KDM6B interacts with TFDP1 to activate P53 signaling in regulating mouse palatogenesis
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Evaluation Summary:
This paper is a tour de force study, with elegant mouse genetics and potentially clinically relevant rescue results using a small molecule inhibitor that can aleriorate cleft palate in a mutant mouse model. The work will be of interest to the craniofacial biology community and to the broader developmental biology community, as well as to all those devoted to the study of the epigenetic and transcriptional regulation of morphogenesis and organogenesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Epigenetic regulation plays extensive roles in diseases and development. Disruption of epigenetic regulation not only increases the risk of cancer, but can also cause various developmental defects. However, the question of how epigenetic changes lead to tissue-specific responses during neural crest fate determination and differentiation remains understudied. Using palatogenesis as a model, we reveal the functional significance of Kdm6b , an H3K27me3 demethylase, in regulating mouse embryonic development. Our study shows that Kdm6b plays an essential role in cranial neural crest development, and loss of Kdm6b disturbs P53 pathway-mediated activity, leading to complete cleft palate along with cell proliferation and differentiation defects in mice. Furthermore, activity of H3K27me3 on the promoter of Trp53 is antagonistically controlled by Kdm6b , and Ezh2 in cranial neural crest cells. More importantly, without Kdm6b , the transcription factor TFDP1, which normally binds to the promoter of Trp53 , cannot activate Trp53 expression in palatal mesenchymal cells. Furthermore, the function of Kdm6b in activating Trp53 in these cells cannot be compensated for by the closely related histone demethylase Kdm6a . Collectively, our results highlight the important role of the epigenetic regulator KDM6B and how it specifically interacts with TFDP1 to achieve its functional specificity in regulating Trp53 expression, and further provide mechanistic insights into the epigenetic regulatory network during organogenesis.
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Evaluation Summary:
This paper is a tour de force study, with elegant mouse genetics and potentially clinically relevant rescue results using a small molecule inhibitor that can aleriorate cleft palate in a mutant mouse model. The work will be of interest to the craniofacial biology community and to the broader developmental biology community, as well as to all those devoted to the study of the epigenetic and transcriptional regulation of morphogenesis and organogenesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Guo et al. report that tissue-specific inactivation of Kdm6b in the neural crest lineage, but not in the epithelium, cause high penetrance of cleft palate and complete penetrance of neonatal lethality. They show that the Wnt1-Cre;Kmd6bfl/fl embryos had apparent defect in palatogenesis by E14.5 and hypoplasia of the palatal processes of the maxillary and palatine bones at later stages.
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Reviewer #2 (Public Review):
The paper aims to understand how palatogenesis is epigenetically controlled by the lysine-specific demethylase Kdm6b, and that loss of Kdm6b in the cranial neural crest (CNC) causes cleft palate by disrupting the p53 pathway. In the authors' Wnt1-Cre;Kdm6bfl/fl mouse model, CNC-specific loss of Kdm6b causes cleft palate with 90% penetrance. The mutant CNC cells (CNCC) migrate properly to the first pharyngeal arch but then exhibit hyperproliferation with evidence of increased DNA damage and inhibited differentiation and osteogenesis. The RNA-seq data indicates p53 pathway involvement, corroborated by the finding that p53 expression is reduced in the mutants' palatal region. Prenatal treatment with Nutlin-3, an MDM2 inhibitor known to increase p53 levels, rescues the cleft palate phenotype of the Wnt1-Cre;Kdm6b…
Reviewer #2 (Public Review):
The paper aims to understand how palatogenesis is epigenetically controlled by the lysine-specific demethylase Kdm6b, and that loss of Kdm6b in the cranial neural crest (CNC) causes cleft palate by disrupting the p53 pathway. In the authors' Wnt1-Cre;Kdm6bfl/fl mouse model, CNC-specific loss of Kdm6b causes cleft palate with 90% penetrance. The mutant CNC cells (CNCC) migrate properly to the first pharyngeal arch but then exhibit hyperproliferation with evidence of increased DNA damage and inhibited differentiation and osteogenesis. The RNA-seq data indicates p53 pathway involvement, corroborated by the finding that p53 expression is reduced in the mutants' palatal region. Prenatal treatment with Nutlin-3, an MDM2 inhibitor known to increase p53 levels, rescues the cleft palate phenotype of the Wnt1-Cre;Kdm6bfl/fl mice. This is a very interesting result with potential future clinical application. Elevated levels of H3K27me3 methylation in the single mutant are decreased to normal levels in the double mutant Wnt1-Cre;Kdm6bfl/fl;Ezh2fl/+, and the cleft palate phenotype is rescued with 70% efficiency in the double mutant, suggesting that the methyltransferase Ezh2 and Kdm6b have opposing functions during palatogenesis and that increased H3K27me3 methylation contributes to cleft palate. The p53 promoter is shown to bind the transcription factor Tfdp1 and to be affected by H3K27me3 methylation. Tfdp1 silencing with siRNA reduces p53 expression in control cells, whereas Tfdp1 overexpression elevates p53 levels in the control but not in the Kdm6b mutant. Kdm6b and Tfdp1 precipitate together in Co-IP (Tfdp1 levels are unchanged in the mutant). The authors conclude that Kdm6b removes the H3K27me3 modification introduced by Ezh2, which allows Tfdp1 to access the p53 promoter and increase p53 expression. Normal levels of p53 are, in turn, required to control CNCC proliferation in the palatal region and to allow differentiation and osteogenesis. These findings illuminate the role of Kdm6b-mediated epigenetic modification in the cleft palate etiology and provide new possible targets for pharmaceutical intervention.
