The DNA methylation-independent role of DNMT1 in cortical neuron development

Mutations in the DNMT1 (DNA methyltransferase 1) gene are associated with severe neurological disorders, such as hereditary sensory and autonomic neuropathy type 1E (HSAN1E) and other neurodegenerative syndromes. However, the molecular mechanisms underlying these pathologies are difficult to explain solely by DNMT1’s classical DNA methylation-maintaining function in dividing progenitors, suggesting additional, yet poorly understood, roles. Recent findings indicate that DNMT1 is not only present in the nucleus but is located in the cytosol. This suggests that, in addition to its classical role in gene expression regulation, DNMT1 may also be involved in cytosolic processes relevant to neuronal development, implying a broader functional spectrum of this key epigenetic player. In this context, the understanding of how DNMT1 contributes to neuronal development beyond catalyzing DNA methylation becomes particularly important. Of note, DNMT1 contains a large N-terminal domain involved in protein-protein interaction, suggesting other modes of action. Indeed, DNMT1 exerts transcriptional control through its interaction with a histone-modifying enzyme in POA (Preoptic area)-derived postmitotic cortical interneurons, maintaining their polarized morphology during long range migration into the developing cortex. Neuronal morphology is tightly controlled across all stages of corticogenesis, encompassing both the migratory phase and the subsequent post-migratory period of morphological refinement. The precise shaping of neuronal morphology depends on dynamic cytoskeletal remodeling. Neurite formation and branching require tightly regulated microtubule stability and are intimately linked to intracellular trafficking. These microtubule dynamics are controlled both by cytosolic microtubule-associated proteins that stabilize or destabilize microtubules and by posttranslational tubulin modifications that fine-tune microtubule stability and motorprotein interactions. DNMT1 had been shown to impact the morphological maturation of cortical excitatory neurons. Dnmt1 hypomorphic cortical neurons show an abnormal branching phenotype. However, the mechanisms underlying this regulation remain largely unexplored. The central aim of this dissertation is therefore to elucidate how DNMT1 controls neuronal morphogenesis during early corticogenesis. To address this question, this thesis first confirmed that DNMT1 influences neuronal branching in the developing cortex in vivo, consistent with previous findings in Dnmt1 hypomorphic cortical neurons. Subsequently, the underlying molecular mechanisms were analyzed in primary cortical neurons in vitro using different knockdown approaches, reproducing the aberrant branching phenotype observed in the initial in vivo experiment. In contrast, blocking DNMT1’s DNA methylation activity alone does not impact the neuronal morphology, suggesting the involvement of non-canonical DNMT1 functions. It was then further shown that DNMT1 is localized not only in the nucleus but also in the cytosol, where it interacts with the microtubule stabilizer DOCK7. Knockdown of either protein indicated that both proteins similarly prevent excessive branching in cortical neurons. In addition, posttranslational microtubule modifications, such as tubulin acetylation indicative of stabilized microtubules, were affected by Dock7 depletion as well as by Dnmt1 knockdown. Moreover, both Dnmt1 and Dock7 knockdown impaired the phosphorylation of the microtubule destabilizer STMN1/Op18 (Stathmin/Oncoprotein 18). Since intracellular trafficking is coupled to microtubule dynamics, mitochondrial transport was examined next. It was demonstrated that mitochondrial trafficking is altered by repression of Dnmt1 or Dock7, whereas mitochondrial activity itself remains unaffected. Further experiments suggest that proper mitochondrial trafficking relies on DNMT1’s cytosolic, DNA-methylation-independent function, similar to its previously verified non-canonical role in regulating neuronal morphology. In sum, this work demonstrates that DNMT1 is required for tubulin acetylation and phosphorylation of STMN1/Op18, both of which correlated with microtubule stability. These effects are likely mediated through its cytosolic interaction with DOCK7, highlighting that DNMT1 fulfills broader and previously unrecognized roles beyond its canonical nuclear function. Accordingly, DNMT1 not only regulates nuclear epigenetic modifications but also carries non-canonical cytosolic functions linked to mitochondrial trafficking and neuronal maturation. These findings offer new insight into DNMT1-associated neurodegenerative syndromes, independent of its DNA-methylation activity.