Journal: Molecular Therapy Oncology

Article Title: Ligand-dependent reprogramming of HNF4A expression and function suppresses multistep hepatocarcinogenesis

doi: 10.1016/j.omton.2026.201246

Figure Lengend Snippet: Antitumor effect of PA is enhanced by fatty acids (A) Quantitation of HNF4A bound to HBEs in DN-like primary HCC cells with no treatment or treated with LA (20 μM), PA (20 μM), ATRA (20 μM), or 9-cis RA (20 μM) for 72 h using ELISA ( n = 3). (B) Quantitation of HNF4A bound to HBEs in DN-like primary HCC cells treated with the indicated concentration of PA for 72 h using ELISA ( n = 3). (C) Quantitation of HNF4A bound to HBEs in PA-treated HLF cells transfected with wild-type (WT) or V255M mutant HNF4A (80 μM, 48 h) using ELISA ( n = 3). (D) Immunoblotting protein levels of HNF4A, RXRα, RXRα ΔN197, RARα, and ACTB in HCC cell lines Huh7, HLE, and HLF. (D) Quantitative reverse transcription polymerase chain reaction analysis of selected genes in DN-like primary HCC cells treated with siRNA control, siRNA HNF4A , or siRNA RXRα for 48 h ( n = 3). (E) Cell proliferation of KH cells transfected with small interfering RNAs treated with PA (40 μM) or ATRA (40 μM) for 96 h ( n = 4). (F) mRNA levels of HNF4A P1 , P2 , ALB , and TTR in DN-like primary HCC cells treated with PA (20 μM) for 1, 3, 6, and 12 h. (G) Protein levels of HNF4A P1 and P2 following treatment with PA (20 μM). (H) H4-Luciferase following 12 h of treatment with PA. (I) Chromatin immunoprecipitation followed by sequencing analysis using an anti-HNF4A antibody in DN-like primary HCC cells treated with 40 μM PA for 12 h, integrated with the RNA sequencing results. (J) Hallmark pathway enrichment analysis of genes activated by HNF4A binding and transcription following treatment with PA. Data are presented as the mean (SD) (in A–B, D–F, and H). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, as determined using one-way analysis of variance. HCC, hepatocellular carcinoma; HBEs, HNF4A-binding elements; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; HNF4A, hepatocyte nuclear factor 4 alpha; TTR, transthyretin.

Article Snippet: Antibodies used included anti-HNF4A P1 K9218 (Perseus Proteomics Inc., Tokyo, Japan), anti-HNF4A P2 H6939 (Perseus Proteomics Inc.), anti-HNF4A All H1415 (Perseus Proteomics Inc.), anti RXRα (Cell Signaling Technology), anti-RXRα ΔN197 (Santa Cruz), anti-RARα C-20 (Santa Cruz), anti-Lamin A/C (Cell Signaling Technology), and anti-ACTB (Santa Cruz).

Techniques: Quantitation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection, Mutagenesis, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Control, Luciferase, Chromatin Immunoprecipitation, Sequencing, RNA Sequencing, Binding Assay

Journal: Molecular Therapy Oncology

Article Title: Ligand-dependent reprogramming of HNF4A expression and function suppresses multistep hepatocarcinogenesis

