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Cited by 113Hippo pathway signaling and YAP/TAZGenomics and Chromatin DynamicsLiver physiology and pathology

Hippo Signaling Influences HNF4A and FOXA2 Enhancer Switching during Hepatocyte Differentiation

Open access
Alder, Olivia, Cullum, Rebecca, Lee, Sam, Kan, Arohumam C., Wei, Wei, Yi, Yuyin, Garside, Victoria C., Bilenky, Misha +10 more·2014·Cell Reports
DOI 10.1016/j.celrep.2014.08.046PMC PMC4612615
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Abstract

Cell fate acquisition is heavily influenced by direct interactions between master regulators and tissue-specific enhancers. However, it remains unclear how lineage-specifying transcription factors, which are often expressed in both progenitor and mature cell populations, influence cell differentiation. Using in vivo mouse liver development as a model, we identified thousands of enhancers that are bound by the master regulators HNF4A and FOXA2 in a differentiation-dependent manner, subject to chromatin remodeling, and associated with differentially expressed target genes. Enhancers exclusively occupied in the embryo were found to be responsive to developmentally regulated TEAD2 and coactivator YAP1. Our data suggest that Hippo signaling may affect hepatocyte differentiation by influencing HNF4A and FOXA2 interactions with temporal enhancers. In summary, transcription factor-enhancer interactions are not only tissue specific but also differentiation dependent, which is an important consideration for researchers studying cancer biology or mammalian development and/or using transformed cell lines.

AI snapshot

Written from the paper’s full text.

TL;DR

HNF4A and FOXA2 occupy thousands of liver enhancers in a differentiation-dependent manner, with 55% of HNF4A and 60% of FOXA2 sites changing between E14.5 hepatoblasts and adult liver. Embryonic-specific enhancers are enriched for TEAD1 motifs and responsive to TEAD2/YAP1, suggesting Hippo signaling influences enhancer switching during hepatocyte differentiation.

Key findings

  • ChIP-seq in E14.5 DLK1+ hepatoblasts identified 11,703 HNF4A-bound and 9,398 FOXA2-bound regions; approximately 30% of FOXA2 binding occurred at HNF4A-occupied sites, and 2,957 genes were identified as putative direct targets.
  • 55% of HNF4A-binding sites and 60% of FOXA2 sites were uniquely occupied in either embryonic or adult liver; co-bound categories included 891 embryonic, 1,050 continuous, and 1,094 adult sites. Embryonic sites showed reduced, monomodal H3K4me1, whereas continuous and adult sites showed bimodal H3K4me1; only 3% and 7% of embryonic enhancers were enriched for H3K27me3 and H3K9me3 in adult liver, and >60% remained accessible.
  • RNA-seq found >4,000 genes more highly expressed in embryonic hepatoblasts and >2,000 enriched in adult hepatocytes; genes associated with embryonic enhancers decreased during development and adult-enhancer genes increased (p < 0.001 and p < 0.01), while association with one or more differentiation-dependent binding sites predicted higher expression at the appropriate stage (p ≤ 0.001).
  • TEAD1 motifs were enriched at embryonic enhancers; Tead2 was the most highly expressed TEAD family member in embryonic liver and most downregulated in adult liver. TEAD2 plus YAP1 induced >2-fold activity for H19, Dlk1, and Sall4 enhancers, all four factors together produced >10-fold induction, and YAP1 induction in transgenic mice decreased HNF4A/FOXA2 occupancy at adult-specific enhancers Ido2 and Prlr (p ≤ 0.05) while increasing embryonic genes such as DIM, H19, and Afp.

Methods

  • Mouse liver development model: E14.5 fetal hepatoblasts purified by DLK1 magnetic-activated cell sorting, with fluorescence-activated cell sorting for RNA-seq; adult perfused liver was compared. YAP1 transgenic mice (ApoE-rtTA; TRE-hYAP) and wild-type controls were treated with 0.2 mg/ml doxycycline for 2 weeks (three transgenic and three wild-type 4-week-old female mice).
  • ChIP-seq was performed for HNF4A, FOXA2, H3K4me1, H3K27me3, H3K9me3, and IgG; RNA-seq used ALEXA-seq; validation included ChIP-qPCR, EMSA, western blotting, and luciferase reporter assays in HEK293T cells.
  • Bioinformatics used mm9 alignment, FindPeaks2 with FDR 0.01, Galaxy/Cistrome, CEAS, SitePro, SeqPos, oPOSSUM, previously established enhancer-promoter units for target-gene association, and ALEXA-seq differential expression with normalized average coverage differing by a factor of 2 and corrected p < 0.05.

