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Cited by 680Epigenetics and DNA MethylationGenomics and Chromatin DynamicsCancer-related gene regulation

Transcription factors as readers and effectors of DNA methylation

Open access
Zhu, Heng, Wang, Guohua, Qian, Jiang·2016·Nature Reviews Genetics
DOI 10.1038/nrg.2016.83PMC PMC5559737
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Abstract

Recent technological advances have made it possible to decode DNA methylomes at single-base-pair resolution under various physiological conditions. Many aberrant or differentially methylated sites have been discovered, but the mechanisms by which changes in DNA methylation lead to observed phenotypes, such as cancer, remain elusive. The classical view of methylation-mediated protein–DNA interactions is that only proteins with a methyl-CpG binding domain (MBD) can interact with methylated DNA. However, evidence is emerging to suggest that transcription factors lacking a MBD can also interact with methylated DNA. The identification of these proteins and the elucidation of their characteristics and the biological consequences of methylation-dependent transcription factor–DNA interactions are important stepping stones towards a mechanistic understanding of methylation-mediated biological processes, which have crucial implications for human development and disease.

AI snapshot

Written from the paper’s full text.

TL;DR

This review/analysis argues that transcription factors lacking methyl-CpG binding domains can act as sequence-specific readers and effectors of methylated DNA, extending the classical MBD-only model. It combines in vitro discovery screens with in vivo ENCODE-based integration to show that many TF binding sites overlap highly methylated CpGs, while stressing unresolved causality and physiological relevance.

Key findings

  • Classical MBD proteins bind methylated DNA without strong sequence specificity, but emerging evidence shows TFs such as Kaiso, CEBPα, ZFP57/KAP1, KLF4 and others can recognize methylated motifs in a sequence-dependent manner.
  • A protein microarray probing 1,321 human TFs and 210 cofactors with 154 methylated motifs found that 150 motifs bound at least one protein; 41 TFs and 6 cofactors bound at least one methylated sequence, with binding spread across families such as zf-C2H2, homeobox, bHLH, forkhead, bZIP and HMG.
  • An MS/MS pulldown from mouse ES nuclear extracts identified 19 proteins preferring methylated over unmethylated probes, including known MBD proteins and TFs; a distinct set was found in neuronal progenitor cells.
  • In H1 human ES cells, 77,124 of 258,188 DNase I hypersensitive sites (29.9%) had average CpG methylation above 80%; for CEBPβ, 6,675 of 15,557 ChIP–seq peaks (42.9%) exceeded 80% methylation, and motif analysis found 3,894 of 15,557 CEBPβ sites (25.0%) carried a highly methylated CpG in the enriched motif, compared with 7.7% for E2F6, 5.2% for RFX5, 3.0% for KLF4 and 1.6% for BACH1.

Methods

  • This is a review/Analysis article synthesizing literature on DNA methylation readers and effectors, focused mainly on mammalian methylated CpG sites.
  • It describes high-throughput discovery approaches including tandem mass spectrometry pulldown, functional protein microarrays, DNA microarrays and ChIP–BS-seq.
  • It also reports an integrative in vivo analysis: ENCODE H1 hESC DNase I hypersensitivity peaks and whole-genome bisulfite methylome data were overlaid; TF ChIP–seq peaks were called with IDR and compared with methylation levels; MEME was used for motif discovery, with NRF1 and TAF1 as negative controls.

Limitations

  • The authors state that causality between DNA methylation and TF binding remains unclear, that a more comprehensive catalogue of readers/effectors is needed, and that in vivo validation and physiological relevance are still major challenges; many interactions rest mainly on in vitro evidence.
  • Method-specific limitations stated include: MS/MS probes are often generic, so sequence specificity is elusive; DNA microarrays require prior knowledge of a candidate TF; ChIP–BS-seq requires prior knowledge and suitable antibodies; and Kaiso in vivo results conflict across cell types, suggesting cell-type specificity.
  • Evident from the text: the in vivo integration is constrained by limited available ChIP–seq data (only six TFs in H1 cells) and uses a stringent >80% methylation cutoff, making the reported overlap estimates conservative; as a review, it does not present a single new experimental cohort.