doi: 10.1016/j.omton.2026.201246

Figure Lengend Snippet: Multistep hepatocarcinogenesis in PDGF-C Tg mice (A) Schematic representation of pathological progression across ages in PDGF-C mice. (B) Representative dynamic magnetic resonance images of PDGF-C Tg mice at 32 and 60 weeks of age. White arrows indicate liver nodules. (C) Immunohistochemistry of Ki-67 and OATP1 in liver tissues with cirrhosis (top panels), DN tissues (middle panels), and HCC (bottom panels). Black arrows indicate liver nodules. Scale bars, 200 μm. (D) Representative MRI images of the liver of PDGF-C Tg mice at different ages. Red arrows indicate liver tumors. (E) Quantitative reverse transcription polymerase chain reaction analysis of the relative mRNA expression of Afp and Slco1a1 (equivalent to human OATP1 ) in the context of liver fibrosis (LC), carcinomatous background liver of 32-week-old PDGF-C Tg mice, DNs, and 60-week-old HCC mice. Data are presented as the mean (SD) ( n = 6). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, as determined using the Mann-Whitney U test. (F) Hematoxylin and eosin and immunohistochemistry staining of OATP1 and AFP in tissues from 32-week-old LC, 32-week-old DN, and 60-week-old HCC mice. Scale bars, 100 μm. (G) Number of somatic mutations in the liver and tumor tissues from 12-week-old LC ( n = 3), 32-week-old DN ( n = 2), and 60-week-old HCC mice ( n = 3). ∗ p < 0.05, as determined using the Mann-Whitney U test. (H) Predicted networks abrogated in HCC tissues. PDGF-C, platelet-derived growth factor-C; Tg, transgenic; HCC, hepatocellular carcinoma; DN, dysplastic nodule; OATP, organic anion transporter polypeptide; HNF4A, hepatocyte nuclear factor 4 alpha.

Article Snippet: Mouse monoclonal anti-human Ki-67 (DAKO); mouse monoclonal anti-human organic anion transporter 1B3 (OATP1B3) MDQ/5F260 (Novus Biologicals, Littleton, CO); rabbit monoclonal anti-human HNF4A (clone C11F12, Cell Signaling Technology, Danvers, MA, USA); an antibody detecting all splicing variants of HNF4A (HNF4A All) mouse monoclonal anti-HNF4A P1 K9218 (Perseus Proteomics Inc., Tokyo, Japan); goat polyclonal anti-human alpha-fetoprotein (AFP) C-19 (Santa Cruz, Dallas, TX, USA); and mouse monoclonal anti-CD31 JC/70A (Abcam, Cambridge, UK) antibodies were used to detect Ki-67, OATP1B3, HNF4A, AFP, and CD31, respectively.

Techniques: Immunohistochemistry, Reverse Transcription, Polymerase Chain Reaction, Expressing, MANN-WHITNEY, Staining, Derivative Assay, Transgenic Assay

Journal: Molecular Therapy Oncology

Article Title: Ligand-dependent reprogramming of HNF4A expression and function suppresses multistep hepatocarcinogenesis

doi: 10.1016/j.omton.2026.201246

Figure Lengend Snippet: PA inhibits the development of HCC from DNs in PDGF-C Tg mice (A) Experimental schedule of PA administration in PDGF-C Tg mice. (B and D) Representative magnetic resonance images of livers from PDGF-C Tg mice before and 8 weeks after PA administration. (C and E) Liver tumor volumes before and after PA administration. Top: treatment starting at 32 weeks of age (control group, n = 7; PA group, n = 8). Bottom: treatment starting at 52 weeks of age (control group, n = 8; PA group, n = 9). ∗ p < 0.05 and ∗∗ p < 0.01, as determined using a paired t test. (F) Heatmap of microarray data showing the expression of 604 genes from tumor and non-tumor tissues treated with PA (orange box) or vehicle (blue box) for 8 weeks ( n = 3 for each condition). Red indicates high expression, whereas green indicates low expression. (G) Predicted transcription factors regulating cluster 1 and 2 genes. (H) Immunohistochemical staining of HNF4A in pre-treatment 32-week-old DN and 52-week-old HCC tissues, together with hematoxylin and eosin, OATP1, and HNF4A P1 staining in tumor tissues following 8 weeks of PA treatment. PDGF-C, platelet-derived growth factor-C; Tg, transgenic; HCC, hepatocellular carcinoma; PA, polyprenoic acid; DN, dysplastic nodule; MRI, magnetic resonance imaging; OATP, organic anion transporter polypeptide; HNF4A, hepatocyte nuclear factor 4 alpha.

