Epigenetic characterization and manipulation of rare circadian neuron subtypes

The molecular circadian clock ticks away similarly in several distinct locations throughout the Drosophila head and body, yet regulation of this clock and specifically that of master transcription factor gene Clock (Clk) and its transcription is largely unknown. ATAC-Seq assays indicate that Clk chromatin appears inaccessible in most head neurons outside the circadian brain network, indicating that circadian clock functionality is gated by access to Clk chromatin. Moreover, distinct Clk enhancers function in circadian neurons versus glia, corresponding to distinct transcripts. To address the chromatin landscape of Clk with more temporal and spatial resolution, we developed a novel, high-purity, nuclear purification method, Elution-based INTACT (El-INTACT). It enabled the first time-resolved, six-timepoint chromatin accessibility landscape of ~120 Drosophila circadian neurons/brain. It moreover allowed chromatin accessibility characterization of the 16 PDF-expressing ventrolateral circadian neurons (LNvs), which identified an additional, previously unidentified Clk enhancer. A CRISPR/Cas9 multiplexed gRNA-based strategy was used to disrupt these enhancers and indicated striking functional specificity. The findings establish a chromatin-level logic for cell type-specific circadian regulation and show that El-INTACT and the multiplexed gRNA-disruption of enhancer function are broadly applicable tools for exploring chromatin regulation in rare cell populations.