Limitations

  • The paper states that mature hepatocyte gene-expression profiles and cell functions are difficult to maintain in vitro and that HEPG2, a hepatocellular carcinoma cell line, is commonly used to represent hepatocytes; it also notes that it has found no evidence to date for a repressive factor masking adult enhancers in embryonic liver.
  • Evident from the text: the in vivo analysis compares mouse E14.5 hepatoblasts with adult liver, so intermediate developmental stages and single-cell heterogeneity are not resolved.
  • Evident from the text: causality of TEAD2/YAP1 in enhancer switching is supported by reporter assays in HEK293T cells and an adult YAP1 transgenic model, not by TEAD2 loss-of-function during embryonic hepatocyte differentiation.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.celrep.2014.08.046

Cites

52 of 52 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

21 of 34 cited sources checked · 10 supported · 1 flagged · accuracy not yet measured

  • Findingown data

    Upon hepatocyte differentiation, HNF4A and FOXA2 binding is lost at many Afp-associated regulatory regions.

    “we confirmed that HNF4A and FOXA2 binding was lost at many of the Afp-associated regulatory regions upon hepatocyte differentiation.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Hoffman et al., 2010 source

      In “Tissue-specific FOXA2 binding and nucleosome displacement determine target gene expression” (page unavailable), the paper reports a developmental change in the H3K4me1 profile of one Afp-upstream locus and loss of Afp expression in adult liver. Figure 5H describes that locus as bound by FOXA2 and HNF4A in adult liver. The paper does not establish that binding by either factor was lost at many Afp-associated regulatory regions during differentiation. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • Rests on an assumption: The HNF4A and FOXA2 antibodies used for ChIP-seq specifically immunoprecipitate their intended TFs in fetal liver.
  • Findingown data

    Only minor contributions from the Hnf4a P2 promoter were detected.

    “Furthermore, we detected only minor contributions from the Hnf4a P2 promoter (Figure S4; Torres-Padilla et al., 2001).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Torres-Padilla et al., 2001 source

      The Discussion reports near-total loss of the HNF4α7 isoform associated with the P2 promoter in adult mouse liver, consistent with a minor P2 contribution there. It also reports abundant HNF4α7 in embryonic and early fetal liver, so the source does not support an unqualified claim that P2 contributions are only minor. The citing authors’ own detection in Figure S4 is not a result this source can verify. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Findingown data

    No statistically significant differential expression of Hnf4a and Foxa2 transcript levels was observed during liver maturation.

    “In contrast to previous studies by Kyrmizi et al. (2006), we did not observe statistically significant differential expression of Hnf4a and Foxa2 transcript levels during liver maturation.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Kyrmizi et al. (2006) source

      The sentence attributes the earlier finding of differential expression—not the citing authors’ nonsignificant result—to Kyrmizi et al. In “mRNA levels of the hepatic regulators during liver development” (page unavailable), the source reports that the measured transcripts, except GATA-6, increased during development, which includes HNF-4α and HNF-3β (Foxa2). The Figure 1 caption describes statistical testing, but the text available in our copy does not establish that the changes for both specific transcripts were statistically significant. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
  • Findingcitation

    More than 60% of embryonic sites remain accessible in adult liver based on public DNase I hypersensitivity data.

    “Furthermore, an analysis of publically available DNase I hypersensitivity data (Shen et al., 2012) confirmed that >60% of embryonic sites remain accessible in the adult liver (data not shown).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Shen et al., 2012 source

      The paper’s study description (section labeled Abstract; page unavailable) describes ChIP-Seq and RNA-Seq mapping in embryonic and adult tissues, but does not report the cited DNase I hypersensitivity data. Searches for DNase, hypersensitivity, and embryonic-to-adult liver accessibility found no passage establishing that more than 60% of embryonic sites remain accessible in adult liver. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • Rests on an assumption: The HNF4A and FOXA2 antibodies used for ChIP-seq specifically immunoprecipitate their intended TFs in fetal liver.
  • Findingown data

    HNF4A/FOXA2 target genes in fetal liver are associated with hepatocyte function processes including lipid metabolic process (p=4.5e-48).

