Modelling chromatin dynamics in neural stem cell quiescence and reactivation

Abstract

Majority of adult mammalian neural stem cells are quiescent, remaining in an actively maintained state of cell cycle arrest. Quiescent and actively proliferating neural stem cells can be distinguished by their transcriptional state, however, the understanding of how these gene signatures are established at the epigenomic level remains lacking. Previous work on the chromatin state of quiescent stem cells in different systems showed variable results, from globally increased heterochromatin to subtle changes in enhancer activation. An increased understanding of the role of chromatin in neural stem cell dynamics could become the basis for potential therapies for neurodegenerative or neurodevelopmental disorders. Drosophila melanogaster neural stem cells recapitulate most behaviours of their mammalian counterparts. The genetic tractability of Drosophila, combined with a wealth of genomic profiling tools such Targeted DamID makes it an ideal model for studying chromatin transitions in vivo. In this project, I used a selection of DamID tools to generate a comprehensive dataset of Drosophila neural stem cell chromatin landscape in early proliferating, quiescent and reactivated cells. I generated a chromatin state annotation of Drosophila neural stem cells with the use of hidden Markov modelling and found that chromatin becomes more accessible upon quiescence induction. Moreover, quiescent cells gain more Trithorax chromatin domains and revert to a less accessible state upon reactivation. I also showed that genes necessary for neurotransmitter-based signalling become upregulated in quiescence by transitioning form H1-bound repressive heterochromatin to an active chromatin state marked by components of the SWI/SNF complex. In contrast, genes necessary for S and M phase progression have lower transcription levels but remain within a permissive euchromatin state. Finally, specific loci are marked by an increase in Polycomb-bound domains in quiescence, suggesting a role for repressive complexes in quiescence. I followed these correlations with functional experiments using hypomorphic mutants or RNA interference to induce knockdown of specific chromatin-modifying enzymes in Drosophila neural stem cells. I discovered that although Trithorax and Polycomb components do not play a role in quiescence establishment, they are necessary for timely reactivation. Finally, I investigated RNA pol II proximal pausing as a possible method for regulation of gene expression during reactivation and found a small group of genes related to RNA splicing that release RNA pol II pausing upon reactivation. Overall, my results show that quiescent neural stem cells undergo an active chromatin remodelling that induces expression of genes necessary for signalling whilst retaining accessible chromatin at loci needed for cell cycle progression. Trithorax Group components are potentially needed for expression of genes necessary to provide a signalling machinery for quiescent neural stem cells that allows them to communicate with other neural stem cells, neurons and glia within their niche. Similarly, Polycomb complex is implicated in regulation of quiescence, potentially via repression of temporal cascade transcription factors or growth factor components.