Clock-dependent chromatin accessibility rhythms regulate circadian transcription

Abstract

Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.

Fig 1. ATAC-sequencing of Drosophila clock neurons.

(A) Schema of the Drosophila molecular clock feedback loops. (B) Experimental schema. Flies were entrained for 5 days in light-dark cycles (ZT0: lights-on/dawn, ZT12: lights-off/dusk) for LD experiments and then optionally released into complete darkness for DD experiments. (C) Overview of clock neuron isolation procedure. Nuclear GFP signal is expressed with Clk-GAL4 driver and cell suspension was prepared from ~60 brains for each sample. Fluorescence activated cell sorting is used to sort live clock neurons with DAPI as viability marker. GFP-negative cells are sorted as non-clock-neuron control. (D) Representative ATAC-signal distribution aligned to transcription start sites (TSS) as reported by ATACseqQC. Fragments are classified into different groups according to alignment length, including the nucleosome-depleted (<100bp) and mono-nucleosome (180~247bp) groups. Clear TSS enrichment of nucleosome-depleted signal and nucleosome ladder are observed. (E) Heatmap of normalized ATAC signal distribution aligned to TSS plotted with ATACseqQC. Normalized ATAC signal at different positions relative to TSS (x-axis) were computed against 34920 annotated TSS’s (y-axis). (F) Representative ATAC signal pile-up tracks at Clock and timeless loci. Signal is normalized to total number of reads for each sample and therefore is comparable among samples. Four different replicates are overlaid. Clock neurons (GFP+, DAPI-) show strong ATAC signal in Clk and tim loci while non-clock cells (GFP-, DAPI-) show significantly lower readout. (G) Comparison of ATAC signal (total number of reads over the full-length gene with promoter) at various clock-neuron neuropeptide gene loci between clock-neurons and non-clock cells. While housekeeping genes (RpL32 and Gapdh1) does not show clock-neuron enrichment, many clock-neuron neuropeptide genes are significantly more accessible in clock neurons. For each neuropeptide, a one-way ANOVA test was used to determine whether clock neuron values are different from non-clock cell values. *P < 0.01, **P < 0.001.

Fig 2. Differential analysis of chromatin accessibility in clock neurons at dawn and dusk.

(A) Binary heatmaps of MACS2 called peaks for light-dark cycling (LD: ZT0, ZT12) and constant darkness (DD2: CT24, CT36) conditions. MACS2 is used to call distinct ATAC signal peaks for each condition. In total, we identified 28.1k/27.6k peaks under LD and DD conditions. A significant portion of the peaks overlap between GFP-positive clock neurons and GFP-negative cells at both timepoints. Black boxes show peaks called in one time-point only in either LD or DD condition. (B) Pairwise correlation heatmap of all samples used in this study. All sample pairs show high correlation (>0.94). Clock neuron samples in ZT0/ZT12 conditions, in CT24/CT36 conditions (including wildtype and per⁰¹) and non-clock cell samples in both ZT and CT conditions form three distinct clusters. (C) Volcano plots of differential peaks under LD and DD conditions. We identified 406 differential peaks under LD and 818 under DD conditions. Peaks corresponding to core clock genes showed higher accessibility at dusk relative to dawn. vri and Pdp1 have more intricate transcriptional regulation as they possess differential peaks at both dawn and dusk. (D, E) Assignment of ATAC peaks to genes by nearest neighbor ranked via chromosome coordinate. The 406 ZT peaks were assigned to 343 distinct genes (~1.2 peaks/gene) and the 818 CT peaks were assigned to 568 genes (~1.4 peaks/gene), showing a mostly bijective mapping. Distribution of dusk (ZT12) peaks (F) and dawn peaks (G) with respect to known genomic features by the ChIPpeakAnno Bioconductor package. ‘imdDS’ refers to genomic regions immediately downstream (within 1kb) of promoter regions. (H) Normalized ATAC signal pile-up tracks of timeless locus under LD and DD conditions. tim locus has three regulatory elements near its promoter that are more accessible during dusk when ATAC signal along its full-length gene body could also be identified.

Fig 3. Transcriptional activity of tim and pdm3 genes quantified by intronic HCR-FISH experiments.

(A) Representative images of tim intron HCR-FISH in lLNv clock neurons at dawn (ZT0) and dusk (ZT12, ZT16) timepoints. A specific intron (‘intron 1’) was probed as it yields a single bright spot of the transcription site. (B) Spot intensity fluorescence of the tim transcription site and percentage of lLNv clock neurons with the transcription spot. Transcription activity of tim is higher at dusk compared with dawn, consistent with the ATAC differential analysis. (C, D) Representative images and quantification of tim intron 1 HCR-FISH at subjective dusk and dawn timepoints. (E) Representative images of pdm3 intron 1 HCR-FISH at subjective dusk and dawn timepoints in DN1 clock neurons. (F) Spot intensity fluorescence of the pdm3 transcription site in DN1 clock neurons. Transcription activity of pdm3 is higher in DN1’s at subjective dawn compared to dusk, consistent with its chromatin accessibility pattern. As pdm3 was found to be a cycling gene specific to DN1 neurons by published single cell RNA-seq dataset [28], pdm3 nascent mRNA was imaged in DN1 neurons instead of lLNvs. The statistical test used was a two-sided Student’s t-test. **P < 0.001. Scale bar: 2μm.

