Journal: bioRxiv

Article Title: Cooperative FOXA1–HNF4A binding emerges from motif spacing and nucleosome architecture

doi: 10.64898/2026.05.27.728252

Figure Lengend Snippet: (a) Top: schematic of the K562 doxycycline-inducible system from Hansen et al. (2022a), in which FOXA1 and HNF4A were induced individually or together in cells lacking endogenous expression of either factor. Bottom: representative CUT&Tag tracks at one peak from each category, showing FOXA1 antibody signal (blue) and HNF4A antibody signal (orange) across the three induction conditions. Co-bound sites (peaks present in both dual-induction antibody tracks; 50% reciprocal overlap on narrowPeak intervals) were classified by their dependence on single-TF expression. FOXA1-enabled (FE, n = 1,510): bound by FOXA1 in the FOXA1-only condition. HNF4A-enabled (HE, n = 2,727): bound by HNF4A in the HNF4A-only condition. Cooperative (CB, n = 1,824): bound by neither factor in either single-TF condition. Redundant (n = 1,875): bound by both factors in their respective single-TF conditions. (b) Per-peak baseline (uninduced) ATAC-seq signal by category. ATAC-seq from GSE182188, same K562 doxycycline-inducible system. (c) Change in per-peak ATAC-seq signal upon dual induction (ΔATAC = induced − uninduced). Dashed line: no change. In b and c, box plots show median (centre line), interquartile range (box), and 1.5×IQR whiskers; violins show the underlying data distribution. Brackets show two-sided Mann–Whitney U tests comparing Cooperative against each other category (****p < 0.0001). (d) Log₂ fold-enrichment of each site category over genome-wide background across seven summary chromatin states consolidated from the Broad 15-state ChromHMM K562 segmentation (wgEncodeBroadHmm). Fold enrichment = (fraction of category overlapping state) / (genomic fraction of state). Cell values are fold enrichments; colour, log₂(fold enrichment). (e) Mean MNase-seq nucleosome occupancy in a ±1 kb window centred on each peak summit, by category. MNase-seq from Mieczkowski et al. 2016 (GEO GSM2083140) . Lines show category means; shaded bands show ±SEM. Sites with usable bigWig coverage (≥50% non-NaN bins): FE n = 1,420; HE n = 2,560; CB n = 1,781; RD n = 1,822. Signal binned at 10 bp and Gaussian-smoothed (σ = 20 bp).

Article Snippet: Recombinant human full-length FOXA1 (Origene TP306045) and HNF4A (Origene TP317863) were used for binding reactions.

Techniques: Expressing, MANN-WHITNEY, Genome Wide

Journal: bioRxiv

Article Title: Cooperative FOXA1–HNF4A binding emerges from motif spacing and nucleosome architecture

doi: 10.64898/2026.05.27.728252

Figure Lengend Snippet: (a) Dual-head binding CNN architecture. One-hot encoded 1,001 bp sequences (summit ± 500 bp) pass through three convolutional blocks (64/128/128 filters; kernel sizes 19/11/7; each: Conv → BatchNorm → ReLU → MaxPool(4) → Dropout 0.25), global average pooling, and two independent task-specific MLP heads with sigmoid output. Training: 138,489 sequences (peaks from all four categories vs. cis-regulatory negatives from uninduced K562 ATAC-seq); chromosome-based splits (test: chr1, chr8, chr9; validation: chr2, chr3). (b) ROC (left) and precision-recall (right) on the held-out test set. FOXA1 head: AUROC = 0.868, AUPRC = 0.792; HNF4A head: AUROC = 0.878, AUPRC = 0.731. (c) DeepLIFT attribution heatmaps by category, after SVA filtering of the HNF4A-Enabled set (see Supplementary Fig. 4): FOXA1-Enabled (n = 1,507), HNF4A-Enabled (n = 2,105; 622 SVA-overlapping sites removed), Co-Bound (n = 1,775), Redundant (n = 1,865). Left: FOXA1 head importance (blue); right: HNF4A head importance (orange). Each row is one site; rows are sorted by position of peak attribution. Each head’s attribution is strongest at its single-TF-enabled category; both heads contribute at Co-Bound sites. (d) Total CNN head attribution within ±250 bp of the peak summit by category. FOXA1 head (left) is most active at FOXA1-Enabled sites (median 3.83 vs. 2.44 at HNF4A-Enabled); HNF4A head (right) is most active at HNF4A-Enabled sites (median 4.49 vs. 2.50 at FOXA1-Enabled). The per-site cognate-head attribution fraction (cognate-head attribution / total attribution) is higher at HNF4A-Enabled than FOXA1-Enabled sites (63.7% vs. 58.4%; two-sided Mann–Whitney p = 2.3 × 10⁻²⁴). (e) FIMO-based motif counts within ±250 bp of the peak summit (FIMO p < 10⁻³; JASPAR MA0148.1, MA0114.2). FOXA1-Enabled sites carry more FOXA1 motifs (median 3) than HNF4A motifs (median 2); HNF4A-Enabled sites show the reverse (median 4 vs. 2). The cognate-motif fraction is correspondingly higher at HNF4A-Enabled sites (71.4% vs. 57.1%; p = 7.8 × 10⁻⁸⁷). In d and e, box plots show median (centre line), interquartile range (box), and 1.5×IQR whiskers; violins show the underlying data distribution.

