whether mechanistic convergence exists between TBRS and other overgrowth-intellectual disability disorders (OGIDs) caused by mutations in EZH2 (Weaver syndrome) or PIK3CA pathway components. Their central conclusion is that GABAergic interneuron development is selectively vulnerable to DNMT3A mutation, where reduced DNA methylation causes premature de-repression of neuronal and synaptic genes, driving precocious neuronal maturation and hyperactivity sufficient to disrupt neuronal network synchrony. This report adds to a growing literature supporting the vulnerability of GABAergic interneurons in NDDs and further provides a mechanistic view of this vulnerability, potentially convergent across OGIDs. The mechanistic claims around H3K27me3 compensation and mTOR-based therapeutic convergence, while promising, rest on more preliminary evidence and would benefit from the distinction between correlation and mechanism being made more explicit in the text. Overall, this is a compelling study with a rigorous experimental design and novel findings with a potential impact on a better understanding of the OGID pathophysiology.

    Strengths:

    (1) A major strength of this work is the breadth and rigor of the disease modeling approach. Four independent TBRS model systems are used in tandem: a patient-derived iPSC line with isogenic CRISPR-corrected control (R882H), a knock-in hESC model (P904L) with its wild-type isogenic, patient deletion iPSC lines (Del1/2), and CRISPRi knockdown models (G1/G2), collectively spanning a range of DNMT3A loss-of-function that correlates with phenotypic severity. This allelic series design substantially strengthens causal inference beyond what any single isogenic pair could provide.

    (2) The multi-omic integration across matched developmental stages provides a strong mechanistic foundation for the cellular phenotyping and provides significantly enhanced novelty. RNA-seq, whole-genome bisulfite sequencing, and H3K27me3 CUT&Tag are combined in the same cell types, and timepoints show that DNMT3A loss reduces CG methylation at neuronal and synaptic gene loci, leading to premature transcriptional activation.

    (3) The selective vulnerability of ventral (GABAergic) versus dorsal (glutamatergic) progenitors is one of the study's most important findings. This lineage specificity is consistently observed across all model systems and in both 2D and organoid formats, where ventral NPCs show increased proliferation, premature neuronal gene expression, and increased neurogenesis, while dorsal NPCs are largely unaffected at the transcriptomic and cellular level despite exhibiting comparable DNA methylation changes. This adds to a body of emerging work showing GABAergic interneuron vulnerability in NDDs where ubiquitously expressed genes such as chromatin modifiers are perturbed, and provides additional molecular insights into potential mechanisms of "resilience" of dorsal populations.

    (4) The functional characterization follows a logical progression from single-neuron electrophysiology (demonstrating GABAergic hyperactivity with increased action potential amplitude and firing rate) to network-level analysis using high-density multi-electrode arrays. The HD-MEA experimental design - pairing TBRS or control GABAergic neurons with a constant background of control iGlut neurons - cleanly isolates GABAergic dysfunction as the driver of network hypersynchrony.

    Weaknesses:

    (1) The concomitant induction of proliferation and differentiation in TBRS V-NPCs is conceptually striking, since these are generally considered antagonistic developmental programs. The authors partially address this tension by noting that DNMT3A LOF alone is insufficient to initiate neuronal differentiation, i.e., V-NPCs upregulate neuronal and synaptic genes while retaining progenitor identity, implying that transcriptomic priming and commitment to differentiation are decoupled. However, the relationship between the proliferative phenotype and the epigenetic priming phenotype remains mechanistically unresolved. The manuscript documents mTOR pathway upregulation at the protein level and identifies shared DEGs that include proliferative regulators, but it does not establish whether mTOR-driven proliferation and mCG-loss-driven neuronal gene de-repression/enhanced differentiation are causally linked or represent two independent consequences of DNMT3A LOF.

    We thank the reviewer for their comment and agree that this phenotype, whereby progenitors exhibited both increased proliferation and hallmarks of gene expression associated with neuronal differentiation is striking and interesting, given that these are typically antagonistic paradigms during normal development.

