New methods for epigenetic characterization and manipulation of rare fly brain neurons

The Drosophila central brain is remarkably heterogeneous. Molecular as well as connectomic data indicate that many of its ca. 65,000 neurons are present in only a few copies per brain. This makes molecular characterization of individual cell types challenging. To address this issue, we developed Elution-based INTACT (El-INTACT), a nuclei-based purification technique that offers much higher purity, yield and efficiency compared to existing methods. El-INTACT successfully isolated two subsets of circadian neuron nuclei, one with [~]120 nuclei per brain and the other with only [~]16 nuclei per brain; the latter are the well-known circadian LNvs or PDF neurons. The method facilitated ATAC-Seq (Assay for Transposase Accessible Chromatin) assays, which revealed temporally regulated enhancers that cycle throughout the day within clock neurons. The ATAC-seq assays also identified cell-type-specific enhancers that correlate with adjacent genes, which are also only expressed in certain neuron types. To test the functional relevance of these enhancers, we developed a second approach, a CRISPR/Cas9 and multiplexed guide-RNA strategy and used it to disrupt individual enhancers of the core circadian gene Clock (Clk). Our results show that these two methods, El-INTACT to identify enhancers and a CRISPR-based strategy to perturb these enhancers, significantly improves the understanding of gene regulation within discrete neuron types.