Article Snippet: Recombinant human full-length FOXA1 (Origene TP306045) and HNF4A (Origene TP317863) were used for binding reactions.

Techniques: Binding Assay, Biomarker Discovery, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cooperative FOXA1–HNF4A binding emerges from motif spacing and nucleosome architecture

doi: 10.64898/2026.05.27.728252

Figure Lengend Snippet: Spacing: centre-to-centre between lowest-p FOXA1 (MA0148.1) and HNF4A (MA0114.2) motifs per peak, summit ± 500 bp, max 500 bp inter-motif (best-score pairing; FIMO p < 10⁻³). (a) Kernel density of per-peak motif spacing by category; lines mark medians. Cooperative is shortest (146 bp) vs FOXA1-enabled (180), HNF4A-enabled (182; SVA-filtered), Redundant (197); n = peaks with both motifs per category. (b) Per-bin log₂(observed/expected) in 5 bp bins against a 1,000-permutation per-peak null (motif positions shuffled within the 1,001 bp window). Dark red, FDR-enriched (BH q < 0.05); dark blue, depleted; pale, n.s. Cooperative shows 12 enriched bins at 15–60 bp; FOXA1-enabled and Redundant show 0; HNF4A-enabled shows 1 (Suppl. Fig. 6 for unfiltered). (c) Same pipeline at endogenously co-bound sites. Top: K562 Cooperative, replotted from (b). Second: HepG2 (FOXA1–HNF4A ChIP-seq, GSE104247; 9,373 motif pairs), 14 enriched bins. Third: HDMA fetal hepatocyte caCREs (Liu et al. 2026, Nature; 44,165 peaks in clusters LI_1/3/4/6 from 29,926 cells, PCW15–22; 24,327 motif pairs), 10 enriched bins concentrated at 15–60 bp. Bottom: HDMA fetal brain caCREs (BR_0–BR_17; 74,035 peaks, 14,015 motif pairs), 0 enriched bins.

Article Snippet: Recombinant human full-length FOXA1 (Origene TP306045) and HNF4A (Origene TP317863) were used for binding reactions.

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: Cooperative FOXA1–HNF4A binding emerges from motif spacing and nucleosome architecture

doi: 10.64898/2026.05.27.728252

Figure Lengend Snippet: (a) Single-site Pioneer-seq library design. A single FOXA1 binding site (blue; TGTTTACTTTG, JASPAR MA0148.1) or a single HNF4A binding site (orange; GAGTCCAAAGTCCAG, JASPAR MA0114.2) was placed at each of 182 centre positions (−85 to +96 bp relative to the dyad) on three reconstituted nucleosomal templates: Widom-601, 5S rDNA, and mouse mammary tumor virus (MMTV)-A. A paired nonspecific control sequence (a partial ETS motif; ACCGGAAGTG, JASPAR MA0098.3) was placed at matched positions on the same templates. Each row of the schematic represents one library member. (b) Relative shift (RS) as a function of binding-site centre position relative to the nucleosome dyad. Top row: FOXA1 (blue) and the paired nonspecific control (grey). Bottom row: HNF4A (orange) and the paired nonspecific control. Points show the mean of n = 3 biological replicates; vertical error bars, SEM. Vertical dashed lines mark the dyad (position 0) and the canonical nucleosome boundaries (±73 bp). RS is defined as −log₂((T / T_NS) / (N / N_NS)), where T and T_NS are read counts of the test and paired nonspecific-control nucleosomes in the unshifted band of the TF-treated lane, and N and N_NS are the corresponding counts in the no-TF (null) lane (Methods). (c) Binding ability per template, defined as the mean excess RS over the nonspecific control, ⟨RS_TF − RS_NS⟩, averaged across all 182 positions. Bars show the mean; error bars, SEM propagated from per-position SEMs. p-values, one-sided paired Wilcoxon signed-rank test (alternative: FOXA1 > HNF4A; 182 paired positions per template); the directional hypothesis was prespecified from cellular observations (Hansen et al., 2022a) of FOXA1’s lower per-motif binding requirement.

Article Snippet: Recombinant human full-length FOXA1 (Origene TP306045) and HNF4A (Origene TP317863) were used for binding reactions.

