Epigenetic characterization and manipulation of rare circadian neuron subtypes
Pranav OjhaiD, Gillian Berglund, Albert D. YuiD, Shlesha RichhariyaiD, Michael RosbashiD
Abstract
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.
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In-depth summary
Claude Opus 5.5 · xhigh effort · 7 Nov 2026 · from the full text of version 3 (21 Sept 2026) · CC BY 4.0
El-INTACT nuclear purification gives clean ATAC-seq from ~120 Drosophila clock neurons and 16 PDF neurons, revealing cell-type-specific Clk enhancers; disrupting two renders flies arrhythmic.
The cis-regulatory basis of Clk transcription was essentially unknown, because Clk has no established elements analogous to the E boxes of per and tim. Dissection plus FACS profiling underrepresents large LNvs, and no time-series chromatin accessibility profile existed for circadian neurons.
Key results
ShownClk chromatin accessibility is strongly cell-type-specific. Three peaks (neuron-specific, common, glia-specific) occur, and pan-neuronal nSyb-Gal4 nuclei show no accessible peak in the Clk region. Neurons and glia use different transcription start sites.
ATAC-seq at ZT02 in Clk856, Repo, nSyb and ninaE Gal4 populations and in heads; RNA-seq at ZT02 and ZT14 shows distinct transcripts in neurons versus glia, and both cycle. · Accessibility of Clk chromatin is highly…; Cis-regulatory control of Clk transcription; Figure legends
ShownEl-INTACT (anti-FLAG bead enrichment, peptide elution, then FACS) strongly enriches rare nuclei and makes ATAC-seq of the 16 LNvs feasible.
Clock neurons rise from ~0.1-0.01% of nuclei to ~10% (100X-1000X enrichment). For LNvs, 3000 heads gave 4000 nuclei in 1 h of sorting, versus ~400 neurons from ~200 dissected brains (3.5 h hands-on); the text reports a 10-fold higher yield. · El-INTACT reliably purifies low copy number…; Purification of LNv nuclei and identification of…; Figure legends
ShownA six-time-point ATAC-seq series from clock neurons shows many cycling peaks. Core clock genes rank among the top cyclers, mostly in ZT02 and ZT14/18 classes, and cycling peaks sit next to CLK:CYC binding sites.
Six timepoints (ZT02 to ZT24), Meta2D p<0.05; per, tim, vri, pdp1 and Clk are among the top by amplitude. Amplitudes of up to 2-4 fold, versus modest differences in a prior 2-timepoint dataset. · ATAC-Seq peaks around the clock; Insights from and advantages of El-INTACT; ATAC-Seq Differential Peak Calling and…
ShownLNv ATAC-seq reveals an additional Clk element (PDF peak) beside the neuron-specific peak. CRISPR disruption of either one makes flies arrhythmic, the neuron-specific peak more strongly; the common and glia-specific peaks have no detectable behavioral effect.
Multiplexed gRNA plus Cas9 driven by elav-Gal4; rhythmicity index in constant darkness (days 2-7); ClkOUT whole-region deletion also arrhythmic. Combining the neuron-specific peak with the common or glia peak resembled the neuron-specific disruption alone. · Purification of LNv nuclei and identification of…; CRISPR-Cas9 mutagenesis identifies a functional…; Circadian Activity Assay; Figure legends
ShownPeak disruptions reduce PER and tim in LNvs, with different sensitivity in large versus small LNvs. Combined PDF and neuron-specific disruption nearly abolishes both in large LNvs.
PER lost in ~25-50% of l-LNvs (PDF peak) and 50-75% (neuron-specific peak); combined, ~100% of l-LNvs and 50% of s-LNvs. tim in situ at ZT14 was abolished in essentially all l-LNvs and s-LNvs with combined disruption. · Molecular consequence of peak disruptions; Figure legends
Methods, data and reproducibility
El-INTACT enriches FLAG-tagged nuclei with magnetic beads, elutes them with FLAG peptide and sorts by tdTomato, then Omni-ATAC-seq is run on clock neurons (six timepoints) and LNvs. Candidate Clk peaks were tested with Gal4-restricted multiplexed gRNA/Cas9 disruption, read out by locomotor rhythms, PER staining and tim RNAScope.
- ATAC-seq of FACS-sorted clock neurons (Clk856), glia (Repo), all neurons (nSyb), photoreceptors (ninaE) and heads, collected at ZT02 · Drosophila melanogaster
- El-INTACT ATAC-seq of Clk856 clock nuclei at ZT02, ZT06, ZT10, ZT14, ZT18, ZT24 · Drosophila melanogaster · n = at least two replicates per timepoint (Fig. 3 legend)
- El-INTACT ATAC-seq of PDF-Gal4 LNv nuclei at ZT02 · Drosophila melanogaster · n = 3000 heads per prep, ~4000 nuclei
- Bulk Smart-seq3 RNA-seq of sorted neurons and glia at ZT02 and ZT14 · Drosophila melanogaster · n = 20-38 million paired-end reads per sample
- Published CLK ChIP-seq time course from heads (reused from a previous publication) · Drosophila melanogaster · n = at least two replicates
- Published embryonic single-cell ATAC-seq peaks (Cusanovich et al. 2018), used for comparison · Drosophila melanogaster
- Methods & tools
- Omni-ATAC-seq, Smart-seq3, fastp, Bowtie2, MACS2, DESeq2, MetaCycle (Meta2D), featureCounts, UROPA, Nitecap, SCAMP, RNAScope
- Reproducibility gaps
- No code repository or sequencing data accession is given. The Tn5 insertion-bias correction used a custom Python script that is not described as released. Strains and gRNAs are listed in Tables S1 and S2.
Limitations (5)
- Multiplexed gRNAs produce heterogeneous, mosaic indels (PCR-validated as mixed products), so each disruption is a mixed population rather than a defined deletion. · stated by the authors
- The proteins occupying the Clk peaks are unknown, and an ATAC-seq peak may reflect binding without any effect on expression. · stated by the authors
- s-LNv results may reflect effects on s-LNv specification as well as on target gene expression, which complicates comparison with l-LNvs. · stated by the authors
- Clk transcript or protein is not measured after peak disruption; readouts are downstream (PER, tim, behavior), and the cell counts are reported without statistical tests in the text. · noted in this summary
- The PDF peak is identified from LNv profiles at a single timepoint (ZT02) and compared with Clk856 profiles at different y-axis scales. · noted in this summary
Who should read:Read this if you profile chromatin in rare, genetically defined fly cell types, or want a Gal4-restricted CRISPR approach for testing enhancers lacking known motifs.
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