Article Snippet: Mouse monoclonal anti-human Ki-67 (DAKO); mouse monoclonal anti-human organic anion transporter 1B3 (OATP1B3) MDQ/5F260 (Novus Biologicals, Littleton, CO); rabbit monoclonal anti-human HNF4A (clone C11F12, Cell Signaling Technology, Danvers, MA, USA); an antibody detecting all splicing variants of HNF4A (HNF4A All) mouse monoclonal anti-HNF4A P1 K9218 (Perseus Proteomics Inc., Tokyo, Japan); goat polyclonal anti-human alpha-fetoprotein (AFP) C-19 (Santa Cruz, Dallas, TX, USA); and mouse monoclonal anti-CD31 JC/70A (Abcam, Cambridge, UK) antibodies were used to detect Ki-67, OATP1B3, HNF4A, AFP, and CD31, respectively.

Techniques: Control, Microarray, Expressing, Immunohistochemical staining, Staining, Derivative Assay, Transgenic Assay, Magnetic Resonance Imaging

Journal: Molecular Therapy Oncology

Article Title: Ligand-dependent reprogramming of HNF4A expression and function suppresses multistep hepatocarcinogenesis

doi: 10.1016/j.omton.2026.201246

Figure Lengend Snippet: PA activates HNF4A P1 signaling and restores hepatocyte homeostasis in DNs of PDGF-C transgenic mice (A) Representative pathological images of two DN cases (left) newly added to the two HCC cases (right) among the 70 HNF4A-stained samples reported by Yamashita et al. (Hepatology, Vol. 60, No. 5, 2014). (B) HNF4A P1 and P2 protein levels in 32-week-old DN and 52-week-old HCC tissues. (C) Protein expression of HNF4A P1 and P2 in fetal and adult liver tissue. (D) Total protein levels of HNF4A P1 and P2 in DN tumor tissues treated with vehicle or PA for 8 weeks. (E) Nuclear HNF4A P1 levels in DN tumor tissues following vehicle or PA treatment, with LAMIN A/C used as controls for nuclear fractions. Data are presented for DN + vehicle ( n = 7) and DN + PA ( n = 6). (F) mRNA expression levels of hepatocyte-maturation-related genes Alb , Ttr , Slco1a1 , Atf7 , Stat6 , Otc1 , and Pck1 in DN + vehicle ( n = 7), DN + PA ( n = 6), HCC + vehicle ( n = 8), and HCC + PA ( n = 8) tumor tissues. (G) mRNA expression levels of the hepatocyte immaturity markers Afp , Vim , and Sall1 in DN + vehicle ( n = 7), DN + PA ( n = 6), HCC + vehicle ( n = 8), and HCC + PA ( n = 8) tumor tissues. (H) Protein expression levels of total ERK1/2, p-ERK1/2 (Thr202/Tyr204), and AKT, p-AKT (Ser473). ACTB was used as a loading control. Data are presented for DN + vehicle ( n = 7) and DN + PA ( n = 6). Data in E–4H are presented as mean ± SD. Statistical significance was assessed using the Mann-Whitney U test (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). PDGF-C, platelet-derived growth factor C; Tg, transgenic; HCC, hepatocellular carcinoma; PA, polyprenoic acid; DN, dysplastic nodule; MRI, magnetic resonance imaging; HNF4A, hepatocyte nuclear factor 4 alpha.

Article Snippet: Mouse monoclonal anti-human Ki-67 (DAKO); mouse monoclonal anti-human organic anion transporter 1B3 (OATP1B3) MDQ/5F260 (Novus Biologicals, Littleton, CO); rabbit monoclonal anti-human HNF4A (clone C11F12, Cell Signaling Technology, Danvers, MA, USA); an antibody detecting all splicing variants of HNF4A (HNF4A All) mouse monoclonal anti-HNF4A P1 K9218 (Perseus Proteomics Inc., Tokyo, Japan); goat polyclonal anti-human alpha-fetoprotein (AFP) C-19 (Santa Cruz, Dallas, TX, USA); and mouse monoclonal anti-CD31 JC/70A (Abcam, Cambridge, UK) antibodies were used to detect Ki-67, OATP1B3, HNF4A, AFP, and CD31, respectively.