Techniques: Binding Assay, Virus, Control, Sequencing

Journal: bioRxiv

Article Title: Cooperative FOXA1–HNF4A binding emerges from motif spacing and nucleosome architecture

doi: 10.64898/2026.05.27.728252

Figure Lengend Snippet: (a) Cobinding Pioneer-seq library design. On each of three nucleosomal templates (Widom-601, 5S rDNA, mouse mammary tumor virus (MMTV)-A; light-to-dark grey shading), a FOXA1 site (blue) and an HNF4A site (orange) were placed adjacently with a fixed 5 bp gap between the two sites, at each of 77 outermost-site positions (bp 21–97 from the dyad). Each row in the schematic represents one library member. (b) Pioneer-seq relative shift (RS) as a function of the outermost site’s distance from the dyad. Green: FOXA1–HNF4A composite (TGTTTACTTTG–N₅–GAGTCCAAAGTCCAG; JASPAR MA0148.1 + MA0114.2). Blue: FOXA1 alone (MA0148.1). Orange: HNF4A alone (MA0114.2). Grey: paired nonspecific control (ACCGGAAGTG; JASPAR MA0098.3). Per-position values are the mean of n = 3 biological replicates with SEM error bars. Vertical dashed line marks the canonical nucleosome edge (bp 73). Large green dots mark positions where the cobinding signal exceeds the sum of single-TF signals on the linear scale (2^FH > 2^F + 2^H; paired z-test with delta-method error propagation; Bonferroni-corrected across the 77 positions per template, α = 0.05). Cartoons at right depict the four binding conditions, colour-matched to the trace lines. (c) Genomic-nucleosome library. Each row represents one of n = 179 nucleosomes selected from K562 Cooperative-category peaks containing exactly one FOXA1 motif (blue) and exactly one HNF4A motif (orange) (FIMO p < 10⁻³, JASPAR MA0148.4 for FOXA1 and MA0114.4 for HNF4A; nucleosome dyads inferred by DANPOS from K562 MNase-seq (Mieczkowski et al. 2016) at occupancy score ≥ 0.7; Methods). (d) Cobinding RS on the genomic-nucleosome library versus the FOXA1 motif’s distance from the inferred dyad (left) and the HNF4A motif’s distance from the inferred dyad (right). Points, individual nucleosomes; line, ordinary least squares fit; grey band, 95% CI. In-panel: Pearson r, two-sided p, and n. Banner: Δr = r_FOXA1 − r_HNF4A and two-sided Fisher z-test comparing the two Pearson correlations.

Article Snippet: Recombinant human full-length FOXA1 (Origene TP306045) and HNF4A (Origene TP317863) were used for binding reactions.

Techniques: Virus, Control, Binding Assay

Journal: Nature

Article Title: Epigenetic memory of colitis promotes tumour growth

doi: 10.1038/s41586-026-10258-4

Figure Lengend Snippet: a, Gene expression of FOX family transcription factors in all stem cells in primary tissue (n = 23 mice). b, Multi-scale footprinting at sample genomic loci. Top, expanded copy of multiscale plot below. The X-axis represents distance in base pairs from the AP-1 motif center and the Y-axis represents radius of footprint being evaluated. The color represents -log10(p-value) from a one-sided binomial test of the predicted footprint at given radius and genomic position. For all in vitro binding scores, a 14 bp footprint radius was used. c, Genomic tracks of in vivo tissue footprint score (top), Tn5 insertions relative to naked DNA alone (middle), and in vitro binding score for given TF combinations. d, All loci and combinations of TFs tested by in vitro binding assay. Columns are positions relative to AP-1 motif site center and rows are each individual genomic locus. Color represents in vitro binding score calculation of -log10(p-value) at 14 bp radius. e, Average in vitro binding scores across all loci in panel (d). f, AlphaFold predicted structures for Fos-Jun dimer, composite motif and either Foxa1, Foxn2 or Foxj2. g, UniProt domain annotation for FOX TFs with black boxes indicating regions predicted to interact with Fos/Jun heterodimer. Numbers indicate amino acid positions. DBD = DNA binding domain, ZF = Zinc finger, LZ = Leucine zipper. h, Predicted interactions with Foxp1 and Jun alone. All error bars are s.e.m.

Article Snippet: In vitro footprinting was performed as described in ref. with the following modifications: briefly, selected sequences (25 ng per reaction) were incubated with various combinations of recombinant JUN (Active Motif, 31116), FOS (OriGene, TP760257), FOXP1 (OriGene, TP313862) and FOXA1 protein (OriGene, TP306045), along with tagmentation buffer (20 mM Tris, 10 mM MgCl 2 and 20% dimethylformamide) and water in a 22.5-μl total volume at room temperature for 1 h. Then, 0.15 μl of preassembled Tn5 (seqWell, Tagify) was combined with 2.35 μl of dilution buffer (50 mM Tris, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% NP-40 and 50% glycerol), and subsequently added to samples (resulting in final TF concentrations of 300 nM each).

Techniques: Gene Expression, Footprinting, In Vitro, Binding Assay, In Vivo