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: The final gRNA row-wise data tables are described in Extended Data Table 4. gRNA oligo pools were ordered from Twist Bioscience.

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) Schematic of the CRISPR-based screening workflow for identifying regulatory elements involved in Th17 differentiation. Naive CD4+ T cells were activated in vitro under Th0 conditions for 24h, followed by transduction with gRNAs targeting open chromatin regions. Cells were then polarized under Th17 conditions for 3 days and prepared for FACS using one of three readouts: i) eGFP expression (i.e Il17a), ii) fixed intracellular staining (i.e RORγt, BATF), or iii) hybridized chain reaction fluorescence in situ hybridization (i.e Il17a Il17f). Cells were finally sorted into high or low expression bins where gRNA abundance was compared. b) Scatter plot comparing sgRNA effect sizes (Log2 fold change hi/lo) between Th17 differentiation noncoding CRISPRi screens with Il17a-eGFP and RORγt readouts (blue = sgRNA significant in both; padj < 0.05). c) Distribution of element-wise effect sizes for gRNA (vertical lines) targeting OCRs in the Il17a- and RORγt-CRISPRi screens (lines = element-targeting gRNA, blue = padj < 0.05). d) Volcano plots depicting sgRNA effect sizes (Log2 fold change) comparing high/low bins for Il17a-eGFP (left) and RORγt(right), with top gRNA labelled (red = padj < 0.05). e ) Mean fluorescence intensity (MFI) of Il17a-eGFP (left) or RORyt (right) from in vitro derived Th17 cells following CRISPRi-mediated repression with individual candidate gRNAs, shown relative to non-targeting control (NTC). Box plots summarize n=5 per targeting gRNA, n=3 for Th0, n=3 for NTC. Statistical analysis was performed using one-way ANOVA with Dunnett’s test versus the NTC and sandwich standard error. Data are shown as mean ± s.e.m. relative to the NTC; * p <0.01; ** p < 0.001

Article Snippet: The final gRNA row-wise data tables are described in Extended Data Table 4. gRNA oligo pools were ordered from Twist Bioscience.

Techniques: CRISPR, In Vitro, Transduction, Expressing, Staining, Fluorescence, In Situ Hybridization, Derivative Assay, Control

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 view of Rorc and surrounding region (200k bp region) integrating 500bp RCMC contact map (ICE balanced, observed/expected normalization) and 3D interactions annotated by dotted lines; ATAC-STARR pooled Input library (blue), Th17 ATAC-STARR-seq activity score (Log2 CPM (RNA / DNA); yellow); CRISPRi- and CRISPRa-RORγt effect size (red = sgRNA FDR < 0.05; grey = tested); and OCR annotations label the direction (+/-) and distance (in Kbp) from nearest gene. b) Scatter plot comparing sgRNA effect sizes (Log2 fold change RORγt high vs low bins) from CRISPRi and CRISPRa screens (green = CRISPRa only; red = CRISPRi only; blue = both; grey = nonsignificant; FDR < 0.05). c) Element-wise distribution of effect sizes for both CRISPRi (left) and CRISPRa (right) (lines = element-tested gRNA; blue = FDR < 0.05) d) Zoomed in RCMC contact map (500bp resolution) focusing on the proximal RORγt locus (16kb window) with 3D contacts annotated as dotted lines, notable contact enrichments labelled with blue triangles, and corresponding Th17 ATAC-seq coverage track (blue) e) Frequency of IL-17a (blue) or MFI of RORγt (green) relative to non-targeting control (NTC) for in vitro derived Th17 cells following CRISPRi-mediated perturbation with top candidate gRNA from RORγt screening. f) Representative stacked histograms depicting RORγt and IL17a flow cytometry signal for Th17 cells transduced (Thy1+; blue/green) or nontransduced (Thy1-; grey) with candidate gRNAs. Statistical analysis was performed using one-way ANOVA with Dunnett’s test versus the NTC and sandwich standard error. Data are shown as mean ± s.e.m. relative to the NTC; * p <0.001; † p < 0.05.

Article Snippet: The final gRNA row-wise data tables are described in Extended Data Table 4. gRNA oligo pools were ordered from Twist Bioscience.

Techniques: Activity Assay, Control, In Vitro, Derivative Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Enhancer hubs govern chromatin topology and Th17 identity

doi: 10.64898/2026.04.02.715458

Figure Lengend Snippet: STARR-seq signal (Log2 FC) at all ATAC-STARR-seq tested OCRs within Batf , Rorc(t), and Il17a/f loci categorized by CRISPR-screen result (untested = no CRISPR gRNA coverage). b) Waterfall plot of Th17 ATAC-STARR-seq signal (Log2 fold change RNA/DNA) for OCRs with at least 1 significant gRNA in Il17a-CRISPRi (blue circle) or Il17f-CRISPRi (orange circle), c) RORγt-CRISPRi (blue circle) and RORγt-CRISPRa (red circle) or d) Batf-CRISPRi (blue circle) and Batf-CRISPRa (red circle)

Article Snippet: The final gRNA row-wise data tables are described in Extended Data Table 4. gRNA oligo pools were ordered from Twist Bioscience.

Techniques: CRISPR

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: The final gRNA row-wise data tables are described in Extended Data Table 4. gRNA oligo pools were ordered from Twist Bioscience.

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