    “Many genes were found to associate with processes integral to hepatocyte function, such as the lipid metabolic process (binominal p value = 4.5 × 10−48).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The HNF4A and FOXA2 antibodies used for ChIP-seq specifically immunoprecipitate their intended TFs in fetal liver.
  • Classified as probably not a substantial claim. 25
  • May combine two claims. 20
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Forced expression of Sall4 inhibits hepatoblast differentiation along the hepatocyte lineage, whereas downregulation encourages bile duct formation.

    “The embryonic target gene Sall4 highlighted in our analysis is of particular interest because forced expression has been shown to inhibit hepatoblast differentiation along the hepatocyte lineage, whereas downregulation encourages bile duct formation (Oikawa et al., 2009).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Oikawa et al., 2009 source

      The Discussion supports the first clause: Sall4a overexpression inhibited hepatoblast maturation along the hepatocyte lineage. But it reports the opposite of the second clause: Sall4 knockdown inhibited, rather than encouraged, formation of bile duct-like structures. (LLM, conf=0.91)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Regions bound by multiple TFs are more likely to function as tissue-specific enhancers.

    “Since regions bound by multiple TFs are more likely to function as tissue-specific enhancers (Hoffman et al., 2010)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Hoffman et al., 2010 source

      The ‘Cooperative binding at bimodal sites drives tissue-specific gene expression’ section reports that genes associated with sites co-occupied by two transcription factors were more tissue-specific than genes associated with singly bound sites. The finding concerns FOXA2–PDX1 sites in islets and FOXA2–HNF4A sites in liver, and measures target-gene expression; it does not establish that multiply bound regions generally are more likely to function as tissue-specific enhancers. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    FOXA TFs can bind condensed chromatin and act as placeholders or recruit factors for transcriptional initiation.

    “FOXAs have a structural similarity to linker histories and are able to bind condensed chromatin, where they can either passively act as placeholders for recruitment of factors later in development or actively recruit other factors for transcriptional initiation (Sekiya et al., 2009; Zaret and Carroll, 2011).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Sekiya et al., 2009 source

      The paper’s Abstract (page unavailable) reports that FoxA can bind highly compacted nucleosome arrays and occupy a target site while the gene remains silent. “Nuclear mobility and pioneer factor function” (page unavailable) suggests that stable FoxA binding allows other factors to engage chromatin, but it does not establish that FoxA actively recruits them for transcriptional initiation. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites Zaret and Carroll, 2011 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Gene transcription is often influenced by TF occupancy at multiple enhancer regions.

    “Gene transcription is often influenced by TF occupancy at multiple enhancer regions (Ong and Corces, 2011).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Ong and Corces, 2011 source

      The opening section says enhancers contain transcription-factor binding sites and that enhancer-bound factors help activate transcription. The long-range interactions section describes multiple enhancer elements interacting with promoters at the Th2 cytokine locus. These passages support the mechanism and an example involving multiple enhancers, but do not establish that transcription is often influenced by factor occupancy at multiple enhancer regions. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Higher levels and a bimodal distribution pattern of H3K4me1 correlate with enhancer function.

    “Previous studies suggested that higher levels and a bimodal distribution pattern of monomethylated histone 3 lysine 4 (H3K4me1) correlate with enhancer function (Hoffman et al., 2010; Rada-Iglesias et al., 2011).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Hoffman et al., 2010 source
    • cites Rada-Iglesias et al., 2011 source

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).

    Premisecitation

    Subtle differences in motifs underlying differentiation-dependent sites enable competitive binding of a repressive TF.

    “In contrast to our analysis, they demonstrated that subtle differences in motifs underlying differentiation-dependent sites enable competitive binding of a repressive TF.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Soleimani et al., 2012 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Nuclear YAP1 is able to induce hepatocyte dedifferentiation into clonogenic progenitors in vivo.

