Fig 2: Digital genomic footprinting reveals that tRF protects diurnal PAR bZip transcription factor activity in islets under conditions of circadian disruption.(A) Overview of transcription factor (TF) footprinting. Regulatory TFs protect against tagmentation, resulting in a signal decrease (footprint), and allow for identification of bound TFs. Differential changes in footprint depth or accessibility are associated with increased/decreased TF activity between conditions. (B) Global changes in TF footprint depth from CT4 to CT16 representing diurnal differential TF activity (y axis) and flanking accessibility from CT4 to CT16 representing diurnal differential global chromatin accessibility around each TF motif tested (x axis) in CON (black), CD (red), and CD-tRF (blue) islets. PAR bZip (DBP, HLF, and TEF) TFs are highlighted in yellow (left). Mean diurnal DBP footprints in CON (gray/black), CD (pink/red), and CD-tRF (light blue/dark blue) islets at CT4 and CT16. Signal is normalized to the total read depth of each sample and is plotted ±50 bp from DBP motif center (FDR = Hotelling’s T squared test with Benjamini-Hochberg correction) (right). (C) ATAC-seq signal at PAR bZip footprints (DBP, HLF, and TEF) detected in both CON and CD-tRF samples and subsequently visualized in CON (gray/black), CD (pink/red), and CD-tRF (light blue/dark blue) samples at CT4 and CT16. Signal is normalized to reads per genomic content and is plotted ±0.5 kb from PAR bZip motif center. Shaded dark regions represent areas of accessible chromatin (top). Enriched GO: Biological Process pathways annotated from common PAR bZip footprints (DBP, HLF, and TEF) detected in both CON and CD-tRF. Key pathways associated with ß cell secretory function are highlighted (bottom). TFBS, Transcription factor binding sites.

Fig 3: Small molecule screen identified ISX-9 as a circadian amplitude enhancer.a A schematic diagram of the compound screen procedures and validation. b Heatmap of the primary screen result of 6777 small molecules (left). Dots represent PER2::LUC bioluminescence at 24-h and 30-h time points, normalized to vehicle control. c Dose-dependent validation of 72 evident circadian-modulating compounds from the primary screen. Each compound was tested with threefold dilution series for six doses, with 10 μM as the highest concentration. d Real-time bioluminescence recording of synchronized mPer2Luc MEF cells treated with indicated small molecules (red curve) that revealed amplitude enhancement activities from the screen. DMSO-treated oscillations were shown as control (black curve). e Amplitude evaluation of 4-day bioluminescence peaks responded to small molecule treatments. Amplitudes of each compound were normalized to its respective day-1 peak. All data were represented as mean ± SD. Two-way ANOVA with Bonferroni correction for multiple comparison was performed, with p value labeled on top. Significances between non-treated and treated cells in each day were shown (ns not significant, #p < 0.05, ###p < 0.001). f Changes of Dbp:Luc and Rev-erbα:Luc expressions responded to small molecule treatments relative to DMSO. One-way ANOVA with Bonferroni correction for multiple comparison was performed (ns not significant, ##p < 0.01, ###p < 0.001), and data were shown as mean ± SD.

Fig 4: Circadian PAR bZip transcription factor activation defines a subset of ß cells in a state of increased insulin production and secretion.(A) Overview of computational approach. All identified ß cells by scATAC-seq were subjected to semisupervised pseudotime ordering by PAR bZip TF activity. ß cells were grouped into quartiles of PAR bZip activity from low (bottom quartile) to intermediate (second and third quartile) and high (top quartile). Accessible chromatin regions and gene activation scores that varied across pseudotime were identified and annotated. (B) Trajectory analysis of PAR bZip TF (DBP, HLF, and TEF) activity along pseudotime in ß cells from CON, CD, and CD-tRF mice isolated at CT4 and CT16 and subjected to scATAC-seq. (C and D) Heatmaps depicting the top 10% of gene activity scores (C) (left) and the top 5% of accessible chromatin regions (D) (left), which varied along pseudotime. Key genes controlling ß cell function, identity, and stress are highlighted in (C) (left). Ontology of enriched GO: Biological Process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from genes (C) (right) and annotated chromatin regions (D) (right) with maximal activity/accessibility in PAR bZiplow ß cells (top in blue; pseudotime, 5 to 50) or in PAR bZiphigh cells (bottom in orange; pseudotime, 70 to 100).

Fig 5: DBP binds to active enhancers critical for the regulation of insulin processing and secretion.(A) Normalized ChIP-seq signal for DBP and input centered ±1 kb around DBP binding sites. HOMER motif enrichment analysis of the D-box motif in DBP binding sites (inset; binomial test, P = 1 × 10−1922). (B) Histogram illustrating the genomic distribution of identified DBP binding sites (relative to TSS). (C) Pie chart illustrating the genomic distribution of identified DBP binding sites against promoter (≤3 kb from TSS), intronic, coding, and intergenic regions. (D) Distribution illustrating the proportion of DBP peaks occurring in H3K27ac active enhancer regions from INS-1 832/13 β cells (GSE126556) (44). DBP peaks found ≤3 kb from TSS were considered proximal. Corresponding visualization of DBP and H3K27ac ChIP-seq signal at DBP peaks (±5 kb) occurring at proximal active enhancers (top), distal active enhancers (center), and non-H3K27ac regions (bottom). (E) Enriched KEGG pathways of genes annotated to DBP-bound proximal active enhancers (top), distal active enhancers (middle), and non-H3K27ac regions (bottom). (F) Genome browser tracks of DBP and H3K27ac within the Grp94 (top), Slc2a2 (middle), and Il6 (bottom) gene loci. (G) Dot plot visualization of DBP target gene expression for genes regulating insulin secretion, circadian rhythm, and protein processing and export KEGG pathways from seven ND/T2DM human islet studies (left). Bar graph summarizing the number of studies where each gene is differentially expressed (FC ≥ 1.2; FDR ≤ 0.25) in ND or T2DM islets (right; P = 2 × 10−4; paired, two-tailed Student’s t test). Note the attenuated expression of DBP target genes in T2DM versus ND human islets.