Techniques: Transgenic Assay, Staining, Expressing, Control, MANN-WHITNEY, Derivative Assay, Magnetic Resonance Imaging

Journal: Molecular Therapy Oncology

Article Title: Ligand-dependent reprogramming of HNF4A expression and function suppresses multistep hepatocarcinogenesis

doi: 10.1016/j.omton.2026.201246

Figure Lengend Snippet: PA serves as a ligand that activates HNF4A (A) Exogenous expressions of HNF4A2 and HNF4A8 in HLF cells, detected via immunoblotting using a FLAG tag. (B–D) H4 luciferase (B), cell growth assay (C), and mRNA expression level of HNF4A P1 , SLCO1B3 , ALB , and TTR (D) in HLF cells with persistent expression of HNF4A P1 and P2. (E) Immunoblot analysis of HNF4A in HLF cells with persistent high expression of HNF4A2 ( HNF4A2 overexpression) compared with HLF cells generated via an empty vector as a negative control. Cell proliferation of control HLF cells or those overexpressing HNF4A treated with the indicated concentration of PA for 72 h ( n = 4). (F) Docking of PA in the X-ray structure of LBD of HNF4A, predicting PA to bind the hydrophobic groove of the LBD in both open (docking score: −11.938 kcal/mol) and closed conformations (docking score: −12.053 kcal/mol). Receptor residues within 4 Å of the PA molecule are shown in line representation. The hydrogen bond/salt bridge to Thr181 and Arg226 is depicted. (G) Quantitation of LA or PA eluted from recombinant HNF4A or RXRA ( n = 4 in each group). (H) Quantitation of PA eluted from recombinant HNF4A with wild-type (WT), V255M, E285Q, or I314F mutations ( n = 7 in each group). (I) Protein levels of HNF4A measured 72 h after transfection of HLF cells with empty (control), HNF4A2 WT, or HNF4A2 V255M. (J) H4 luciferase activity values 48 h after the addition of 40 and 80 μM of PA to HLF cells overexpressing control, HNF4A2 WT, and HNF4A2 V255M ( n = 3 in each group). (K) DR1 WT luciferase activity and DR1 mut-luciferase activity after 48 h of overexpression of control, HNF4A2 WT, and HNF4A2 V255M in HLF cells, followed by the addition of 40 and 80 μM of PA ( n = 3 in each group). Data are presented as the mean (SD) (in B–E and G–J); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, as determined using the one-way analysis of variance. LBD, ligand-binding domain; PA, polyprenoic acid; HNF4A, hepatocyte nuclear factor 4 alpha; DN, dysplastic nodule; WT, wild-type; RXR, retinoid X receptor.

Article Snippet: Mouse monoclonal anti-human Ki-67 (DAKO); mouse monoclonal anti-human organic anion transporter 1B3 (OATP1B3) MDQ/5F260 (Novus Biologicals, Littleton, CO); rabbit monoclonal anti-human HNF4A (clone C11F12, Cell Signaling Technology, Danvers, MA, USA); an antibody detecting all splicing variants of HNF4A (HNF4A All) mouse monoclonal anti-HNF4A P1 K9218 (Perseus Proteomics Inc., Tokyo, Japan); goat polyclonal anti-human alpha-fetoprotein (AFP) C-19 (Santa Cruz, Dallas, TX, USA); and mouse monoclonal anti-CD31 JC/70A (Abcam, Cambridge, UK) antibodies were used to detect Ki-67, OATP1B3, HNF4A, AFP, and CD31, respectively.

