Journal: bioRxiv

Article Title: Enhancer hubs govern chromatin topology and Th17 identity

doi: 10.64898/2026.04.02.715458

Figure Lengend Snippet: a) Genome browser plot of the Il17a / Il17f locus (70kb window) integrating 500bp resolution region capture Micro-C (RCMC; ICE balanced, normalized by observed/expected), with 3D contacts annotated by dashed line and Il17a-5 enhancer contacts indicated by blue triangles; ATAC-STARR-seq pooled input DNA library coverage track containing DNA fragments from Th0 Th1 Th2 Th17 and Treg ATAC-seq (grey); ATAC-STARR-seq activity score (Log2 fold change CPM) from Th0 (blue), Th1 (orange), Th2 (red), Th17 (yellow) and Treg (green) RNA versus Input DNA; Effect sizes for gRNA in CRISPRi for Il17a and Il17f (grey = tested; red = FDR < 0.05). OCRs are labeled with direction (+/-) and distance (in Kbp) relative to nearest gene. b) Scatter plot comparing sgRNA effect sizes (Log2 fold change high vs low bin) for CRISPRi screens using Il17a and Il17f reporters (green = only Il17f, red = only Il17a, blue = both, grey = non-significant; FDR < 0.05). c) Distribution of elementwise sgRNA effect sizes grouped by top functional OCRs in both Il17a (left) and Il17f (right) CRISPRi screens (lines = tested gRNA per element, blue = FDR < 0.05). Density plot (top) shows distribution of effect sizes for all gRNA. d) Flow cytometry analysis summarizing frequency of IL-17a+ cells or e) geometric MFI of Il17f (HCR-FlowFish) expression from in vitro derived Th17 cells following CRISPRi-mediated perturbation with candidate gRNAs. f) Representative stacked histograms to show distribution of in vitro derived Th17 cell Il17a and Il17f signal (red) relative to non-transduced (grey) following CRISPRi-mediated repression with top candidate single gRNA. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-hoc test versus NTC and sandwich standard error ( d ) or one-sample t-tests with Benjamini-Hochberg correction (e) . Data are shown as mean ± s.e.m. for gRNA-transduced (Thy1.1 + ) relative to non-transduced (Thy1.1-) cell signal; *** p<0.001; ** p<0.0001; * p<0.05.

Article Snippet: Region-capture probes were designed by Twist Bioscience to multiplex-capture the Il17a/f (chr1:20198461-22383378), Rorc (chr3:93261362-95108248), and Batf (chr12:84681315-86479642) loci using end-to-end tiling with 80-120mer non-overlapping probes, and low off-target stringency settings.

Techniques: Activity Assay, Labeling, Functional Assay, Flow Cytometry, Expressing, In Vitro, Derivative Assay

Journal: bioRxiv

Article Title: Enhancer hubs govern chromatin topology and Th17 identity

doi: 10.64898/2026.04.02.715458

Figure Lengend Snippet: a) Multimodal view of the Batf locus (100k bp window). Top: Region-capture Micro-C (RCMC) contact map (200bp resolution; ICE balanced), with interactions annotated by dotted lines. Tracks display Th17 ATAC-seq coverage by condition (non-targeting control [NTC] = grey; +19kb CRISPRi = red), Th17 ATAC-STARR-seq activity (Log2 CPM RNA / DNA; yellow), and CRISPRi/CRISPRa screen effect sizes (points indicate tested sgRNA, red = FDR < 0.05). Enhancers are annotated by distance (kb) and direction (+/-) relative to the Batf TSS. b) Scatter plot comparing CRISPRi versus CRISPRa effect sizes (Log2 fold change) for all tested sgRNA. Points coloured by significance (FDR < 0.05). c) Distribution of sgRNA effect sizes at selected elements from CRISPRi (left) and CRISPRa (right) screens (blue = significant; grey = tested) d) Comparison of RCMC contact frequency (500bp resolution) at the Batf locus following transduction with Batf +19kb-targeting (top) or NTC (bottom) sgRNAs in dCas9-KRAB Th17 cells. e) Differential contact map showing Log2 fold-change in interaction frequency (Batf +19kb sgRNA / NTC) f) Aggregate Peak Analysis quantifying contact frequency of interactions between the Batf-TSS (P), Batf +19kb (E1) and Batf +43kb (E2) elements in CRISPRi-mediated Batf +19kb perturbed Th17 cells (red) versus NTC (grey). g) Quantitative comparison of transcriptomic changes measured by RNA-seq (Log2 fold-changes relative to control) or h) chromatin accessibility changes by ATAC-seq (Log2 fold-change relative to control) in Batf-/-(BATF-KO) and CRISPRi-mediated Batf +19kb enhancer perturbation (Batf-gRNA) of in vitro derived Th17 cells (RNA Pearson’s r = 0.78; ATAC Pearson’s r = 0.774). i) MFI of BATF (red) or RORyt (green), and frequency of IL-17A+ (blue) from in vitro derived Th17 cells following CRISPRi-mediated repression of candidate OCRs with single gRNA relative to non-targeting control. Box plots summarise n=3 biological replicates j) Representative stacked histograms for BATF (red) IL-17a (blue) and RORγt (green) protein levels in Th17 cells following CRISPRi-mediated repression of Batf +19kb enhancer compared to nontargeting control (grey). Statistical analysis was performed using one-way ANOVA with Dunnett’s test versus the NTC and sandwich standard errors. Data are shown as mean ± s.e.m. relative to the NTC; * p <0.001.

Article Snippet: Region-capture probes were designed by Twist Bioscience to multiplex-capture the Il17a/f (chr1:20198461-22383378), Rorc (chr3:93261362-95108248), and Batf (chr12:84681315-86479642) loci using end-to-end tiling with 80-120mer non-overlapping probes, and low off-target stringency settings.

Techniques: Control, Activity Assay, Comparison, Transduction, RNA Sequencing, In Vitro, Derivative Assay