    “only very recent studies using a mosaic YAP1 induction model have shown that nuclear YAP1 is able to induce hepatocyte dedifferentiation into clonogenic progenitors in vivo (Yimlamai et al., 2014).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Yimlamai et al., 2014 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Sall4 was recently identified as a "targetable marker" for an aggressive subset of HCC.

    “Furthermore, Sall4 was recently identified as a “targetable marker” for an aggressive subset of HCC (Yong et al., 2013).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Yong et al., 2013 source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    TEAD2 functions as a nuclear transcription factor in the Hippo signaling pathway, which can regulate cell death, proliferation, and differentiation.

    “TEAD2 functions as a nuclear TF in the Hippo signaling pathway, which can regulate cell death, proliferation, and differentiation (reviewed in Yu and Guan, 2013).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Yu and Guan, 2013 source

  • Classified as probably not a substantial claim.

    Premisecitation

    In the absence of Hippo pathway activity (e.g., at low cell density), YAP1 is shuttled to the nucleus, which enhances the ability of TEAD2 to induce target gene expression.

    “In the absence of Hippo pathway activity (e.g., at low cell density), YAP1 is shuttled to the nucleus, which enhances the ability of TEAD2 to induce target gene expression (Schlegelmilch et al., 2011).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Schlegelmilch et al., 2011 source

      The Results section, “TEAD interaction is critical for the role of Yap1 in the epidermis” (page unavailable), reports that Yap1 increases TEAD-reporter activity and that the signal decreases at high cell density. The Discussion section, “14-3-3 and Yap1 phosphorylation in the skin” (page unavailable), describes Yap1 entering the nucleus, binding TEADs, and activating gene expression when its inhibitory regulation is lost. The paper discusses TEAD proteins collectively, however, and does not establish the claimed effect specifically for TEAD2. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The pluripotency-associated pioneer factor OCT4 dramatically switches enhancers upon transition from an embryonic stem cell to an epiblast-like cell state.

    “For example, the pluripotency-associated pioneer factor OCT4 dramatically switches enhancers upon transition from an embryonic stem cell to an epiblast-like cell state (Buecker et al., 2014).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Buecker et al., 2014 source

  • Classified as probably not a substantial claim.

    Premisecitation

    It is difficult to propagate or even maintain hepatocytes in a functional differentiated state in vitro.

    “It is notoriously difficult to propagate or even maintain hepatocytes in a functional differentiated state in vitro.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Raju et al., 2013 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Investigators have achieved partial success in propagating/maintaining hepatocytes in vitro by developing 3D tissue culture models and using a multitude of extracellular matrix components in the form of Matrigel.

    “Investigators have achieved partial success by developing 3D tissue culture models and using a multitude of extracellular matrix (ECM) components in the form of Matrigel (Raju et al., 2013).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Raju et al., 2013 source

      The Introduction describes difficulty maintaining primary hepatocytes in culture, and its Adult liver stem cells section describes a decellularized liver biomatrix used for differentiation. Searches for Matrigel and 3D tissue culture models found no passage saying those approaches achieved the claimed partial success. (LLM, conf=0.72)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    ChIP-seq data sets were limited by applying thresholds defined by an FDR cutoff of 0.01 and by trimming at 20% of maximal height to remove flanks and separate composite regions.

    “The data sets were limited by applying thresholds as defined by an FDR cutoff of 0.01 and by trimming at 20% of maximal height to remove flanks and separate composite regions (Fejes et al., 2008).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Fejes et al., 2008 source

      The Summary (p. 1729–1730) describes FindPeaks as a tool for identifying enriched regions in ChIP-seq data, but does not specify the cited thresholds. Searches for an FDR cutoff of 0.01 and trimming at 20% of maximal height found no passage in our copy addressing those settings or their stated purpose. (LLM, conf=0.74)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    MYOD can change binding sites between primary myoblast and induced myotube states.

    “Soleimani et al. (2012) showed that MYOD, a TF that is well known to induce muscle cell fate, can change binding sites between primary myoblast and induced myotube states.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Soleimani et al., 2012 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The FXR motif, more highly expressed in adult, is enriched at adult-specific sites.