    We documented that these phenotypes involve upregulated expression of both neuronal/synaptic and proliferative genes in V-NPCs (Figure 2d), with concomitant loss of repressive DNA methylation at regulatory elements associated with these genes (Figure 2f, Supplemental Data 5). In this work, DNMT3A mutation had a more prominent role in de-repressing neuronal and synaptic gene expression to promote hallmarks of neuron differentiation, while playing a relatively less central role in direct regulation of proliferation genes, as seen from the relative prominence of neuronal/synaptic- versus proliferation-related GO terms in our Supplemental Data 5 table.

    To examine the mechanisms underlying increased V-NPC proliferation in our TBRS models, we assessed a potential relationship with the PIK3/AKT/mTOR pathway, as this is implicated in increased proliferation resulting from DNMT3A-associated mutation in myeloid leukemia (Dai et al., 2017, PMID: 28461508). In our work, DNMT3A mutation increased the expression and/or phosphorylation of mTOR signaling pathway targets specifically in V-NPCs (Figure 1q-r, Supplemental Figure S3a-d). However, while TBRS mutation directly affected repressive DNA methylation at a suite of cell proliferation-related genes, these did not include the PIK3/AKT/mTOR pathway genes themselves, suggesting an indirect relationship between altered DNA methylation and increased mTOR signaling.

    Text Edits: We will incorporate further discussion of how DNMT3A-mediated gene repression and levels of PIK3/AKT/mTOR pathway signaling may be interacting, providing a framework for future studies to identify how these related OGID gene mutations may converge mechanistically.

    (2) Relatedly, the rapamycin rescue experiment is a valuable proof-of-concept for the PIK3/AKT/mTOR convergence but is limited to a single dose in a single model (882) with a single readout (Ki67+ proliferation). Given the prominence of mTOR pathway convergence in the manuscript as a potential shared therapeutic avenue across OGIDs, the data supporting this claim are somewhat preliminary. It remains unknown whether mTOR inhibition rescues downstream phenotypes (neurogenesis, gene expression, neuronal maturation) or whether less severe TBRS models respond similarly. This might also help tackle the first comment above. e.g., if mTOR inhibition rescued proliferation but not the transcriptomic priming, that would support two independent mechanisms.

    We thank the reviewer for their comment. We explored both the overall levels and phosphorylation of proteins involved in PIK3/AKT/mTOR signaling in the 882, 904, Del1, Del2, and KO V-NPC models (Figure 1q-r, Supplementary Figure S3a-d), finding specific increases of all proteins. We showed that rapamycin addition reversed the increased proportion of KI67+ proliferating cell nuclei resulting from 882 mutation in V-NPCs in main Figure 1s, while demonstrating that rapamycin also reduced the proportion of KI67+ nuclei observed in both less severe 904 and Del1 V-NPC models (Supplementary Figure S3e-f).

    We agree that understanding whether rapamycin treatment can rescue TBRS neuronal phenotypes would be very interesting, as previous work on Tuberous Sclerosis Complex has utilized rapamycin and other mTOR inhibitors to effectively reverse TSC-related alterations of neuronal morphology and neuronal hyperexcitability (Buttermore et al., 2025, PMID: 40792287). Future studies examining convergent mechanisms and therapeutics for OGIDs should examine how similarly targeting this and related pathways rescues altered neuronal morphology, maturation, and function, as we have demonstrated that TBRS mutation has subsequent consequences for V-IN differentiation, maturation, and function. This point has been detailed in the discussion section on pages 15-16.

    (3) The claim that H3K27me3 compensates for mCG loss is an important mechanistic point, but the current data do not distinguish between active compensation, in which EZH2 is recruited in response to methylation loss, and functional redundancy, in which H3K27me3 is independently established and becomes the dominant repressive mark once DNA methylation is reduced. The EZH2 knockdown/inhibition experiments show that H3K27me3 is sufficient to maintain repression at hypo-DMR sites, but they do not establish that H3K27me3 gain is itself a response to methylation loss. Because H3K27me3 profiling was performed only in the severe 882 model, it is also unclear whether H3K27me3 gain scales with DNMT3A LOF severity, as a compensatory model would predict. Finally, the EZH2 overexpression rescue is performed in V-NPCs, whereas the compensation model is developed primarily in D-NPCs, making it difficult to assess whether the same mechanism operates in the lineage where it was originally inferred.

