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( A ) Schematic of experimental design to infect K562 cells with FOXA1- or <t>HNF4A-lentivirus</t> and then perform functional assays on dox-induced cells. In CUT&Tag, a protein A-protein G fusion (pA/G) increases the binding spectrum for Fc-binding and allows Tn5 recruitment to antibody-labeled transcription factor (TF) binding sites. In ATAC-seq, Tn5 homes to any accessible site. And in RNA-seq, polyA RNA is captured and sequenced. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1-HNF4A compared to the number actually activated. Both liver- (p<10 –38 ) and intestinal enrichment (p<10 –13 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C ) Genome browser view of a representative liver-specific locus ( ALB ) in FOXA1-HNF4A clonal line that shows uninduced and induced accessibility, FOXA1 binding, and HNF4A binding. ( D ) Heatmap showing uninduced and induced accessibility at all FOXA1-HNF4A co-bound sites within 50 kb of each FOXA1-HNF4A-activated liver-specific gene (n = 53). ( E ) Meta plot showing average signal across each site from ( D ).

Hnf4a Orf, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A test of the pioneer factor hypothesis using ectopic liver gene activation"

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

Journal: eLife

doi: 10.7554/eLife.73358

( A ) Schematic of experimental design to infect K562 cells with FOXA1- or HNF4A-lentivirus and then perform functional assays on dox-induced cells. In CUT&Tag, a protein A-protein G fusion (pA/G) increases the binding spectrum for Fc-binding and allows Tn5 recruitment to antibody-labeled transcription factor (TF) binding sites. In ATAC-seq, Tn5 homes to any accessible site. And in RNA-seq, polyA RNA is captured and sequenced. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1-HNF4A compared to the number actually activated. Both liver- (p<10 –38 ) and intestinal enrichment (p<10 –13 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C ) Genome browser view of a representative liver-specific locus ( ALB ) in FOXA1-HNF4A clonal line that shows uninduced and induced accessibility, FOXA1 binding, and HNF4A binding. ( D ) Heatmap showing uninduced and induced accessibility at all FOXA1-HNF4A co-bound sites within 50 kb of each FOXA1-HNF4A-activated liver-specific gene (n = 53). ( E ) Meta plot showing average signal across each site from ( D ).

Figure Legend Snippet: ( A ) Schematic of experimental design to infect K562 cells with FOXA1- or HNF4A-lentivirus and then perform functional assays on dox-induced cells. In CUT&Tag, a protein A-protein G fusion (pA/G) increases the binding spectrum for Fc-binding and allows Tn5 recruitment to antibody-labeled transcription factor (TF) binding sites. In ATAC-seq, Tn5 homes to any accessible site. And in RNA-seq, polyA RNA is captured and sequenced. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1-HNF4A compared to the number actually activated. Both liver- (p<10 –38 ) and intestinal enrichment (p<10 –13 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C ) Genome browser view of a representative liver-specific locus ( ALB ) in FOXA1-HNF4A clonal line that shows uninduced and induced accessibility, FOXA1 binding, and HNF4A binding. ( D ) Heatmap showing uninduced and induced accessibility at all FOXA1-HNF4A co-bound sites within 50 kb of each FOXA1-HNF4A-activated liver-specific gene (n = 53). ( E ) Meta plot showing average signal across each site from ( D ).

Techniques Used: Functional Assay, Binding Assay, Labeling, RNA Sequencing Assay

qPCR measurements made from RNA extracted from either the FOXA1 clonal line ( A–D ) or the HNF4A clonal line ( E–H ) that was treated with either increasing doxycycline concentrations or longer time periods. Expression is displayed as log10 fold induction over either 0 µg/ml doxycycline control (for concentration titration) or time 0 (for time titration). Each sample primer was normalized to the HPRT housekeeping gene. Doxycycline concentration titration measurements were made at 0, 0.01, 0.05, 0.1, 0.5, 2, and 5 µg/ml. Doxycycline treatment time measurements were made at 0, 6, 12, 24, 48, 72, and 96 hr.

Figure Legend Snippet: qPCR measurements made from RNA extracted from either the FOXA1 clonal line ( A–D ) or the HNF4A clonal line ( E–H ) that was treated with either increasing doxycycline concentrations or longer time periods. Expression is displayed as log10 fold induction over either 0 µg/ml doxycycline control (for concentration titration) or time 0 (for time titration). Each sample primer was normalized to the HPRT housekeeping gene. Doxycycline concentration titration measurements were made at 0, 0.01, 0.05, 0.1, 0.5, 2, and 5 µg/ml. Doxycycline treatment time measurements were made at 0, 6, 12, 24, 48, 72, and 96 hr.

Techniques Used: Expressing, Concentration Assay, Titration

( A ) The number of genome-wide FOXA1 or HNF4A transcription factor binding sites (TFBS) in the induced (+dox) cells that overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘accessible binding site’) or that do not overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘inaccessible binding site’). ( B ) The number of inaccessible binding sites from ( A ) that overlap with an ATAC-seq peak in the induced (+dox) cells (‘opened’) or that do not overlap with an ATAC-seq peak (‘remained closed’). ( C ) The number of FOXA1 or HNF4A binding sites within 50 kb of each FOXA1-HNF4A co-activated gene characterized as either a ‘HepG2 binding site,’ where the TFBS overlaps a TFBS of FOXA1 or HNF4A in HepG2 liver cells, or as a ‘Novel K562 binding site,’ where the TFBS does not overlap with a HepG2 binding site.

Figure Legend Snippet: ( A ) The number of genome-wide FOXA1 or HNF4A transcription factor binding sites (TFBS) in the induced (+dox) cells that overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘accessible binding site’) or that do not overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘inaccessible binding site’). ( B ) The number of inaccessible binding sites from ( A ) that overlap with an ATAC-seq peak in the induced (+dox) cells (‘opened’) or that do not overlap with an ATAC-seq peak (‘remained closed’). ( C ) The number of FOXA1 or HNF4A binding sites within 50 kb of each FOXA1-HNF4A co-activated gene characterized as either a ‘HepG2 binding site,’ where the TFBS overlaps a TFBS of FOXA1 or HNF4A in HepG2 liver cells, or as a ‘Novel K562 binding site,’ where the TFBS does not overlap with a HepG2 binding site.

Techniques Used: Genome Wide, Binding Assay

( A ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1 compared to the number actually activated. Liver enrichment (p<10 –4 ) is significant. There are 242 total liver-enriched genes. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by HNF4A compared to the number actually activated. Liver- (p<10 –8 ) and intestine enrichment (p<10 –15 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C ) 242 liver genes characterized as activated by Foxa1, HNF4A, both, or neither. ( D ) 122 intestine genes characterized as activated by FOXA1, HNF4A, both, or neither.