    “Interestingly, we found that the motif related to the nuclear receptor FXR, which is more highly expressed in the adult, was enriched at adult specific sites (data not shown).”
    Quote in the paperNot a substantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    ChIP-qPCR experiments on 24 sites validated distinct enrichment patterns; embryonic genes Cdknlc, Cit, Dlk1, Sall4, Gpc3, H19, Hmga2, Igf2, Igf2bp3, Klf7, Mycn, and Zfhx3 showed enriched HNF4A and FOXA2 binding in embryonic but not adult ChIP-seq.

    “Cdknlc, Cit, Dlk1, Sall4, Gpc3, H19, Hmga2, Igf2, Igf2bp3, Klf7, Mycn, and Zfhx3 showed enriched binding of both HNF4A and FOXA2 in our embryonic, but not adult, chIP-seq data sets.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The HNF4A and FOXA2 antibodies used for ChIP-seq specifically immunoprecipitate their intended TFs in fetal liver.

  • May combine two claims.

    Findingown data

    TEAD2 protein is robustly detected in embryonic liver and its levels are decreased in adult liver.

    “TEAD2 protein was robustly detected in embryonic liver and its levels were decreased in adult liver (Figure 5E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Tead2 is the most highly expressed TEAD family member in embryonic liver, expressed at a level similar to Foxa2, and is the most dramatically downregulated in adult liver.

    “Tead2 was the most highly expressed family member in embryonic liver, being expressed at a level similar to that observed for Foxa2, and was the most dramatically downregulated in adult liver.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    No strong differences in sequence conservation or underlying HNF4A or FOXA2 motifs were found between sites bound in a differentiation-dependent manner.

    “We found no strong differences in either sequence conservation or the underlying HNF4A or FOXA2 motifs between sites bound in a differentiation-dependent manner (Figure S5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cell cycle is significantly enriched in proliferating embryonic hepatoblasts, while oxidation reduction and metabolic process dominate the mature hepatocyte transcriptome.

    “As anticipated, “cell cycle” was significantly enriched in proliferating embryonic hepatoblasts, whereas pathways including “oxidation reduction” and “metabolic process” dominated the transcriptome of mature hepatocytes.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    Sites were categorized as embryonic (891), continuous (1,050), or adult (1,094) based on HNF4A and FOXA2 enrichment.

    “We categorized sites as embryonic (HNF4A and FOXA2 enrichment in embryonic data sets only [891 sites]), continuous (HNF4A and FOXA2 enrichment in all data sets [1,050 sites]), or adult (HNF4A and FOXA2 enrichment in adult data sets only [1,094 sites] Figure 2D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The HNF4A and FOXA2 antibodies used for ChIP-seq specifically immunoprecipitate their intended TFs in fetal liver.

  • May combine two claims.

    Interpretationown data

    H3K4me1 and H4ac signatures are distinct at differentiation-dependent binding sites and support the existence of widely dispersed, developmentally regulated, functional enhancers differentially bound by HNF4A and FOXA2 in liver.

    “In summary, our results show that H3K4me1 and H4ac signatures are distinct at differentiation-dependent binding sites, and further support the existence of widely dispersed, developmentally regulated, functional enhancers that are differentially bound by HNF4A and FOXA2 in the liver.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The HNF4A and FOXA2 antibodies used for ChIP-seq specifically immunoprecipitate their intended TFs in fetal liver.

  • May combine two claims.

    Premisecitation

    HNF4A is necessary for hepatocyte differentiation; in Hnf4a null embryos rescued by tetraploid methodology, liver specification occurs but hepatocyte precursors fail to differentiate.

    “In this context, liver specification occurs in Hnf4a null embryos; however, hepatocyte precursors fail to differentiate (Parviz et al., 2003).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Parviz et al., 2003 source

  • May combine two claims.

    Study design and conductown data

    Unthresholded data analysis found no evidence that TF enrichment was artificially masked by imposed height cutoffs or that TFs bound alternative sites in close proximity, confirming differentiation-dependent regions.

    “We found no evidence to suggest that TF enrichment was artificially masked by imposed height cutoffs or that the TFs were binding to alternative sites in close proximity, confirming that we had identified differentiation-dependent regions of TF-DNA interactions (Figure 2D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The HNF4A and FOXA2 antibodies used for ChIP-seq specifically immunoprecipitate their intended TFs in fetal liver.

