Analyzing DOT1L-dependent hippocampus development using single-cell high throughput sequencing

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Epigenetic processes are implicated in central nervous system development and function. However, there is still limited insight into the role of individual chromatin modifiers and their targets in physiological and patho-physiological conditions in the central nervous system. It is largely unknown how chromatin modifications impact on hippocampus development. The histone methyltransferase Disruptor of Telomeric Silencing 1-Like (DOT1L) confers histone H3 lysine 79 (H3K79) methylations. Our preliminary data indicate that studying the function of DOT1L will give unique opportunity to narrow the gap of knowledge of how this epigenetic modifier affects hippocampal development and function. Importantly, our data indicate that DOT1L deficiency interferes with the correct exertion of fundamental processes during hippocampus development. We observe altered development of the dentate gyrus (DG) as well as the cornu ammonis (CA) fields. Our current insights suggest that the derailed developmental program is reflected by disturbed neuronal differentiation that occurs prematurely and confers incorrect cell identities.We here propose to extend a preliminary single-cell RNA data set from embryonic day (E) 16.5 control and DOT1L-deficient mouse hippocampus to further developmental time points, spanning from E12.5 until birth. Characterising the emerging data set covering various stages of development shall allow gaining comprehensive knowledge about differentiation trajectories from diverse, but partly unknown stem cell populations to the neurons of the hippocampus. Further insights into molecular alterations underlying derailed transcriptional programs in DOT1L deficient hippocampus shall come from studying H3K79 methylation and accessibility of regulatory regions, especially enhancers. We propose to use a diverse repertoire of bioinformatics analyses to explore the emerging comprehensive data set to 1) decipher the impact of H3K79 methylation in control of transcriptional programs necessary to specify the heterogeneous neuronal subpopulation, 2) infer transcriptional programs that contribute to hippocampal fissure and DG formation, 3) resolve different subsets of neural stem cells contributing to differentiation of DG and CA fields, and 4) determine gene activities needed and fine-tuned at the boundary of the subiculum and the CA neuroepithelium. We aim to decipher master regulators that determine differentiation of the diverse neurons in the hippocampus and these candidate genes shall be functionally validated in vivo through gain- or loss-of-function experiments.