Figure Legend Snippet: ( A ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1 compared to the number actually activated. Liver enrichment (p<10 –4 ) is significant. There are 242 total liver-enriched genes. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by HNF4A compared to the number actually activated. Liver- (p<10 –8 ) and intestine enrichment (p<10 –15 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C ) 242 liver genes characterized as activated by Foxa1, HNF4A, both, or neither. ( D ) 122 intestine genes characterized as activated by FOXA1, HNF4A, both, or neither.

Techniques Used:

Figure Legend Snippet: ( A ) Genome browser view of a representative liver-specific locus ( ARG1 ) in FOXA1 clonal line showing uninduced and induced accessibility and FOXA1 binding. ( B ) Genome browser view of a representative liver-specific locus ( APOC3 ) in HNF4A clonal line showing uninduced and induced accessibility and HNF4A binding. ( C ) Heatmap of uninduced and induced accessibility at all FOXA1 binding sites within 50 kb of each FOXA1-activated liver-specific genes (n = 59). ( D ) Heatmap of uninduced and induced accessibility at all HNF4A binding sites within 50 kb of each HNF4A-activated liver-specific genes (n = 76). ( E ) Meta plot showing average signal across each site from ( C ). ( F ) Meta plot showing average signal across each site from ( D ). ( G ) Human FOXA1 and HNF4A sequence logo from JASPAR. ( H ) FOXA1 or HNF4A motif count within 500 bp centered upon FOXA1 or HNF4A binding sites within 50 kb of each FOXA1- or HNF4A-activated liver-specific genes, respectively. Motifs were called with FIMO using 1e-3 a p-value threshold. For each boxplot, the center line represents the median, the box represents the first to third quartiles, and the whiskers represent any points within 1.5× the interquartile range.

Techniques Used: Binding Assay, Sequencing

Figure Legend Snippet: ( A ) The number of genome-wide FOXA1 or HNF4A transcription factor binding sites (TFBS) in the induced (+dox) cells that overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘aAccessible binding site’) or that do not overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘inaccessible binding site’). ( B ) The number of inaccessible binding sites from ( A ) that overlap with an ATAC-seq peak in the induced (+dox) cells (‘opened’) or that do not overlap with an ATAC-seq peak (‘remained closed’). ( C ) The number of FOXA1 or HNF4A binding sites within 50 kb of each FOXA1- or HNF4A-activated gene characterized as either a ‘HepG2 binding site,’ where the TFBS overlaps a TFBS of FOXA1 or HNF4A in HepG2 liver cells, or as a ‘Novel K562 binding site,’ where the TFBS does not overlap with a HepG2 binding site.

Techniques Used: Genome Wide, Binding Assay

( A ) FIMO scans at p-value threshold 1e-3 for four most common proposed K562 pioneer factors (PFs) in either FoxA1 inaccessible binding sites (red), Hnf4a inaccessible binding sites (blue), or random equally lengthed binding sites (gray).

Figure Legend Snippet: ( A ) FIMO scans at p-value threshold 1e-3 for four most common proposed K562 pioneer factors (PFs) in either FoxA1 inaccessible binding sites (red), Hnf4a inaccessible binding sites (blue), or random equally lengthed binding sites (gray).

Techniques Used: Binding Assay

( A ) 1000 random 200 bp fragments were generated using BEDTools and then scanned for FOXA1 and HNF4A motifs with FIMO using 1e-3 a p-value threshold. Total motif count was divided by the number of non-N-containing random sequences (924) to identify motifs per random 200 bp fragment.

Figure Legend Snippet: ( A ) 1000 random 200 bp fragments were generated using BEDTools and then scanned for FOXA1 and HNF4A motifs with FIMO using 1e-3 a p-value threshold. Total motif count was divided by the number of non-N-containing random sequences (924) to identify motifs per random 200 bp fragment.

Techniques Used: Generated

( A ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1 at a lower doxycycline concentration (0.05 µg/ml) compared to the number actually activated. There are 242 total liver-enriched genes. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by HNF4A at a lower doxycycline concentration (0.05 µg/ml) compared to the number actually activated. Liver- (p<10 –5 ) and intestine enrichment (p<10 –14 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C, D ) Genome-wide FOXA1 ( C ) or HNF4A ( D ) binding sites classified as either events that occurred at sites that were accessible or inaccessible in the uninduced (-dox) state at 0.5 and 0.05 µg/ml doxycycline induction.

Figure Legend Snippet: ( A ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1 at a lower doxycycline concentration (0.05 µg/ml) compared to the number actually activated. There are 242 total liver-enriched genes. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by HNF4A at a lower doxycycline concentration (0.05 µg/ml) compared to the number actually activated. Liver- (p<10 –5 ) and intestine enrichment (p<10 –14 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C, D ) Genome-wide FOXA1 ( C ) or HNF4A ( D ) binding sites classified as either events that occurred at sites that were accessible or inaccessible in the uninduced (-dox) state at 0.5 and 0.05 µg/ml doxycycline induction.

Techniques Used: Concentration Assay, Genome Wide, Binding Assay

$( A ) Venn diagram of all liver genes categorized as either activated by FOXA1, HNF4A, FOXA1-HNF4A, some combination, or by none of the three cocktails. ( B ) Genome browser view of a representative liver-specific locus ( AMDHD1 ) showing examples of a co-bound site that is ‘FOXA1 pioneered’ (FP), ‘HNF4A pioneered’ (HP), and ‘cooperatively bound’ (CB). The first two tracks are FOXA1 and HNF4A binding in the FOXA1-HNF4A co-expression clone, and the last two tracks are FOXA1 and HNF4A binding in their individual expression clones. ( C ) List of the 31 liver genes that are only activated by FOXA1-HNF4A co-expression. The columns indicate how many co-bound FP, HP, or CB peaks exist within 100 kb of the gene. ( D ) Venn diagram of all genome-wide co-bound peaks categorized as either bound by FOXA1 individually (FP), HNF4A individually (HP), by both, or by neither (CB). ( E ) Overlap of FP, HP, and CB sites from ( D ) with ChromHMM annotations showing the fraction of each co-binding site type in each chromatin region.$
Figure Legend Snippet: ( A ) Venn diagram of all liver genes categorized as either activated by FOXA1, HNF4A, FOXA1-HNF4A, some combination, or by none of the three cocktails. ( B ) Genome browser view of a representative liver-specific locus ( AMDHD1 ) showing examples of a co-bound site that is ‘FOXA1 pioneered’ (FP), ‘HNF4A pioneered’ (HP), and ‘cooperatively bound’ (CB). The first two tracks are FOXA1 and HNF4A binding in the FOXA1-HNF4A co-expression clone, and the last two tracks are FOXA1 and HNF4A binding in their individual expression clones. ( C ) List of the 31 liver genes that are only activated by FOXA1-HNF4A co-expression. The columns indicate how many co-bound FP, HP, or CB peaks exist within 100 kb of the gene. ( D ) Venn diagram of all genome-wide co-bound peaks categorized as either bound by FOXA1 individually (FP), HNF4A individually (HP), by both, or by neither (CB). ( E ) Overlap of FP, HP, and CB sites from ( D ) with ChromHMM annotations showing the fraction of each co-binding site type in each chromatin region.

