3D environment influences DNA methylation and DNMTs regulation during motor neuron differentiation

Introduction This database includes the raw data linked with the paper “3D environment influences DNA methylation and DNMTs regulation during motor neuron differentiation” published on “Frontiers in Cell and Developmental Biology”. In this paper, we reported the data about the global 5-methylcytosine levels and the expression of key DNA methyltransferases in iPSC-derived neural systems cultured in conventional 2D conditions and in organoids, across successive stages of motor neuron differentiation. Primary aim of this study was to assess how the microenvironment affects cells epigenetic features. Moreover, we compared Ctrl and sALS cells to assess potential disease-associated differences in DNA methylation and DNMTs expression. Methods Induced pluripotent stem cells (iPSCs) from controls and sALS subjects were cultured and differentiated into MNOs. DNA methylation was analyzed in both 2D cultures and organoids at various stages of differentiation. Global methylation levels were measured using ELISA. In addition, the expression of DNMT1, DNMT3A, DNMT3B was assessed at the mRNA level by Real-Time qPCR and at the protein level by western blot. Results Global DNA methylation analysis revealed reduced 5-methylcytosine levels in organoids compared to 2D cultures at the mature motor neuron stage in both control and sporadic ALS conditions, suggesting a potential influence of the microenvironment on the epigenetic state. Gene expression profiling showed increased DNMT1 and DNMT3A expression in organoids relative to 2D cultures, whereas DNMT3B displayed an opposite trend. Notably, these transcriptional differences were not consistently reflected at the protein level, highlighting a partial uncoupling between mRNA and protein expression levels. When comparing control and sporadic ALS conditions, no significant differences in global DNA methylation were detected; however, a disease-associated gene upregulation of DNMT1 was observed at early differentiation stages, while DNMT3A and DNMT3B remained largely unchanged. Our findings suggest that DNA methylation and DNMTs expression are influenced by the cellular context and differentiation stage. Only modest disease-associated alterations were observed between control and sporadic ALS conditions. These results highlight the relevance of organoid systems for studying epigenetic regulation during neural differentiation and suggest that disease-associated epigenetic alterations may involve more specific or localized changes rather than global DNA methylation levels.