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Integrity signals

Registry

Crossref · 10.1038/nrg.2016.83

Cites

146 of 146 references verified0 citation errors · 0 not checkable

Statistics

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Claims the paper makes

0 of 134 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Of 15,557 CEBPβ ChIP-seq peaks, 6,675 (42.9%) had a methylation level greater than 80%.

    “of the 15,557 ChIP–seq peaks identified for CEBPβ, 6,675 (42.9%) had a methylation level greater than 80%.”
    Quote in the paperSubstantial claim
  • Findingown data

    In H1 human embryonic stem cells, 77,124 of 258,188 DNase I hypersensitivity sites had an average CpG methylation level greater than 80%.

    “Overall, 77,124 (29.9%) of the 258,188 DHSs detected in H1 cells had an average methylation level greater than 80% at CpG sites (FIG. 2a)”
    Quote in the paperSubstantial claim
  • Findingcitation

    KLF4 binds methylated enhancer regions in embryonic stem cells.

    “Based on the analysis of KLF4 binding in ES cells, we also found that KLF4 binds to methylated enhancer regions53”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [53] Hu, S. et al. DNA methylation presents distinct binding sites for human transcription factors. eLife 2, e00726 (2013). This study identifies the transcription factors that preferentially bind to methylated DNA using a protein microarray-based approach and verified that endogenous KLF4 binds to methylated DNA in human ES cells. source

      Not checked in the free run.

  • Findingown data

    A motif reconstructed from 3,894 binding peaks represents the methylated motif for CEBPβ.

    “A motif could be reconstructed with these 3,894 binding peaks, which represented the methylated motif for CEBPβ (FIG. 2c).”
    Quote in the paperSubstantial claim
  • Findingown data

    Among 6,675 high-methylation CEBPβ peaks, 3,894 carried a highly methylated (>80%) CpG site within the enriched motifs.

    “Among the 6,675 peaks with a high methylation level, 3,894 carried a highly methylated (>80%) CpG site within the enriched motifs.”
    Quote in the paperSubstantial claim
  • May combine two claims. 38
  • Classified as probably not a substantial claim. 7

  • May combine two claims.

    Findingown data

    NRF1 and TAF1, used as negative controls, do not show a bimodal methylation distribution, indicating that they bind only regions with low methylation levels.

    “neither NRF1 (FIG. 2a) nor TAF1 (Supplementary information S1 (figure)) showed a bimodal distribution, demonstrating that these TFs only bind to regions with low levels of methylation.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: NRF1 and TAF1 do not interact with methylated DNA.

  • May combine two claims.

    Findingown data

    The percentages of binding sites that were highly methylated were 25.0% for CEBPβ, 7.7% for E2F6, 5.2% for RFX5, 3.0% for KLF4 and 1.6% for BACH1.

    “25.0% (3,894 out of 15,557), 7.7% (1,103 out of 14,396), 5.2% (88 out of 1,695), 3.0% (115 out of 3,793) and 1.6% (186 out of 11,457) of binding sites were highly methylated for CEBPβ, E2F6, RFX5, KLF4 and BACH1, respectively (FIG. 2c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The herpesvirus protein Zta binds methylated regulatory elements and controls the epigenetic landscape during the latency-to-lytic transition.

    “a bZIP herpesvirus protein, Zta, was found to bind to methylated regulatory elements and control the epigenetic landscape during the latency-to-lytic phase transition in infected mammalian cells45”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] Karlsson, Q. H., Schelcher, C., Verrall, E., Petosa, C. & Sinclair, A. J. Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein. PLoS Pathog. 4, e1000005 (2008). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    CEBPα, ZFP57 and its cofactor KAP1 interact with specific methylated sequences.

    “the basic leucine zipper (bZIP) CCAAT/enhancer-binding protein-α (CEBPα)42, the zinc-finger protein ZFP57 and its cofactor KRAB-associated protein 1 (KAP1; also known as TIF1β)43,44 were shown to interact with specific methylated sequences.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [42] Rishi, V. et al. CpG methylation of half-CRE sequences creates C/EBPα binding sites that activate some tissue-specific genes. Proc. Natl Acad. Sci. USA 107, 20311–20316 (2010). source

      Not checked in the free run.