Techniques Used: Binding Assay, Expressing, Clone Assay, Genome Wide

( A ) Venn diagram of all FOXA1-HNF4A-induced differentially accessible peaks categorized by whether the peak was also induced in the FOXA1 clone, HNF4A clone, neither, or both.

Figure Legend Snippet: ( A ) Venn diagram of all FOXA1-HNF4A-induced differentially accessible peaks categorized by whether the peak was also induced in the FOXA1 clone, HNF4A clone, neither, or both.

Techniques Used:

( A ) FOXA1 or HNF4A motif count at all genomic occurrences of the respective transcription factor’s (TF’s) accessible or inaccessible binding sites. ( B ) FOXA1 or HNF4A motif count in genome-wide inaccessible binding sites versus length-matched random inaccessible DNA sequences. ( C ) Receiver operating characteristic (ROC) curves for predictive power of using sequence motif content to predict accessible (left panels) or inaccessible (right panels) binding sites from random sequence. ( D ) Total FOXA1 and HNF4A motif count at all genomic occurrences of inaccessible co-binding versus length-matched random inaccessible DNA sequences. ( E ) FOXA1 or HNF4A motif count in respective FOXA1 or HNF4A pioneered sites versus in cooperative binding sites (where neither TF bound individually). ( F ) ROC curves for predictive power of using sequence motif content to predict accessible or inaccessible co-binding events from random sequence (top panels) or to predict FOXA1 or HNF4A pioneered events from cooperative binding events. All FIMO scans used 1e-3 as p-value threshold and were conducted on 500 bp of sequence centered upon the binding site.

Figure Legend Snippet: ( A ) FOXA1 or HNF4A motif count at all genomic occurrences of the respective transcription factor’s (TF’s) accessible or inaccessible binding sites. ( B ) FOXA1 or HNF4A motif count in genome-wide inaccessible binding sites versus length-matched random inaccessible DNA sequences. ( C ) Receiver operating characteristic (ROC) curves for predictive power of using sequence motif content to predict accessible (left panels) or inaccessible (right panels) binding sites from random sequence. ( D ) Total FOXA1 and HNF4A motif count at all genomic occurrences of inaccessible co-binding versus length-matched random inaccessible DNA sequences. ( E ) FOXA1 or HNF4A motif count in respective FOXA1 or HNF4A pioneered sites versus in cooperative binding sites (where neither TF bound individually). ( F ) ROC curves for predictive power of using sequence motif content to predict accessible or inaccessible co-binding events from random sequence (top panels) or to predict FOXA1 or HNF4A pioneered events from cooperative binding events. All FIMO scans used 1e-3 as p-value threshold and were conducted on 500 bp of sequence centered upon the binding site.

Techniques Used: Binding Assay, Genome Wide, Sequencing

Figure Legend Snippet:

Techniques Used: Infection, Plasmid Preparation, SYBR Green Assay, Software

Image Search Results

HNF4A is methylated and suppressed in pancreatic cancer. (A) Global DNA methylation analysis (Illumina Human Methylation 450K array) in 20 human pancreatic cancer and 11 normal tissues (Stanford University Medical Center, USA). Heatmap of methylation beta values for the top 7242 differentially regulated loci in cancer vs control ( P < .05, FC ≥ 1.5). (B) Venn diagram showing 2717 unique methylation sites identified in our study. (C) Heatmap of methylation beta values across the HNF4A locus. The HNF4A promoter area with increased DNA methylation (high beta values) corresponds to probes covering the proximal promoter area (from −200 nt to −3 nt, TSS200). (D) HNF4A expression as assessed by RT-qPCR, in pancreatic cancer and control tissues. Expression was normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to control tissues (set as 1). (E) Immunohistochemical analysis for HNF4A in normal (N) and pancreatic cancer tissues (red, HNF4A; blue, nuclei staining). Scale bar: 1 mm and 100 μm (left and right panel, respectively).

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: HNF4A is methylated and suppressed in pancreatic cancer. (A) Global DNA methylation analysis (Illumina Human Methylation 450K array) in 20 human pancreatic cancer and 11 normal tissues (Stanford University Medical Center, USA). Heatmap of methylation beta values for the top 7242 differentially regulated loci in cancer vs control ( P < .05, FC ≥ 1.5). (B) Venn diagram showing 2717 unique methylation sites identified in our study. (C) Heatmap of methylation beta values across the HNF4A locus. The HNF4A promoter area with increased DNA methylation (high beta values) corresponds to probes covering the proximal promoter area (from −200 nt to −3 nt, TSS200). (D) HNF4A expression as assessed by RT-qPCR, in pancreatic cancer and control tissues. Expression was normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to control tissues (set as 1). (E) Immunohistochemical analysis for HNF4A in normal (N) and pancreatic cancer tissues (red, HNF4A; blue, nuclei staining). Scale bar: 1 mm and 100 μm (left and right panel, respectively).

Article Snippet: HNF4A sequence, tagged with MYC-Flag, was obtained from HNF 4 alpha (HNF4A) (HNF4alpha1, NM_178849) Human Tagged ORF Clone Lentiviral Particle (RC214914, Origene) and cloned into the pLenti CMV/TO Puro DEST vector (17293, Addgene).