Techniques: Western Blot, FLAG-tag, Luciferase, Growth Assay, Expressing, Over Expression, Generated, Plasmid Preparation, Negative Control, Control, Concentration Assay, Quantitation Assay, Recombinant, Transfection, Activity Assay, Ligand Binding Assay

Journal: Molecular Therapy Oncology

Article Title: Ligand-dependent reprogramming of HNF4A expression and function suppresses multistep hepatocarcinogenesis

doi: 10.1016/j.omton.2026.201246

Figure Lengend Snippet: Antitumor effect of PA is enhanced by fatty acids (A) Quantitation of HNF4A bound to HBEs in DN-like primary HCC cells with no treatment or treated with LA (20 μM), PA (20 μM), ATRA (20 μM), or 9-cis RA (20 μM) for 72 h using ELISA ( n = 3). (B) Quantitation of HNF4A bound to HBEs in DN-like primary HCC cells treated with the indicated concentration of PA for 72 h using ELISA ( n = 3). (C) Quantitation of HNF4A bound to HBEs in PA-treated HLF cells transfected with wild-type (WT) or V255M mutant HNF4A (80 μM, 48 h) using ELISA ( n = 3). (D) Immunoblotting protein levels of HNF4A, RXRα, RXRα ΔN197, RARα, and ACTB in HCC cell lines Huh7, HLE, and HLF. (D) Quantitative reverse transcription polymerase chain reaction analysis of selected genes in DN-like primary HCC cells treated with siRNA control, siRNA HNF4A , or siRNA RXRα for 48 h ( n = 3). (E) Cell proliferation of KH cells transfected with small interfering RNAs treated with PA (40 μM) or ATRA (40 μM) for 96 h ( n = 4). (F) mRNA levels of HNF4A P1 , P2 , ALB , and TTR in DN-like primary HCC cells treated with PA (20 μM) for 1, 3, 6, and 12 h. (G) Protein levels of HNF4A P1 and P2 following treatment with PA (20 μM). (H) H4-Luciferase following 12 h of treatment with PA. (I) Chromatin immunoprecipitation followed by sequencing analysis using an anti-HNF4A antibody in DN-like primary HCC cells treated with 40 μM PA for 12 h, integrated with the RNA sequencing results. (J) Hallmark pathway enrichment analysis of genes activated by HNF4A binding and transcription following treatment with PA. Data are presented as the mean (SD) (in A–B, D–F, and H). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, as determined using one-way analysis of variance. HCC, hepatocellular carcinoma; HBEs, HNF4A-binding elements; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; HNF4A, hepatocyte nuclear factor 4 alpha; TTR, transthyretin.

Article Snippet: Mouse monoclonal anti-human Ki-67 (DAKO); mouse monoclonal anti-human organic anion transporter 1B3 (OATP1B3) MDQ/5F260 (Novus Biologicals, Littleton, CO); rabbit monoclonal anti-human HNF4A (clone C11F12, Cell Signaling Technology, Danvers, MA, USA); an antibody detecting all splicing variants of HNF4A (HNF4A All) mouse monoclonal anti-HNF4A P1 K9218 (Perseus Proteomics Inc., Tokyo, Japan); goat polyclonal anti-human alpha-fetoprotein (AFP) C-19 (Santa Cruz, Dallas, TX, USA); and mouse monoclonal anti-CD31 JC/70A (Abcam, Cambridge, UK) antibodies were used to detect Ki-67, OATP1B3, HNF4A, AFP, and CD31, respectively.

Techniques: Quantitation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection, Mutagenesis, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Control, Luciferase, Chromatin Immunoprecipitation, Sequencing, RNA Sequencing, Binding Assay

Journal: Molecular Therapy Oncology

Article Title: Ligand-dependent reprogramming of HNF4A expression and function suppresses multistep hepatocarcinogenesis