Fig 4. Identification of motifs and analysis of differential peaks.

(A) De novo motif analysis of differential ATAC peaks. Peaks more accessible at dusk (ZT12/CT36) are highly enriched in E-box sequences, indicating that the corresponding regulatory elements might be directly regulated by the CLK feedback loop. Peaks more accessible at dawn (ZT0/CT24) shows different motif enrichment. ZT0 peaks are enriched in VP-box sequences while CT24 peaks are instead enriched in HMG-domain transcription factor SoxNeuro motif. (B) mRNA-seq expression profile of identified motif proteins in different clock neuron groups (LNv, DN1, LNd) and a control non-clock cell group (TH). Analysis was performed with publicly available data (32). (C) Illustration of E-box and VP-box motif sites along core clock genes tim, vri, and Clk. RNA polymerase II (Pol II) and CLK binding sites [42] and ATAC differential peaks (from this study) are shown. (D) Overlap analysis of differential ATAC peaks against known regulatory elements. ATAC peaks are first overlapped with a public database of Drosophila cis-regulatory elements [43] (REDfly). For the peaks that do not overlap, they are then overlapped with CLK-binding regions identified by a previous microarray study [42]. Finally, the unannotated peaks are overlapped with promoter regions. Overall, ~50% of differential ATAC peaks correspond to known regulatory elements.

Fig 5. Correlation between gene expression and chromatin accessibility in clock neurons.

(A) Table showing overlap between scRNA-seq data set [28] and our ATAC-seq dataset. For genes with any ATAC peak (i.e., genes that are accessible in clock neurons), a small percentage of them are found to be cycling in scRNA-seq dataset (10.9% and 7.4% under LD and DD conditions, respectively). For genes with differential ATAC peaks, overlap percentage increases to 36%/45% under LD and 27% under DD conditions. Analysis was performed with publicly available data [28]. (B) Distribution of genes with differential ATAC peaks under LD and DD conditions among all the scRNA-seq clock neuron clusters. (C) Distribution of phases for peak expression for genes that were more accessible at dawn or dusk. Dawn-accessible genes tend to reach peak transcript levels around ZT0, while dusk-accessible genes tend to reach peak transcript levels around ZT12, indicating strong correlation between chromatin accessibility and transcription. Individual genes may be cycling in multiple clock neuron clusters with different phases and all phases are plotted. (D) Ontology enrichment of genes that were more accessible at dusk or dawn. (E) Ontology enrichment of genes that were more accessible at subjective dusk or subjective dawn. To remove trivial enrichment terms from pan-neuronal genes, a custom background set of all clock-neuron accessible genes is used.

Fig 6. Chromatin accessibility rhythms are abolished in arrhythmic per⁰¹ null mutants.

(A) ATAC signal pile-up tracks of Clk and per gene loci in clock neurons from per⁰¹ mutants at subjective dawn and dusk. As showcased here and by a genome-wide differential analysis, no differential accessibility is identified between subjective dawn and subjective dusk in per⁰¹ mutants. Peaks that are differentially accessible in wildtype are marked by black boxes. (B-C) Volcano plots of differential peaks comparing per⁰¹ to wildtype at subjective dawn and subjective dusk timepoints. (D) ATAC signal pile-up tracks of tim locus in clock neurons from per⁰¹ mutants at subjective dawn and dusk. (E-F) Representative images of tim intron HCR-FISH (E) and quantification of fluorescence intensity and percentage of lLNv clock neurons with the transcription spot in per⁰¹ mutants at dawn (ZT0) and dusk timepoints (ZT12, ZT16) (F). The statistical test used was a two-sided Student’s t-test, no significance was detected. Scale bar: 2μm.

Fig 7. Differential chromatin accessibility in non-clock cells and model.

(A) Summary table of differential peaks identified in non-clock cells at dusk compared to dawn. (B) ATAC signal pile-up tracks of eIF5B and Eip75B gene loci in non-clock cells at dawn and dusk. Differentially accessible peaks are marked in black boxes. (C) Summary of our model. Chromatin accessibility of regulatory elements of clock-regulated genes varies at dawn and suck timepoints over the circadian cycle. E-box containing genes, such as per and tim, were typically more accessible at dusk, while VP-box containing genes, such as Clk, were typically more accessible at dawn.