Techniques: Methylation, DNA Methylation Assay, Control, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Verification of HNF4A CpG methylation sites through bisulfite sequencing. (A) HNF4A expression as assessed by RT-qPCR, in 8 pancreatic cancer cell lines. Expression was normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to the low HNF4A expressing cell line, MIA PaCa-2 (set as 1). (B) Diagram illustrating the generation of HNF4A isoforms through transcriptional regelation by P1 and P2 promoters and alternative splicing. Different primers were designed to recognize the 4 subgroups of the 12 HNF4A isoforms. (C) HNF4A P1a isoforms expression in pancreatic and liver (SNU-475 and Hep-3B) cancer cell lines as assessed by RT-qPCR. Expression was normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to the high HNF4A expressing cell line, Hep-3B (set as 1). (D) Diagram illustrating the experimental design for the evaluation of HNF4A methylation through bisulfite sequencing. (E) Heatmaps of the methylation ratio across the HNF4A locus, at the single CpG site level, for untreated (Cont) or 5-AZA-CdR–treated MIA PaCa-2 cells (5-Aza). Cells were treated with 5-Aza (1μM), for 48 h. Analysis was performed in 40 CpG sites spanning from a distal locus upstream of +1 position to exon 3.

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: Verification of HNF4A CpG methylation sites through bisulfite sequencing. (A) HNF4A expression as assessed by RT-qPCR, in 8 pancreatic cancer cell lines. Expression was normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to the low HNF4A expressing cell line, MIA PaCa-2 (set as 1). (B) Diagram illustrating the generation of HNF4A isoforms through transcriptional regelation by P1 and P2 promoters and alternative splicing. Different primers were designed to recognize the 4 subgroups of the 12 HNF4A isoforms. (C) HNF4A P1a isoforms expression in pancreatic and liver (SNU-475 and Hep-3B) cancer cell lines as assessed by RT-qPCR. Expression was normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to the high HNF4A expressing cell line, Hep-3B (set as 1). (D) Diagram illustrating the experimental design for the evaluation of HNF4A methylation through bisulfite sequencing. (E) Heatmaps of the methylation ratio across the HNF4A locus, at the single CpG site level, for untreated (Cont) or 5-AZA-CdR–treated MIA PaCa-2 cells (5-Aza). Cells were treated with 5-Aza (1μM), for 48 h. Analysis was performed in 40 CpG sites spanning from a distal locus upstream of +1 position to exon 3.

Techniques: CpG Methylation Assay, Methylation Sequencing, Expressing, Quantitative RT-PCR, Alternative Splicing, Methylation

DNA methylation at the proximal promoter area regulates HNF4A transcription in pancreatic cancer. (A) HNF4A expression restoration following pancreatic cancer cell treatment with 5-AZA-CdR (5-Aza). Low HNF4A-expressing cells (MIA PaCa-2 and PANC-1) were treated with different concentrations (1 and 2 μM) for 48 or 96 h. HNF4A expression was assessed through RT-qPCR, normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to MIA PaCa-2 untreated cells (set as 1). (B and C) HNF4A isoforms expression in MIA PaCa-2 and BxPC-3, respectively. Cells were treated with different concentrations (1 and 2 μM) of 5-Aza, for 48 h. RT-qPCR data were normalized to GAPDH and β-actin levels. (D) HNF4A expression in high (Capan-1 and HPAF-II) HNF4A-expressing pancreatic cancer cell lines. Cells were treated with different concentrations (1 and 2 μM) of 5-Aza, for 48 h. HNF4A expression was assessed through RT-qPCR, normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to untreated cells (set as 1). (E–G) Pearson’s correlation analyses between HNF4A site-specific DNA methylation and gene expression, in human pancreatic cancer tissues. Correlations in TSS200 (proximal promoter area, from −200 nt to −3 nt), distal promoter, and the gene body. (H) Pearson’s correlation analyses between HNF4A site-specific DNA methylation (TSS200 area) and gene expression, in pancreatic cancer cell lines. DNA methylation is expressed in normalized beta values and gene expression assessed by RT-qPCR is expressed in comparison to MIA PaCa-2 cells (set as 1). (I) Methylation of the HNF4A promoter area using the pCpGfree-basic- Lucia reporter plasmid. Upper panel : Diagram illustrating the HNF4A cloned promoter fragment containing 8 CpG sites, highlighted in red on the sequence (represented as lollipops). M.SssI and M.HpaII enzymes were used to methylate the HNF4A promoter region. Lower panel : HEK293T cells were transiently transfected with unmethylated, M.SssI or M.HpaII methylated reporter constructs and luciferase activity was measured in cell supernatants at 48 and 72 h after transfection. The cells were co-transfected with pCMV-Cypridina Luc Vector which secretes a variant of Cypridina luciferase and was used for normalization. Results were expressed as mean ± SEM of Lucia / Cypridina activity compared to the unmethylated (Un) respective control (set as 1). Asterisks denote statistically significant differences, ∗∗∗ P < .001, Student’s t -test.

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: DNA methylation at the proximal promoter area regulates HNF4A transcription in pancreatic cancer. (A) HNF4A expression restoration following pancreatic cancer cell treatment with 5-AZA-CdR (5-Aza). Low HNF4A-expressing cells (MIA PaCa-2 and PANC-1) were treated with different concentrations (1 and 2 μM) for 48 or 96 h. HNF4A expression was assessed through RT-qPCR, normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to MIA PaCa-2 untreated cells (set as 1). (B and C) HNF4A isoforms expression in MIA PaCa-2 and BxPC-3, respectively. Cells were treated with different concentrations (1 and 2 μM) of 5-Aza, for 48 h. RT-qPCR data were normalized to GAPDH and β-actin levels. (D) HNF4A expression in high (Capan-1 and HPAF-II) HNF4A-expressing pancreatic cancer cell lines. Cells were treated with different concentrations (1 and 2 μM) of 5-Aza, for 48 h. HNF4A expression was assessed through RT-qPCR, normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to untreated cells (set as 1). (E–G) Pearson’s correlation analyses between HNF4A site-specific DNA methylation and gene expression, in human pancreatic cancer tissues. Correlations in TSS200 (proximal promoter area, from −200 nt to −3 nt), distal promoter, and the gene body. (H) Pearson’s correlation analyses between HNF4A site-specific DNA methylation (TSS200 area) and gene expression, in pancreatic cancer cell lines. DNA methylation is expressed in normalized beta values and gene expression assessed by RT-qPCR is expressed in comparison to MIA PaCa-2 cells (set as 1). (I) Methylation of the HNF4A promoter area using the pCpGfree-basic- Lucia reporter plasmid. Upper panel : Diagram illustrating the HNF4A cloned promoter fragment containing 8 CpG sites, highlighted in red on the sequence (represented as lollipops). M.SssI and M.HpaII enzymes were used to methylate the HNF4A promoter region. Lower panel : HEK293T cells were transiently transfected with unmethylated, M.SssI or M.HpaII methylated reporter constructs and luciferase activity was measured in cell supernatants at 48 and 72 h after transfection. The cells were co-transfected with pCMV-Cypridina Luc Vector which secretes a variant of Cypridina luciferase and was used for normalization. Results were expressed as mean ± SEM of Lucia / Cypridina activity compared to the unmethylated (Un) respective control (set as 1). Asterisks denote statistically significant differences, ∗∗∗ P < .001, Student’s t -test.

Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR, Gene Expression, Comparison, Methylation, Plasmid Preparation, Clone Assay, Sequencing, Transfection, Construct, Luciferase, Activity Assay, Variant Assay, Control

HNF4A loss-of-function and gain-of-function studies in pancreatic cancer cell lines. (A–C) Stable HNF4A knockdown was achieved by means of 2 different shRNAs (shHNF4A_1 and shHNF4A_2) in Capan-1 and HPAF-II, through lentiviral transduction. Cells transduced with shGFP were used as the control. (A) HNF4A protein levels were determined through western blot analysis and loading was assessed using an antibody against CREB. (B and C) Cell growth was assessed by the MTT and CellTiter-Glo luminescent cell viability assay. Data were expressed as mean ± SEM (the respective control cells, at day 2, were set as 100%). (D–F) HNF4A was stably overexpressed, through lentiviral transduction (HNF4A) in PANC-1 and MIA PaCa-2 cells. Cells transduced with the empty retroviral vector tagged with GFP (EV-GFP) were used as the control. (D) HNF4A protein levels were determined through western blot analysis and loading was assessed using an antibody against CREB. (E and F) Cell growth was assessed by the MTT and CellTiter-Glo luminescent cell viability assay. Data were expressed as mean ± SEM (the respective control cells, at day 2, were set as 100%). (G and H) Anchorage-independent cell growth was assessed by soft agar assays for 5 days. Data were expressed as the mean number of colonies ± SEM (respective control cells set as 100). Representative images were acquired at a 10× and 4× magnification, respectively, using an Evos microscope. (I) Spheroid formation assays using the ultra-low attachment 96-well plate method for PANC-1 (1000 cells/well) and the hanging drop method for MIA PaCa-2 (30,000 cells/20 μL drop). Representative images were acquired on day 7, at a 10× magnification, using an Evos microscope. Asterisks denote statistically significant differences, ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, Student’s t -test.

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: HNF4A loss-of-function and gain-of-function studies in pancreatic cancer cell lines. (A–C) Stable HNF4A knockdown was achieved by means of 2 different shRNAs (shHNF4A_1 and shHNF4A_2) in Capan-1 and HPAF-II, through lentiviral transduction. Cells transduced with shGFP were used as the control. (A) HNF4A protein levels were determined through western blot analysis and loading was assessed using an antibody against CREB. (B and C) Cell growth was assessed by the MTT and CellTiter-Glo luminescent cell viability assay. Data were expressed as mean ± SEM (the respective control cells, at day 2, were set as 100%). (D–F) HNF4A was stably overexpressed, through lentiviral transduction (HNF4A) in PANC-1 and MIA PaCa-2 cells. Cells transduced with the empty retroviral vector tagged with GFP (EV-GFP) were used as the control. (D) HNF4A protein levels were determined through western blot analysis and loading was assessed using an antibody against CREB. (E and F) Cell growth was assessed by the MTT and CellTiter-Glo luminescent cell viability assay. Data were expressed as mean ± SEM (the respective control cells, at day 2, were set as 100%). (G and H) Anchorage-independent cell growth was assessed by soft agar assays for 5 days. Data were expressed as the mean number of colonies ± SEM (respective control cells set as 100). Representative images were acquired at a 10× and 4× magnification, respectively, using an Evos microscope. (I) Spheroid formation assays using the ultra-low attachment 96-well plate method for PANC-1 (1000 cells/well) and the hanging drop method for MIA PaCa-2 (30,000 cells/20 μL drop). Representative images were acquired on day 7, at a 10× magnification, using an Evos microscope. Asterisks denote statistically significant differences, ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, Student’s t -test.

Techniques: Knockdown, Transduction, Control, Western Blot, Cell Viability Assay, Stable Transfection, Retroviral, Plasmid Preparation, Microscopy

HNF4A loss is an early event and promotes PDAC growth in vivo. (A–C) Effect of HNF4A on in vivo xenograft tumour growth. HNF4A was stably overexpressed, through lentiviral transduction (HNF4A) in MIA PaCa-2 and PANC-1 cells. Cells transduced with the empty retroviral vector tagged with GFP (GFP) were used as the control. Stable HNF4A knockdown was achieved by means of shRNA in HPAF-II cells, through lentiviral transduction and cells transduced with shControl were used as the control. Cells were injected subcutaneously in NOD-SCID mice and tumor growth was monitored for a total period of 4 weeks. Tumor volumes were calculated by the equation V (mm 3 ) = a × b 2 /2, where a is the largest diameter and b is the perpendicular diameter. (D) Representative images of tumours extracted from mice at the end of the experiment. (E) HNF4A is suppressed at early stages of pancreatic cancer growth in the KPC ( LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre ) mouse model. Tissues extracted from different stages of pancreatic cancer development were subjected to HNF4A immunohistochemical analysis (brown, HNF4A; blue, haematoxylin). PanIN, pancreatic intraepithelial neoplasia; PDAC, pancreatic ductal adenocarcinoma. For PANC-1, N = 3 mice/group. For MIA PaCa-2 and HPAF-II, N = 8 mice/group. Asterisks denote statistically significant differences, ∗ P < .05, ∗∗ P < .01, Student’s t -test.

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: HNF4A loss is an early event and promotes PDAC growth in vivo. (A–C) Effect of HNF4A on in vivo xenograft tumour growth. HNF4A was stably overexpressed, through lentiviral transduction (HNF4A) in MIA PaCa-2 and PANC-1 cells. Cells transduced with the empty retroviral vector tagged with GFP (GFP) were used as the control. Stable HNF4A knockdown was achieved by means of shRNA in HPAF-II cells, through lentiviral transduction and cells transduced with shControl were used as the control. Cells were injected subcutaneously in NOD-SCID mice and tumor growth was monitored for a total period of 4 weeks. Tumor volumes were calculated by the equation V (mm 3 ) = a × b 2 /2, where a is the largest diameter and b is the perpendicular diameter. (D) Representative images of tumours extracted from mice at the end of the experiment. (E) HNF4A is suppressed at early stages of pancreatic cancer growth in the KPC ( LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre ) mouse model. Tissues extracted from different stages of pancreatic cancer development were subjected to HNF4A immunohistochemical analysis (brown, HNF4A; blue, haematoxylin). PanIN, pancreatic intraepithelial neoplasia; PDAC, pancreatic ductal adenocarcinoma. For PANC-1, N = 3 mice/group. For MIA PaCa-2 and HPAF-II, N = 8 mice/group. Asterisks denote statistically significant differences, ∗ P < .05, ∗∗ P < .01, Student’s t -test.