doi: 10.1016/j.omton.2026.201246

Figure Lengend Snippet: HNF4α mediates sensitivity to PA in an in vivo DN model (A) Experimental schedule of LNP-small interfering RNA and PA administration in 32-week-old male PDGF-C Tg mice. (B) Representative magnetic resonance images of PDGF-C Tg livers treated with siRNA control or siRNA Hnf4a before and after treatment with PA. (C) Tumor volumes in the liver before and after PA treatment for siRNA control ( n = 5) and siRNA Hnf4a ( n = 4). (∗ p < 0.05, as determined using paired t test.) (D) Quantitative reverse transcription polymerase chain reaction and immunoblot analyses of PDGF-C Tg mouse liver treated with siRNA Control or siRNA Hnf4a All ( n = 3). (E) Immunoblot analysis of livers from PDGF-C Tg mice following siRNA and PA administration according to the schedule shown in (A). (F) Number of mutated genes in liver tumors of mice treated with siRNA control ( n = 2) or siRNA Hnf4a All ( n = 2) groups. (G and H) Representative photomicrographs of hematoxylin and eosin staining and immunohistochemistry for Ki-67, OATP1, AFP, CD31, and HNF4A in tumors of PDGF-C Tg mice treated with siRNA control and PA (G) siRNA Hnf4a All and PA for 8 weeks (H). Scale bars, 100 μm. (I) mRNA expression levels of Ttr , Slco1a1 , Alb , and Afp in liver tumors from PDGF-C Tg mice receiving PA with continuous tail injection of siRNA control ( n = 5) or siRNA Hnf4a All ( n = 4). Data are presented as the mean (SD). ∗ p < 0.05, as determined using the Mann-Whitney U test. PDGF-C, platelet-derived growth factor C; PA, polyprenoic acid; HNF4A, hepatocyte nuclear factor 4 alpha; Tg, transgenic.

Article Snippet: Mouse monoclonal anti-human Ki-67 (DAKO); mouse monoclonal anti-human organic anion transporter 1B3 (OATP1B3) MDQ/5F260 (Novus Biologicals, Littleton, CO); rabbit monoclonal anti-human HNF4A (clone C11F12, Cell Signaling Technology, Danvers, MA, USA); an antibody detecting all splicing variants of HNF4A (HNF4A All) mouse monoclonal anti-HNF4A P1 K9218 (Perseus Proteomics Inc., Tokyo, Japan); goat polyclonal anti-human alpha-fetoprotein (AFP) C-19 (Santa Cruz, Dallas, TX, USA); and mouse monoclonal anti-CD31 JC/70A (Abcam, Cambridge, UK) antibodies were used to detect Ki-67, OATP1B3, HNF4A, AFP, and CD31, respectively.

Techniques: In Vivo, Small Interfering RNA, Control, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Staining, Immunohistochemistry, Expressing, Injection, MANN-WHITNEY, Derivative Assay, Transgenic Assay

Journal: Molecular Therapy Oncology

Article Title: Ligand-dependent reprogramming of HNF4A expression and function suppresses multistep hepatocarcinogenesis

doi: 10.1016/j.omton.2026.201246

Figure Lengend Snippet: Reduced expression of HNF4A in liver tumors abrogates the antitumor effects of PA (A) Experimental timeline of the treatment course of 32-week-old male hPDGF-C Tg/Alb-creERT2/ Hnf4a flox/+ (heterozygous) mice, with intraperitoneal administration of 1 mg TAM/mice and oral 80 mg/kg/day. (B) EOB-magnetic resonance images before and after PA oral administration (vehicle n = 9, PA n = 8). Red arrows indicate a liver tumor. (C) Tumor mass measurements before and after PA oral administration. Data are presented as the mean (SD) (∗ p < 0.05, as determined using paired t test). (D) Protein levels of HNF4A P1, P2, and ACTB in liver tissues of mice administered vehicle or PA. (E) mRNA levels of Afp , Alb , Slco1a1 , and Ttr in liver tumor tissues from vehicle and PA-treated groups. Data are presented as the mean (SD) ( n = 8) (∗ p < 0.05, as determined using Mann-Whitney U test.). (F) Immunohistochemistry analysis of hematoxylin and eosin, HNF4A, OATP1, and Ki67 in liver tissues from vehicle- and PA-treated groups. Scale bars, 100 μm. PA, polyprenoic acid; HNF4A, hepatocyte nuclear factor 4 alpha; Tg, transgenic.