    We thank the reviewer for the opportunity to clarify our findings and experimental reasoning. A previous study using a conditional Dnmt3a knockout mouse model (Li et al., 2022, PMID: 35604009) demonstrated increased expression of multiple PRC2 components following the loss of Dnmt3a. This study demonstrated that sites which lost DNA methylation gained H3K27me3 in postnatal neurons upon Dnmt3a loss. Therefore, we hypothesize that the gain of H3K27me3 likely occurs in response to loss of DNMT3A methylation.

    While we did not perform CUT&Tag for H3K27me3 in our less severe models, we did validate gene expression changes following EZH2 knockdown and inhibition in both the R882H (Figure 4g-h) and P904L (Supplementary Figure S8b) models, finding that gene expression was unchanged in the model with the less severe DNMT3A mutation (P904L). Based upon these findings, we hypothesized that compensatory H3K27me3 may occur only upon severe DNMT3A loss, as seen in the dominant-negative R882H model. Furthermore, as H3K27me3 compensation was more prominent in D-NPCs, we hypothesized that this might be sufficient to prevent de-repression and aberrant neuronal gene repression upon loss of DNMT3A-mediated repression in D-NPCs. However, since TBRS mutation caused the most prominent de-repression of neuronal gene expression in V-NPCs, we also tested whether EZH2 overexpression could reverse this, finding that it partially suppressed this dysregulated neuronal gene expression. To better clarify this logic and the findings, we will make text edits to this results section.

    Text edits: We will clarify the reasoning for performing the EZH2 overexpression experiments in V-NPCs and reference Li et al., 2022 in both the results (pg. 9-10) and discussion.

    (4) The narrative framing of dorsal neuron development as unaffected by DNMT3A LOF is somewhat at odds with the data presented. The 882 D-NPCs show substantial DNA methylation changes, and TBRS D-INs exhibit what the authors describe as "substantive transcriptomic differences" involving persistent expression of pluripotency and progenitor genes, which seems to be a distinct but potentially significant phenotype. The impact of DNMT3A loss between ventral and dorsal lineages might be more accurately framed as divergent in nature rather than specific to a certain population.

    We thank the reviewer for their comment. While TBRS mutations appear to have a significantly stronger effect on V-NPCs and subsequently V-INs, both transcriptomic and methylation alterations do also occur upon TBRS mutation in D-NPCs and D-INs, as noted in Supplemental Figure S4d, S11, and Supplemental Data 2. However, we observed substantially greater molecular alterations in V-NPCs/V-INs, a lack of overt cellular phenotypes in D-NPCs where assayed, and a lack of functional consequences in matured D-INs, suggesting a more significant requirement for DNMT3A in regulating the differentiation and subsequent maturation of cortical inhibitory interneurons during embryonic and early pre-natal development, the developmental periods that we can readily model in hPSC-derived neurons.

    It should also be noted that these hPSC differentiation models do not recapitulate post-natal deposition of non-CpG (mCA) DNA methylation, a mechanism disrupted postnatally by TBRS-associated mutations in our prior work in murine models (Harrison Gabel; e.g. Beard et al., 2023, PMID: 37952155). Therefore, we hypothesize that if we could sufficiently mature D-INs to a state that modeled postnatal development and recapitulated this non-CpG methylation, we might be able to detect cellular and functional phenotypes in later stage D-INs. To avoid misinterpretation, we will alter the language in the results section to confirm that there are both transcriptomic and methylation changes in our D-NPCs/D-INs, but that these are not accompanied by cellular phenotypes or neuronal dysfunction.

    Text edits: We will better clarify that there are transcriptomic and methylation changes in D-NPCs/D-INs, but that these changes are minimal compared to those in V-NPCs/V-INs, as supported by the lack of cellular and functional phenotypes seen in D-NPCs/D-INs.