Techniques: In Vivo, Stable Transfection, Transduction, Retroviral, Plasmid Preparation, Control, Knockdown, shRNA, Injection, Immunohistochemical staining

Discovery cohort of 168 pancreatic cancer patients reveals that HNF4A loss is an early event and correlates with poor overall survival. HNF4A expression was assessed by immunohistochemical analysis, in 168 pancreatic cancer and 38 normal (uninvolved) tissues (QMC: Queen’s Medical Centre, Nottingham, UK). (A) Staining and scoring of tissues was performed in Histopathology Department of QMC and results were expressed as mean ± SEM compared to normal tissues (set as 1). (B) Assessment of HNF4A staining in 168 pancreatic cancer tissues according to their tumor stage. Results were expressed as mean ± SEM compared to normal tissues (set as 1). (C) Survival analysis in 153 patients divided into low, intermediate (interm), and high HNF4A expression subgroups. Survival estimates were generated using the Kaplan-Meier method and compared using log-rank tests.

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: Discovery cohort of 168 pancreatic cancer patients reveals that HNF4A loss is an early event and correlates with poor overall survival. HNF4A expression was assessed by immunohistochemical analysis, in 168 pancreatic cancer and 38 normal (uninvolved) tissues (QMC: Queen’s Medical Centre, Nottingham, UK). (A) Staining and scoring of tissues was performed in Histopathology Department of QMC and results were expressed as mean ± SEM compared to normal tissues (set as 1). (B) Assessment of HNF4A staining in 168 pancreatic cancer tissues according to their tumor stage. Results were expressed as mean ± SEM compared to normal tissues (set as 1). (C) Survival analysis in 153 patients divided into low, intermediate (interm), and high HNF4A expression subgroups. Survival estimates were generated using the Kaplan-Meier method and compared using log-rank tests.

Techniques: Expressing, Immunohistochemical staining, Staining, Histopathology, Generated

Effect of HNF4A Expression on Overall Survival was Assessed in 153 Patients (QMC Cohort), Using the Univariate and Multivariate Cox Proportional Hazard Analyses

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: Effect of HNF4A Expression on Overall Survival was Assessed in 153 Patients (QMC Cohort), Using the Univariate and Multivariate Cox Proportional Hazard Analyses

Techniques: Expressing

HNF4A loss indicates an increased risk of death in a validation cohort of 145 pancreatic cancer patients. HNF4A expression was assessed by immunohistochemical analysis, in 145 pancreatic cancer tissues (UCLA, USA). (A) Staining and scoring of the HNF4A immunostained tissues was performed in the Department of Pathology at UCLA Medical Center. Assessment of HNF4A staining in pancreatic cancer tissues according to their tumor stage. (B) Survival analysis in patients divided into low, intermediate (interm), and high HNF4A expression subgroups. Survival estimates were generated using the Kaplan-Meier method and compared using log-rank tests.

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: HNF4A loss indicates an increased risk of death in a validation cohort of 145 pancreatic cancer patients. HNF4A expression was assessed by immunohistochemical analysis, in 145 pancreatic cancer tissues (UCLA, USA). (A) Staining and scoring of the HNF4A immunostained tissues was performed in the Department of Pathology at UCLA Medical Center. Assessment of HNF4A staining in pancreatic cancer tissues according to their tumor stage. (B) Survival analysis in patients divided into low, intermediate (interm), and high HNF4A expression subgroups. Survival estimates were generated using the Kaplan-Meier method and compared using log-rank tests.

Techniques: Biomarker Discovery, Expressing, Immunohistochemical staining, Staining, Generated

Effect of HNF4A Expression on Overall Survival Was Assessed in 145 Patients (UCLA Cohort), Using the Univariate and Multivariate Cox Proportional Hazard Modelling

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: Effect of HNF4A Expression on Overall Survival Was Assessed in 145 Patients (UCLA Cohort), Using the Univariate and Multivariate Cox Proportional Hazard Modelling

Techniques: Expressing

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Techniques: Knockdown, Transduction, Control, Western Blot, Cell Viability Assay, Stable Transfection, Retroviral, Plasmid Preparation, Microscopy

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Techniques: In Vivo, Stable Transfection, Transduction, Retroviral, Plasmid Preparation, Control, Knockdown, shRNA, Injection, Immunohistochemical staining

HEK293A cells overexpressing mouse HA‐tagged wild‐type Prox1 (wt), K556R mutant (KR) or E558A mutant (EA) were treated with cycloheximide (CHX) for 6 and 8 h. The cell lysates were analyzed by immunoblotting using anti‐HA and anti‐β actin antibodies. HepG2 cells overexpressing mouse YFP‐tagged wild‐type Prox1 (wt) or K556R mutant were analyzed by fluorescence microscopy (magenta), DNA was stained with DAPI (blue); (representative images; scale bar = 20 μm). The nuclei from HepG2 cells were isolated and used for protein extraction. Multiple nuclear extracts were prepared using different concentrations of NaCl (110, 230, 350 or 420 mM). Total samples collected before cell lysis (T), the cytoplasmic extracts (CE) and the nuclear extracts (NE) were analyzed by immunoblotting using anti‐Prox1 antibodies. The chromatin binding factor RCC1 and tubulin were used as controls. Prox1/PXR and Prox1/HNF4α co‐immunoprecipitation in HEK293A cells overexpressing HA‐tagged Prox1 wild‐type (wt) or K556R mutant (KR) and Myc‐tagged PXR or HNF4α.

Journal: EMBO Reports

Article Title: Fasting‐sensitive SUMO‐switch on Prox1 controls hepatic cholesterol metabolism

doi: 10.15252/embr.202255981

Figure Lengend Snippet: HEK293A cells overexpressing mouse HA‐tagged wild‐type Prox1 (wt), K556R mutant (KR) or E558A mutant (EA) were treated with cycloheximide (CHX) for 6 and 8 h. The cell lysates were analyzed by immunoblotting using anti‐HA and anti‐β actin antibodies. HepG2 cells overexpressing mouse YFP‐tagged wild‐type Prox1 (wt) or K556R mutant were analyzed by fluorescence microscopy (magenta), DNA was stained with DAPI (blue); (representative images; scale bar = 20 μm). The nuclei from HepG2 cells were isolated and used for protein extraction. Multiple nuclear extracts were prepared using different concentrations of NaCl (110, 230, 350 or 420 mM). Total samples collected before cell lysis (T), the cytoplasmic extracts (CE) and the nuclear extracts (NE) were analyzed by immunoblotting using anti‐Prox1 antibodies. The chromatin binding factor RCC1 and tubulin were used as controls. Prox1/PXR and Prox1/HNF4α co‐immunoprecipitation in HEK293A cells overexpressing HA‐tagged Prox1 wild‐type (wt) or K556R mutant (KR) and Myc‐tagged PXR or HNF4α.