Article Snippet: Mouse monoclonal anti-human Ki-67 (DAKO); mouse monoclonal anti-human organic anion transporter 1B3 (OATP1B3) MDQ/5F260 (Novus Biologicals, Littleton, CO); rabbit monoclonal anti-human HNF4A (clone C11F12, Cell Signaling Technology, Danvers, MA, USA); an antibody detecting all splicing variants of HNF4A (HNF4A All) mouse monoclonal anti-HNF4A P1 K9218 (Perseus Proteomics Inc., Tokyo, Japan); goat polyclonal anti-human alpha-fetoprotein (AFP) C-19 (Santa Cruz, Dallas, TX, USA); and mouse monoclonal anti-CD31 JC/70A (Abcam, Cambridge, UK) antibodies were used to detect Ki-67, OATP1B3, HNF4A, AFP, and CD31, respectively.

Techniques: Expressing, MANN-WHITNEY, Immunohistochemistry, Transgenic Assay

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: npj Metabolic Health and Disease

Article Title: A multi-omic approach reveals iron availability influences cell fate fidelity

doi: 10.1038/s44324-026-00102-8

Figure Lengend Snippet: A Volcano plot of differentially expressed genes (DEGs) identified from RNA-Seq analysis comparing HepG2 cells cultured in Plasmax with HepG2 cells cultured in EMEM, n = 3. Genes significantly downregulated and upregulated in Plasmax are highlighted in blue and red, respectively. B Gene set enrichment analysis (GSEA) plots derived from RNA-Seq analysis comparing HepG2 cells cultured in Plasmax with HepG2 cells cultured in EMEM demonstrating signatures associated with hepatocyte cell state and function. Hepatocyte signature, Gene set AIZARANI_LIVER_C14_HEPATOCYTES_2; HNF4A Target genes, Gene set OHGUCHI_LIVER_HNF4A_TARGETS_DN. C Representative immunoblot analysis of HNF4A expression in HepG2 cells cultured in EMEM or Plasmax. Actin is included as a loading control. D Heatmap of hepatocyte and hepatoblast gene expression in HepG2 cells culture in EMEM or Plasmax as determined by RNA-Seq analysis, n = 3. E Representative confocal images of HepG2 cells cultured in EMEM or Plasmax and stained with BODIPY (green) and DAPI (blue). White scale bars represent 50 µm. Yellow scale bars represent 10 µm. F Intensity of BODIPY staining per cell, determined by confocal microscopy, in HepG2 cells cultured in EMEM or Plasmax. Data are shown as median and interquartile ranges, n = 3. G Viability of HepG2 cells cultured in EMEM or Plasmax following treatment with ethanol for 24 h as determined by a CellTiter-Glo Assay. Data are shown as mean ± SEM, n = 4. For all experiments, * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: Membranes were probed with primary antibodies recognising HNF4A (Cell Signaling Technology, 3113, 1:1000 dilution), BACH1 (Proteintech, 14018-1-AP, 1:500 dilution), and β-Actin (Cell Signaling Technology, 3700, 1:5000 dilution).

Techniques: RNA Sequencing, Cell Culture, Derivative Assay, Western Blot, Expressing, Control, Gene Expression, Staining, Confocal Microscopy, Glo Assay

Journal: npj Metabolic Health and Disease

Article Title: A multi-omic approach reveals iron availability influences cell fate fidelity

doi: 10.1038/s44324-026-00102-8

Figure Lengend Snippet: A Representative immunoblot analysis of HNF4A expression in HepG2 cells cultured in EMEM, Plasmax, Plasmax devoid of trace elements (TE), or Plasmax devoid of supplemental metabolites (SM). Actin is included as a loading control. B GSEA plots derived from RNA-Seq analysis comparing HepG2 cells cultured in Plasmax with HepG2 cells cultured in EMEM or comparing HepG2 cells cultured in Plasmax-TE with HepG2 cells cultured in EMEM demonstrating signatures associated with hepatocyte cell state and function. Hepatocyte signature, Gene set AIZARANI_LIVER_C14_HEPATOCYTES_2; HNF4A Target genes, Gene set OHGUCHI_LIVER_HNF4A_TARGETS_DN. C Volcano plot of DEGs identified from RNA-Seq analysis comparing HepG2 cells cultured in Plasmax-TE with HepG2 cells cultured in Plasmax, n = 3. Hepatocyte differentiation markers are annotated and highlighted in maroon. Significantly downregulated and upregulated genes are highlighted in blue and red, respectively. D SOX4 transcript expression (counts per million, CPM) based on RNA-Seq analysis of HepG2 cells cultured in EMEM, Plasmax, or Plasmax-TE, n = 3. For all experiments, ns not significant, *** P < 0.001.