  • May combine two claims.

    Interpretationown data

    The Sall4 enhancer showed a more robust response to TEAD2 and YAP1 than to HNF4A and FOXA2, suggesting it is highly dependent on Hippo signaling.

    “Interestingly, the enhancer associated with Sall4 showed a more robust response to TEAD2 and YAP1 than to HN4A and FOXA2, suggesting that it is highly dependent on Hippo signaling.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    TEAD transcriptional activity is dependent on interactions with YAP1, a downstream component of the Hippo signaling cascade.

    “TEAD transcriptional activity is dependent on interactions with YAP1, a downstream component of the Hippo signaling cascade.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The Hippo pathway controls liver size and is commonly amplified in human hepatocellular carcinoma.

    “This pathway controls liver size and is commonly amplified in human hepatocellular carcinoma (Camargo et al., 2007; Zender et al., 2006).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Camargo et al., 2007 source
    • cites Zender et al., 2006 source

  • May combine two claims.

    Interpretationreasoning

    Hippo signaling influences HNF4A and FOXA2 enhancer switching, thereby affecting target gene expression and the status of hepatocyte differentiation.

    “Furthermore, we highlight how Hippo signaling influences HNF4A and FOXA2 enhancer switching, thereby affecting target gene expression and the status of hepatocyte differentiation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Transcription factor-enhancer interactions are not only tissue specific but also differentiation dependent.

    “In summary, transcription factor-enhancer interactions are not only tissue specific but also differentiation dependent”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The presence of YAP1 in the nucleus influences HNF4A and FOXA2 binding-site choice and target gene expression.

    “Together, these results suggest that the presence of YAP1 in the nucleus influences HNF4A and FOXA2 binding-site choice and target gene expression.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Ectopic expression of YAP1 in the transgenic mouse model mimics the nuclear localization of YAP1 that occurs upon reduced cell density or disrupted cell-cell and cell-ECM contacts.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Foxa2 null mice fail to form regions of definitive endoderm that give rise to liver.

    “Foxa2 null mice fail to form regions of definitive endoderm that give rise to liver (Lee et al., 2005)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lee et al., 2005 source

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Input control samples (hepatoblasts and adult liver) were sequenced and analyzed using similar limits, and any regions of enrichment identified in input control samples were subtracted from the data sets.

    “Input control samples (hepatoblasts and adult liver) were sequenced and analyzed using similar limits. Any regions of enrichment identified in input control samples were subtracted from the data sets.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The synergy uncovered between TEAD2 and HNF4A could also be applicable to human liver development and HCC.

    “suggesting that the synergy we uncovered between TEAD2 and HNF4A could also be applicable to human liver development and HCC.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: HEPG2 cells are a suitable model to represent hepatocytes for inferring human liver development and HCC.
    • Rests on an assumption: TEAD4 in HEPG2 cells is functionally analogous to TEAD2 in mouse hepatocytes.
    • Rests on an assumption: Co-occupancy of HNF4A and TEAD4 at enhancers in HEPG2 cells indicates functional synergy between these factors.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    HNF4A has previously been implicated in regulating tissue remodeling, which is required for normal liver formation.

    “HNF4A has previously been implicated in regulating tissue remodeling, which is required for normal liver formation.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Battle et al., 2006 source
    • cites Parviz et al., 2003 source

  • May combine two claims.

    Interpretationreasoning

    Reduced cell densities and disrupted cell-cell and cell-ECM contacts, all of which are required for in vitro propagation, likely cause nuclear localization of YAP1, which in turn influences HNF4A and FOXA2 binding-site choice.

    “We propose a model in which reduced cell densities and disrupted cell-cell and cell-ECM contacts, all of which are required for in vitro propagation, likely cause nuclear localization of YAP1, which in turn influences HNF4A and FOXA2 binding-site choice.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Ectopic expression of YAP1 in the transgenic mouse model mimics the nuclear localization of YAP1 that occurs upon reduced cell density or disrupted cell-cell and cell-ECM contacts.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ablation of HNF4A specifically in the embryonic liver causes loss of cohesive tissue architecture with disruption of cell-cell junction assembly.