Article Snippet: HEK293A cells (Thermo Fischer Scientific—R70507) were transfected with mouse HA‐tagged wild‐type Prox1 (wt) or K556R mutant together with mouse Myc‐tagged HNF4α (Origene—MR227662), Myc‐tagged LRH‐1 (Origene—MR225371) or Myc‐tagged PXR (Origene—MR226044) using Lipofectamine 3000 with Plus Reagent (Thermo Fisher Scientific) according to the manufacturer's protocol.

Techniques: Mutagenesis, Western Blot, Fluorescence, Microscopy, Staining, Isolation, Protein Extraction, Lysis, Binding Assay, Immunoprecipitation

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) Schematic of experimental design to infect K562 cells with FOXA1- or HNF4A-lentivirus and then perform functional assays on dox-induced cells. In CUT&Tag, a protein A-protein G fusion (pA/G) increases the binding spectrum for Fc-binding and allows Tn5 recruitment to antibody-labeled transcription factor (TF) binding sites. In ATAC-seq, Tn5 homes to any accessible site. And in RNA-seq, polyA RNA is captured and sequenced. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1-HNF4A compared to the number actually activated. Both liver- (p<10 –38 ) and intestinal enrichment (p<10 –13 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C ) Genome browser view of a representative liver-specific locus ( ALB ) in FOXA1-HNF4A clonal line that shows uninduced and induced accessibility, FOXA1 binding, and HNF4A binding. ( D ) Heatmap showing uninduced and induced accessibility at all FOXA1-HNF4A co-bound sites within 50 kb of each FOXA1-HNF4A-activated liver-specific gene (n = 53). ( E ) Meta plot showing average signal across each site from ( D ).

Article Snippet: Strain, strain background ( H. sapiens ) , FOXA1-HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying FOXA1 ORF and then HNF4A ORF .

Techniques: Functional Assay, Binding Assay, Labeling, RNA Sequencing Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: qPCR measurements made from RNA extracted from either the FOXA1 clonal line ( A–D ) or the HNF4A clonal line ( E–H ) that was treated with either increasing doxycycline concentrations or longer time periods. Expression is displayed as log10 fold induction over either 0 µg/ml doxycycline control (for concentration titration) or time 0 (for time titration). Each sample primer was normalized to the HPRT housekeeping gene. Doxycycline concentration titration measurements were made at 0, 0.01, 0.05, 0.1, 0.5, 2, and 5 µg/ml. Doxycycline treatment time measurements were made at 0, 6, 12, 24, 48, 72, and 96 hr.

Techniques: Expressing, Concentration Assay, Titration

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) The number of genome-wide FOXA1 or HNF4A transcription factor binding sites (TFBS) in the induced (+dox) cells that overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘accessible binding site’) or that do not overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘inaccessible binding site’). ( B ) The number of inaccessible binding sites from ( A ) that overlap with an ATAC-seq peak in the induced (+dox) cells (‘opened’) or that do not overlap with an ATAC-seq peak (‘remained closed’). ( C ) The number of FOXA1 or HNF4A binding sites within 50 kb of each FOXA1-HNF4A co-activated gene characterized as either a ‘HepG2 binding site,’ where the TFBS overlaps a TFBS of FOXA1 or HNF4A in HepG2 liver cells, or as a ‘Novel K562 binding site,’ where the TFBS does not overlap with a HepG2 binding site.

Techniques: Genome Wide, Binding Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1 compared to the number actually activated. Liver enrichment (p<10 –4 ) is significant. There are 242 total liver-enriched genes. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by HNF4A compared to the number actually activated. Liver- (p<10 –8 ) and intestine enrichment (p<10 –15 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C ) 242 liver genes characterized as activated by Foxa1, HNF4A, both, or neither. ( D ) 122 intestine genes characterized as activated by FOXA1, HNF4A, both, or neither.

Techniques:

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) Genome browser view of a representative liver-specific locus ( ARG1 ) in FOXA1 clonal line showing uninduced and induced accessibility and FOXA1 binding. ( B ) Genome browser view of a representative liver-specific locus ( APOC3 ) in HNF4A clonal line showing uninduced and induced accessibility and HNF4A binding. ( C ) Heatmap of uninduced and induced accessibility at all FOXA1 binding sites within 50 kb of each FOXA1-activated liver-specific genes (n = 59). ( D ) Heatmap of uninduced and induced accessibility at all HNF4A binding sites within 50 kb of each HNF4A-activated liver-specific genes (n = 76). ( E ) Meta plot showing average signal across each site from ( C ). ( F ) Meta plot showing average signal across each site from ( D ). ( G ) Human FOXA1 and HNF4A sequence logo from JASPAR. ( H ) FOXA1 or HNF4A motif count within 500 bp centered upon FOXA1 or HNF4A binding sites within 50 kb of each FOXA1- or HNF4A-activated liver-specific genes, respectively. Motifs were called with FIMO using 1e-3 a p-value threshold. For each boxplot, the center line represents the median, the box represents the first to third quartiles, and the whiskers represent any points within 1.5× the interquartile range.

Techniques: Binding Assay, Sequencing

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) The number of genome-wide FOXA1 or HNF4A transcription factor binding sites (TFBS) in the induced (+dox) cells that overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘aAccessible binding site’) or that do not overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘inaccessible binding site’). ( B ) The number of inaccessible binding sites from ( A ) that overlap with an ATAC-seq peak in the induced (+dox) cells (‘opened’) or that do not overlap with an ATAC-seq peak (‘remained closed’). ( C ) The number of FOXA1 or HNF4A binding sites within 50 kb of each FOXA1- or HNF4A-activated gene characterized as either a ‘HepG2 binding site,’ where the TFBS overlaps a TFBS of FOXA1 or HNF4A in HepG2 liver cells, or as a ‘Novel K562 binding site,’ where the TFBS does not overlap with a HepG2 binding site.