Overall this is a real tour de force study, with elegant mouse genetics and potentially clinically relevant rescue results.
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Reviewer #3 (Public Review):
In the present study, Tingwei Guo et al use the mouse secondary palate as a model to assess the function of Kdm6b, a H3K27me3 demethylase, in the regulation of embryonic development. Guo's study shows that Kdm6b plays an essential role in neural crest development, and that loss of Kdm6b perturbs p53 pathway-mediated activity, leading to complete clefting of the secondary palate along with cell proliferation and differentiation defects.
In addition, the study reveals that Kdm6b and Ezh2 control p53 expression in cranial neural crest cells and that Kdm6b renders chromatin accessible to the transcription factor TFDP1 to activate p53 expression during palatogenesis. Together, the findings presented in this manuscript highlight the important role of the epigenetic regulator KDM6B and how it cooperates with TFDP1 …
Reviewer #3 (Public Review):
In the present study, Tingwei Guo et al use the mouse secondary palate as a model to assess the function of Kdm6b, a H3K27me3 demethylase, in the regulation of embryonic development. Guo's study shows that Kdm6b plays an essential role in neural crest development, and that loss of Kdm6b perturbs p53 pathway-mediated activity, leading to complete clefting of the secondary palate along with cell proliferation and differentiation defects.
In addition, the study reveals that Kdm6b and Ezh2 control p53 expression in cranial neural crest cells and that Kdm6b renders chromatin accessible to the transcription factor TFDP1 to activate p53 expression during palatogenesis. Together, the findings presented in this manuscript highlight the important role of the epigenetic regulator KDM6B and how it cooperates with TFDP1 to achieve its functional specificity in controlling p53 expression, and further provide mechanistic insights into the epigenetic regulatory network during secondary palate organogenesis.
Over the last years, it has been reported by multiple groups that among the various layers of epigenetic regulation, DNA methylation and histone methylation are key drivers of diverse cellular events and developmental processes. In addition, it has been demonstrated that demethylation also plays important roles during development. For instance, demethylation of H3K4 is required for maintaining pluripotency in embryonic stem cells, and the demethylases KDM6A and KDM6B are required for proper gene expression. Indeed, the concept that failure to maintain epigenomic integrity can cause deleterious consequences for embryonic development has been extensively explored by various groups and is not novel per se. In addition, both lysine methyltransferase Kmt2a and demethylase Kdm6a have been recently shown to be essential for cardiac and neural crest development. For example, Shpargel reported that mice carrying neural crest deletion of Kdm6a exhibit craniofacial defects, including cleft or arched palate, cardiac abnormalities, and postnatal growth retardation, modeling the clinical features of Kabuki syndrome (Shpargel et al. PNAS, 2017). In summary, roles of these demethylases in neural crest development are already known. However, how these epigenetic changes lead to tissue-specific responses during neural crest fate determination and differentiation remains poorly understood and understudied, making the current manuscript of interest and timely.
The study is robust, detailed, and comprises a wealth of original results and data of high quality, illustrated through many elegant figures. There are only some points of concern that need to be addressed, mainly related to additional quantitative analyses that are required for some of the experiments discussed in the manuscript and the need for clarifications regarding the regulation of the p53 pathway.
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