The human neuronal development is an intricate process orchestrated through epigenetic regulation of gene expression. Combination of transcription factors, chromatin remodellers, histone modifications, DNA methylation and other factors determine the gene expression programs during the differentiation of neuronal cell types. Technologies with single-cell resolution, such as scRNA-seq gave us powerful tools to characterize and better describe the molecular architecture of the developing brain. Nevertheless, the spectrum of epigenetic modalities that can be profiled at single-cell resolution is limited. We have recently developed a novel method that can be in scalable way used to profile histone modifications in single cells, thousands of cells at the same time (Bartosovic et al., 2021, Nat. Biotech., Bartosovic & Castelo-Branco, Nat. Biotech., 2022). In this project I aim to use our newly developed set of technologies to multimodally profile several histone modifications, transcription factors and open chromatin in single cells simultaneously. Multimodal epigenomic profiles can be used to predict the most relevant regulatory regions in the at unprecedented resolution. In contrast to profiling of single modalities, the multimodal data can be used to get insights into non-trivial interactions between various chromatin features such in bivalency, chromatin opening, or epigenetic priming. Moreover, by combining the single-cell multimodal profiling with spatial barcoding technology, we can for the first time investigate epigenetic dynamics in spatially and temporally resolved way. I will apply these new technologies to investigate in an integrative way the epigenetic regulation of the human brain in development and in disease. Finally, we will perturb the most important candidate regulatory elements and probe their function to gain further insights into gene regulatory networks.