    “Ablation of HNF4A specifically in the embryonic liver causes loss of cohesive tissue architecture with disruption of cell-cell junction assembly (Battle et al., 2006; Parviz et al., 2003).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Battle et al., 2006 source
    • cites Parviz et al., 2003 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The ENCODE Project has conducted chIP-seq to examine HNF4A and TEAD4 in HEPG2 cells.

    “Interestingly, the ENCODE Project has conducted chIP-seq to examine HNF4A and TEAD4 in HEPG2 cells (https://www.encodeproject.org/experiments/ENCSROOOBRP/).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites https://www.encodeproject.org/experiments/ENCSROOOBRP/

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationreasoning

    Consequently, genes associated with embryonic liver are activated and the expression levels of genes associated with fully differentiated, functional hepatocytes decline.

    “Consequently, genes associated with embryonic liver are activated and the expression levels of genes associated with fully differentiated, functional hepatocytes decline.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Transcripts were considered differentially expressed if NAC values differed by a factor of 2 and their corrected p value was <0.05 (by Fisher's exact test).

    “Transcripts were considered differentially expressed if NAC values differed by a factor of 2 and their corrected p value was <0.05 (by Fisher's exact test).”
    Quote in the paperSubstantial claim
40 of 40

  • The paper relies on this without support.

    The HNF4A and FOXA2 antibodies used for ChIP-seq specifically immunoprecipitate their intended TFs in fetal liver.

    “We sequenced DNA immunoprecipitated by HNF4A or FOXA2 antibodies (as described in Experimental Procedures)”

  • The paper relies on this without support.

    TEAD motifs are sufficiently similar among family members that TEAD1 motif enrichment can be used to infer TEAD2 binding.

    “Since TEAD motifs are similar among family members, we verified the transcript levels of all four family members in embryonic and adult liver (Figure 5C).”

  • The paper relies on this without support.

    The dominant-negative HNF4A and TEAD2 constructs specifically inhibit HNF4A and TEAD2 activity.

    “The additive effect was diminished when a dominant-negative version of HNF4A or TEAD2 was substituted.”

  • The paper relies on this without support.

    HEPG2 cells are a suitable model to represent hepatocytes for inferring human liver development and HCC.

    “Due to the difficulty of propagating hepatocytes in vitro, HEPG2, a hepatocellular carcinoma (HCC) cell line, is commonly used to represent hepatocytes.”

  • The paper relies on this without support.

    TEAD4 in HEPG2 cells is functionally analogous to TEAD2 in mouse hepatocytes.

    “Furthermore enhancers co-occupied by HNF4A and TEAD4 in HEPG2 cells were found at “embryonic” target genes, such as Sall4 and Dlk1, suggesting that the synergy we uncovered between TEAD2 and HNF4A could also be applicable to human liver development and HCC.”

  • The paper relies on this without support.

    Co-occupancy of HNF4A and TEAD4 at enhancers in HEPG2 cells indicates functional synergy between these factors.

    “Furthermore enhancers co-occupied by HNF4A and TEAD4 in HEPG2 cells were found at “embryonic” target genes, such as Sall4 and Dlk1, suggesting that the synergy we uncovered between TEAD2 and HNF4A could also be applicable to human liver development and HCC.”

  • The paper relies on this without support.

    Ectopic expression of YAP1 in the transgenic mouse model mimics the nuclear localization of YAP1 that occurs upon reduced cell density or disrupted cell-cell and cell-ECM contacts.

    “we used an inducible transgenic mouse model in which YAP1 is expressed in the liver in response to doxycycline (Figure S6; Dong et al., 2007).”
7 of 7

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • HNF4A bound regions (HBRs) are flanked by nucleosomes marked with H3K4me1.

    “HNF4A bound regions (HBRs) are flanked by nucleosomes marked with H3K4me148–50, leading us to question whether HNF4A contributes to the establishment and maintenance of H3K4me1 at HBRs.”

    From HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements (2024) · cites this paper as [49]

    The Results section (“Differentiation-Dependent Binding Sites Function as Temporal Enhancers”; page not provided) reports H3K4me1 enrichment on both sides of HNF4A/FOXA2 binding sites that remained occupied or were occupied in adult liver. It does not establish this pattern for all HNF4A-bound regions: sites bound only in the embryo instead showed a central H3K4me1 signal. The passage describes histone-mark signal, not nucleosomes directly. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • HNF4A cooperates with the FOXA family and GATA6 of pioneer factors.