Article Snippet: Membranes were probed with primary antibodies recognising HNF4A (Cell Signaling Technology, 3113, 1:1000 dilution), BACH1 (Proteintech, 14018-1-AP, 1:500 dilution), and β-Actin (Cell Signaling Technology, 3700, 1:5000 dilution).

Techniques: Western Blot, Expressing, Cell Culture, Control, Derivative Assay, RNA Sequencing

Journal: npj Metabolic Health and Disease

Article Title: A multi-omic approach reveals iron availability influences cell fate fidelity

doi: 10.1038/s44324-026-00102-8

Figure Lengend Snippet: A Representative immunoblot analysis of HNF4A expression in HepG2 cells cultured in Plasmax, Plasmax-TE, Plasmax-TE+Fe, Plasmax-TE supplemented with copper salts (0.005 µM cupric sulfate), or Plasmax-TE supplemented with iron and copper salts (0.12 µM ferric nitrate, 1.04 µM ferric sulfate, 0.005 µM cupric sulfate). Actin is included as a loading control. B GSEA plots derived from RNA-Seq analysis comparing HepG2 cells cultured in Plasmax-TE with HepG2 cells cultured in Plasmax and comparing HepG2 cells cultured in Plasmax-TE+Fe with HepG2 cells cultured in Plasmax demonstrating signatures associated with hepatocyte cell state and function. Hepatocyte signature, Gene set AIZARANI_LIVER_C14_HEPATOCYTES_2; HNF4A Target genes, Gene set OHGUCHI_LIVER_HNF4A_TARGETS_DN. C Heatmap of hepatocyte and hepatoblast gene expression in Plasmax, Plasmax-TE, or Plasmax-TE+Fe as determined by RNA-Seq analysis, n = 3. D Volcano plot of differentially expressed proteins identified from proteomic analysis comparing HepG2 cells cultured in Plasmax-TE+Fe with HepG2 cells cultured in Plasmax-TE, n = 5. BACH1 targets are highlighted in maroon. Significantly downregulated and upregulated proteins are highlighted in blue and red, respectively. E Representative immunoblot analysis of BACH1 and HNF4A expression in HepG2 cells cultured in EMEM or Plasmax. Actin is included as a loading control. F Schematic demonstrating the reciprocal relationship between iron availability, HNF4A activity, and hepatic fate.

Article Snippet: Membranes were probed with primary antibodies recognising HNF4A (Cell Signaling Technology, 3113, 1:1000 dilution), BACH1 (Proteintech, 14018-1-AP, 1:500 dilution), and β-Actin (Cell Signaling Technology, 3700, 1:5000 dilution).

Techniques: Western Blot, Expressing, Cell Culture, Control, Derivative Assay, RNA Sequencing, Gene Expression, Activity Assay

Journal: Cells

Article Title: From Patient Liver Tissue to Organoids: Establishment of a Translational Platform Using Healthy, Steatotic, and Cirrhotic Tissue Sources

doi: 10.3390/cells15050432

Figure Lengend Snippet: Relative expression of HNF4A , MKI67 , LGR5 , and ALB in human organoids as determined by qPCR. The bars represent the mean ± SD (N = 3). An ordinary one-way ANOVA was conducted for statistical analysis (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: The human HNF4a qPCR primer pair (# HP200437 , NM_000457 , Origene), human MKI67 (# HP206104 , NM_002417.5 , Origene), human LGR5 ( NM_001277226.2 , [ ]), and human ALB ( NM_000477.7 , [ ]) were used.

Techniques: Expressing