Techniques: Genome Wide, Binding Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) FIMO scans at p-value threshold 1e-3 for four most common proposed K562 pioneer factors (PFs) in either FoxA1 inaccessible binding sites (red), Hnf4a inaccessible binding sites (blue), or random equally lengthed binding sites (gray).

Techniques: Binding Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) 1000 random 200 bp fragments were generated using BEDTools and then scanned for FOXA1 and HNF4A motifs with FIMO using 1e-3 a p-value threshold. Total motif count was divided by the number of non-N-containing random sequences (924) to identify motifs per random 200 bp fragment.

Techniques: Generated

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1 at a lower doxycycline concentration (0.05 µg/ml) compared to the number actually activated. There are 242 total liver-enriched genes. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by HNF4A at a lower doxycycline concentration (0.05 µg/ml) compared to the number actually activated. Liver- (p<10 –5 ) and intestine enrichment (p<10 –14 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C, D ) Genome-wide FOXA1 ( C ) or HNF4A ( D ) binding sites classified as either events that occurred at sites that were accessible or inaccessible in the uninduced (-dox) state at 0.5 and 0.05 µg/ml doxycycline induction.

Techniques: Concentration Assay, Genome Wide, Binding Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) Venn diagram of all liver genes categorized as either activated by FOXA1, HNF4A, FOXA1-HNF4A, some combination, or by none of the three cocktails. ( B ) Genome browser view of a representative liver-specific locus ( AMDHD1 ) showing examples of a co-bound site that is ‘FOXA1 pioneered’ (FP), ‘HNF4A pioneered’ (HP), and ‘cooperatively bound’ (CB). The first two tracks are FOXA1 and HNF4A binding in the FOXA1-HNF4A co-expression clone, and the last two tracks are FOXA1 and HNF4A binding in their individual expression clones. ( C ) List of the 31 liver genes that are only activated by FOXA1-HNF4A co-expression. The columns indicate how many co-bound FP, HP, or CB peaks exist within 100 kb of the gene. ( D ) Venn diagram of all genome-wide co-bound peaks categorized as either bound by FOXA1 individually (FP), HNF4A individually (HP), by both, or by neither (CB). ( E ) Overlap of FP, HP, and CB sites from ( D ) with ChromHMM annotations showing the fraction of each co-binding site type in each chromatin region.

Techniques: Binding Assay, Expressing, Clone Assay, Genome Wide

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) Venn diagram of all FOXA1-HNF4A-induced differentially accessible peaks categorized by whether the peak was also induced in the FOXA1 clone, HNF4A clone, neither, or both.

Techniques:

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) FOXA1 or HNF4A motif count at all genomic occurrences of the respective transcription factor’s (TF’s) accessible or inaccessible binding sites. ( B ) FOXA1 or HNF4A motif count in genome-wide inaccessible binding sites versus length-matched random inaccessible DNA sequences. ( C ) Receiver operating characteristic (ROC) curves for predictive power of using sequence motif content to predict accessible (left panels) or inaccessible (right panels) binding sites from random sequence. ( D ) Total FOXA1 and HNF4A motif count at all genomic occurrences of inaccessible co-binding versus length-matched random inaccessible DNA sequences. ( E ) FOXA1 or HNF4A motif count in respective FOXA1 or HNF4A pioneered sites versus in cooperative binding sites (where neither TF bound individually). ( F ) ROC curves for predictive power of using sequence motif content to predict accessible or inaccessible co-binding events from random sequence (top panels) or to predict FOXA1 or HNF4A pioneered events from cooperative binding events. All FIMO scans used 1e-3 as p-value threshold and were conducted on 500 bp of sequence centered upon the binding site.

Techniques: Binding Assay, Genome Wide, Sequencing

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet:

Techniques: Infection, Plasmid Preparation, SYBR Green Assay, Software

Effect of combined expression of Foxa2, Hnf4a and Sall1 on hepatic differentiation of DFAT cells. (a) DFAT cells transfected with 3 genes (Foxa2 (F), Hnf4a (H) and Sall1 (S)) or infected with the corresponding GFP vector (Ctrl) were exposed to induction medium of differentiation for 14 days. The expression levels of Foxa2, Hnf4a, Sall1 and liver genes (Alb and Afp) were measured using RT‐PCR. Gapdh was used as an internal control. (b) Real‐time PCR analyses showed the presence of hepatic marker genes for Alb and Afp in FH‐ and FHS‐expressing DFAT cells after hepatic differentiation. Mean values ± SEM . were then normalized to Gapdh and expressed relative to normal hepatocytes from adult mice. p ‐Value was calculated using Student's t test (** p < .01). (c) Phase‐contrast microscopy showing DFAT cells and morphological changes of the cells after hepatic induction. Arrows show binucleated FHS‐DFAT cells. Scale bars represent 50 μm. All quantitative data are mean ± SD . ( n = 3 experiments)

Journal: Genes to Cells

Article Title: Generation of metabolically functional hepatocyte‐like cells from dedifferentiated fat cells by Foxa2, Hnf4a and Sall1 transduction

doi: 10.1111/gtc.12814

Figure Lengend Snippet: Effect of combined expression of Foxa2, Hnf4a and Sall1 on hepatic differentiation of DFAT cells. (a) DFAT cells transfected with 3 genes (Foxa2 (F), Hnf4a (H) and Sall1 (S)) or infected with the corresponding GFP vector (Ctrl) were exposed to induction medium of differentiation for 14 days. The expression levels of Foxa2, Hnf4a, Sall1 and liver genes (Alb and Afp) were measured using RT‐PCR. Gapdh was used as an internal control. (b) Real‐time PCR analyses showed the presence of hepatic marker genes for Alb and Afp in FH‐ and FHS‐expressing DFAT cells after hepatic differentiation. Mean values ± SEM . were then normalized to Gapdh and expressed relative to normal hepatocytes from adult mice. p ‐Value was calculated using Student's t test (** p < .01). (c) Phase‐contrast microscopy showing DFAT cells and morphological changes of the cells after hepatic induction. Arrows show binucleated FHS‐DFAT cells. Scale bars represent 50 μm. All quantitative data are mean ± SD . ( n = 3 experiments)

Article Snippet: Mouse cDNAs for full‐length Foxa2, Hnf4a and Sall1 were obtained by digestion of OriGene plasmids (MR227662, MR227354 and MC203471) with NotI and BamHI, and the resulting fragments were cloned into the NotI and BamHI sites of retroviral plasmid pMEFs (kindly provided by H. Nobusue).

Techniques: Expressing, Transfection, Infection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Marker, Microscopy

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