Postdoc Centre de recherche Azrieli du CHU Ste-Justine Montreal, Quebec, Canada
Abstract Authors: Karine Doiron1, Diego A. Camacho-Hernandez1,2, Anthony Lemieux1, and Serge McGraw1,3
1. Centre de Recherche du CHU Ste-Justine, Montréal, Québec, Canada
2. Département de Biochimie et Médecine Moléculaire, Université de Montréal, Québec, Canada
3. Département d’Obstétrique et Gynécologie, Université de Montréal, Québec, Canada
Abstract Text: DNMT3A (DNA methyltransferase 3A) is essential for establishing DNA methylation; an epigenetic mechanism implicated in gene regulation and crucial for development and cellular identity. During brain development, DNMT3A adds DNA methylation marks to the promoters of germline- and pluripotency- specific genes, repressing their expression in progenitor cells. Conversely, DNA methylation is lost at promoters of neuron-specific genes, activating their expression. Alterations in these processes are linked to many neurological diseases. In mice, complete lack of Dnmt3a is lethal, whereas brain samples of mice with heterozygous Dnmt3a loss-of-function mutations show alterations in DNA methylation and transcription profiles. In humans, heterozygous loss-of-function mutations in the DNMT3A gene are associated with a range of conditions linked to overgrowth and neurodevelopmental disorders. Interestingly, altered DNA methylation patterns and changes in the expression of genes crucial for neural development and differentiation was found in blood cells from individuals with DNMT3A loss-of-function mutations. This intriguing connection underscores the intricate relationship between DNMT3A function, genetic regulation, and neurodevelopmental outcomes. Despite existing knowledge about the role of DNMT3A in brain development, our understanding of how DNMT3A loss-of-function mutations impact the differentiation of neural progenitor cells into specific neuronal cell types and how that contributes to neurological disorders is almost inexistent because of the lack of targeted mechanistic studies, especially in patient-derived brain cells.
We hypothesize that DNMT3A loss-of-function disrupts DNA methylation marks and affects the normal programming of progenitors into terminally differentiated neurons, ultimately leading to neurodevelopmental disorders. To test this, we generated induced pluripotent stem cells (iPSC) from an individual presenting with a DNMT3A loss-of-function mutation leading to overgrowth and intellectual disabilities. We differentiated these human iPSC into neuronal progenitor cells (NPC), cortical neurons and 3D organoids.
Our study reveals that DNMT3A loss-of-function in iPSC does not compromise pluripotency or the capacity to differentiate into all three germ layers. Although this loss-of-function does induce substantial changes in DNA methylation profiles within iPSC, the subsequent gene expression alterations are limited in number at this stage. Nevertheless, as cells traverse the neurogenesis pathway, transitioning from iPSC to NPC and cortical neurons, discernible changes in both DNA methylation and gene expression become increasingly apparent. Throughout our dataset, DNMT3A loss-of-function induces a reduction in DNA methylation at gene promoters in iPSC, setting the stage for the loss of H3K27me3 and an increase in gene expression during the NPC and neuron stages. Notably, we discern that DNMT3A loss-of-function prompts alterations in the expression of genes pivotal for neurogenesis and cell fate commitment (e.g., WNT7A, NEUROD1), reminiscent of characteristic phenotypes observed in various neurodevelopmental disorders. Interestingly, DNMT3A loss-of-function leads to larger expansion of derived 3D cortical organoids, which show cortical disorganization due to dysregulated fate specification.
Our study reveals that DNMT3A loss-of-function in human pluripotent stem cells disrupts the establishment of precise DNA methylation profiles during neural lineage specification. This interference triggers alterations in gene expression, impacting cell fate commitment and potentially resulting in aberrant functioning of brain cells.