    “HNF4A is known to cooperate with the FOXA family and GATA6 of pioneer factors12,49,55.”

    From HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements (2024) · cites this paper as [49]

    The Results section, “Widespread Combinatorial Role for HNF4A and FOXA2 in the Embryonic Liver,” describes cooperation and shared binding between HNF4A and FOXA2. The cited paper does not establish cooperation with the FOXA family as a whole or with GATA6; a search for GATA6 found no relevant passage in our copy. Page numbers were not available. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • HNF4A binding is highly dynamic and bound regions can change based on tissue and differentiation state.

    “We have shown that HNF4A binding is highly dynamic and that bound regions can change based on tissue and differentiation state (11).”

    From Hepatocyte Nuclear Factor 4‐Alpha Is Essential for the Active Epigenetic State at Enhancers in Mouse Liver (2019) · cites this paper as [11]

    In Results, under “HNF4A and FOXA2 Bind the Genome in a Differentiation-Dependent Manner” (page unavailable), the paper reports that 55% of HNF4A-binding sites were occupied uniquely in either embryonic or adult liver. This supports dynamic binding across differentiation states, but the passages do not establish that HNF4A-bound regions change between different tissues. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • 95% of our ~12,000 HBRs matched regions identified as bound by HNF4A in Reizel et al. data.

    “we compared the adult mouse liver HNF4A ChIP-seq data from Reizel et al.52 to our HNF4A ChIP-seq data48,49 and observed that 95% of our ~12,000 HBRs matched regions identified as bound by HNF4A in their data (Supplementary Fig. 1d).”

    From HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements (2024) · cites this paper as [49]

  • There is a significant overlap between HNF4A and FOXA2 bound regions in hepatocytes.

    “We previously reported a significant overlap between HNF4A and FOXA2 bound regions (FBRs) in hepatocytes (11).”

    From Hepatocyte Nuclear Factor 4‐Alpha Is Essential for the Active Epigenetic State at Enhancers in Mouse Liver (2019) · cites this paper as [11]

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Authors
Alder, O., Cullum, R., Lee, S., Kan, A. C., Wei, W., Yi, Y., Garside, V. C., Bilenky, M., Griffith, M., Morrissy, A. S., Robertson, G. A., Thiessen, N., Zhao, Y., Chen, Q., Pan, D., Jones, S. J. M., Marra, M. A., & Hoodless, P. A.
Affiliations
  • BC Cancer Agency (CA)
  • Johns Hopkins University (US)
Corresponding author
Hoodless, Pamela A.
ORCID
Venue
Cell Reports
Publisher
Elsevier BV
Published
2014-10
Volume
9
Issue
1
Pages
261-271
ISSN
2211-1247 (ISSN-L)
DOI
10.1016/j.celrep.2014.08.046
PMC
PMC4612615
OpenAlex
W1983601196
Cited by
113
References
52
Citations by year
2026: 6 · 2025: 11 · 2024: 14 · 2023: 7 · 2022: 13 · 2021: 5 · 2020: 10 · 2019: 10 · 2018: 10 · 2017: 7 · 2016: 11 · 2015: 7 · 2014: 1 · 2013: 1
FWCI
3.77
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by-nc-nd)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Minerva Access (University of Melbourne) (submittedVersion, cc-by-nc-nd)
Funders
  • Howard Hughes Medical Institute
  • Canadian Institutes of Health Research (FRN259575)
  • National Cancer Institute (P30CA 006973)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Hippo pathway signaling and YAP/TAZ
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
Keywords
Hippo signaling pathway, HNF4α, FOXA2, hepatocyte differentiation, liver development, TEAD2, YAP, chromatin remodeling, lineage specification
MeSH terms
Hippo Signaling Pathway, Animals, Cell Differentiation, Female, Mice, Inbred C57BL, Mice, Transgenic, Signal Transduction, Gene Expression, Protein Serine-Threonine Kinases, Hepatocytes, Mice, Hepatocyte Nuclear Factor 3-beta, Hepatocyte Nuclear Factor 4

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-23.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.

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