    (5) SST stainings are not entirely convincing. They appear mostly nuclear, and some instances localized to rosettes in organoids, whereas the protein is largely confined to processes and is expected to be found outside progenitor-rich zones like rosettes.

    We agree that the perinuclear SST staining detected in these young ventral telencephalic-patterned organoids at day 30 differs somewhat from the more process-localized and cytosolic signal seen in later stage organoids in other studies. This may be related to the use of different commercial SST antibodies across studies but also likely reflects SST immunoreactivity in newborn neurons near the onset of SST expression. For example, immature SST-immunoreactive neurons in the early postnatal rat cortex exhibit predominant SST staining in perinuclear cytoplasm and short processes (e.g. Fig. 3 in Lee et al, PMID: 9664223) while acquiring more cytosolic and process-localized staining as postnatal neuron maturation occurs. Evaluation of immunopositivity for other markers of neurogenesis (ASCL1) and immature neurons (TUJ1) is also congruent with these findings for SST, with TBRS-associated mutations increasing in the fraction of cells in V-NPCs/V-ORGs that express these three markers.

    Reviewer #3 (Public review):

    Summary:

    In this manuscript, the authors investigated TBRS etiology by using new human pluripotent stem cell models, modeling varying levels of TBRS-associated loss of DNMT3A function. They identified increased lineage-specific proliferation of precursors in TBRS ventral MGE-like progenitors, which they propose was related to increased signaling through the PIK3/AKT/mTOR pathway. Furthermore, they show that reduced DNA methylation during MGE-like progenitor differentiation into GABAergic interneurons can cause a premature expression of neuronal and synaptic genes, triggering precocious neuronal maturation. In conclusion, they propose that TBRS-derived GABAergic neurons exhibit hyperactivity that can alters the development and structure of neuronal networks.

    Strengths:

    Overall, the data presented is convincing, from an early developmental point of view, given that the iPSC-derived 2D cultures or organoids used do not get to reach a mature state. Nonetheless, the data clearly show the effects that deleterious mutations in TBRS can cause during the period of neurogenesis, which was missing in the field.

    Weaknesses:

    (1) Li et al., 2022 (referred to in the manuscript) seems to already show the interplay between H3K27me3 and Dnmt3a discussed in this study i.e., that in the absence of DNA methylation, there is an expansion of polycomb-like repression. These data should be better acknowledged in the paragraph 'Repressive H3K27me3 compensates for severe loss of DNA methylation' (page 9), given it supports the data presented in this manuscript and suggests this as a common mechanism in the interplay between these two repressive marks, as it is well established in the literature.

    We thank the reviewer for this suggestion and will incorporate this reference into both the results and the discussion when discussing the respective roles of DNMT3A and PCR2-mediated repression.

    Text edits: We will add Li et al., 2022 to both the results section (pg. 9-10) and our discussion section.

    (2) The authors should acknowledge that the omics data come from a mixed population of cells.

    We thank the reviewer for their comment. We have validated that the established 2-D differentiation methods we used in this study generate cell populations with >85-90% enrichment for the desired progenitor and neuronal cell type, based upon marker expression, but acknowledge that these are bulk -omics data obtained from cells that may represent a mixed population and have now detailed this in the methods section under “Sequencing”.

    Text edits: we will add language acknowledging that our omics data (bulk) was generated from mixed populations of cells.

    (3) The authors are encouraged to further discuss whether the overgrowth observed in ventral GABAergic cultures or organoids compares to the overgrowth observed in diseased patients. One expects MRIs to have been performed in patients and that these could be harnessed to discern if overgrowth occurs in the cortex or ventral regions of the brain.

    We thank the reviewer for their suggestion and do note that at least one published study documents increased cortical thickness in the MRIs of TBRS patients (Jiménez de la Peña et al., 2024, PMID: 37795572); however, to our knowledge studies have not examined regional or cell type-selective overgrowth of cortical tissue in TBRS patients. Future clinical studies examining the nature of the neuronal progenitor overgrowth and resulting consequences for patient brain imaging would be of interest to better understand TBRS-associated etiology of brain overgrowth and its manifestations.

  8. Version published to 10.1101/2025.06.24.661324 on bioRxiv