    • cites [43] Quenneville, S. et al. In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions. Mol. Cell 44, 361–372 (2011). This paper demonstrates that ZFP57 and its cofactor KAP1 affect chromatin by interacting with methylated ICRs in embryonic stem cells. source

      Not checked in the free run.

    • cites [44] Liu, Y., Toh, H., Sasaki, H., Zhang, X. & Cheng, X. An atomic model of Zfp57 recognition of CpG methylation within a specific DNA sequence. Genes Dev. 26, 2374–2379 (2012). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    In neuronal progenitor cells, a large and distinct set of proteins shows preferential binding to methylated CpG sites, suggesting that methyl-CpG interactions are dynamic and vary under different physiological conditions.

    “The authors applied the same approach to neuronal progenitor cells and found that a large and distinct set of proteins showed preferential binding to mCpG sites, suggesting that the interaction with mCpG is dynamic and thus varies under different physiological conditions.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [51] Spruijt, C. G. et al. Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 (2013). This paper describes the identification of proteins that interact with mCpG sites, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in ES cells and neuronal progenitor cells using a MS/MS-based approach. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Reduction of DNA methylation leads to a decrease in H3K27ac marks, suggesting an active role of DNA methylation in regulating enhancer activity.

    “the reduction of DNA methylation led to a decrease in H3K27ac marks, suggesting an active role of DNA methylation in regulating enhancer activity.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [79] Charlet, J. et al. Bivalent regions of cytosine methylation and H3K27 acetylation suggest an active role for DNA methylation at enhancers. Mol. Cell 62, 422–431 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    CEBPα binds the sequence 5′-TGACGTCA, and when the CpG is methylated it recognizes the half motif 5′-mCGTCA.

    “CEBPα is known to bind a particular sequence element, 5′-TGACGTCA42. However, when the CpG is methylated, CEBPα can effectively recognize half of the motif: 5′-mCGTCA42.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [42] Rishi, V. et al. CpG methylation of half-CRE sequences creates C/EBPα binding sites that activate some tissue-specific genes. Proc. Natl Acad. Sci. USA 107, 20311–20316 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The crystal structure of mouse ZFP57 with methylated DNA shows that its two zinc-fingers interact with methylated DNA and that Arg178 plays a crucial part in mCpG binding.

    “the recent crystal structure of mouse ZFP57 in complex with a methylated DNA sequence demonstrated that its two zinc-fingers interact with methylated DNA, and that an arginine (Arg178), which is involved in hydrophobic interactions, plays a crucial part in mCpG binding44.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] Liu, Y., Toh, H., Sasaki, H., Zhang, X. & Cheng, X. An atomic model of Zfp57 recognition of CpG methylation within a specific DNA sequence. Genes Dev. 26, 2374–2379 (2012). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Approximately 6.7% of the mouse genome is differentially methylated, mostly at distal cis-regulatory regions.

    “approximately 6.7% of the mouse genome is differentially methylated, mostly at distal cis-regulatory regions77.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [77] Hon, G. C. et al. Epigenetic memory at embryonic enhancers identified in DNA methylation maps from adult mouse tissues. Nat. Genet. 45, 1198–1206 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductreasoning

    The MS/MS-based approach uses generic DNA probes, so sequence specificity of observed interactions remains elusive.

    “the DNA probes used in this approach are typically generic and, therefore, sequence specificity of observed interactions remains elusive.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductreasoning

    The DNA microarray approach requires prior knowledge of a candidate transcription factor and is cumbersome for surveying an entire transcription factor family.

    “prior knowledge of a candidate TF is required because it can be cumbersome to survey an entire TF family.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Most of the factors that bind methylated sequences bind only a few such sequences, suggesting that the interactions are both methylation-dependent and sequence-specific.

    “Most of these factors were found to bind to only a few methylated sequences, suggesting that the interactions are not only methylation-dependent but also sequence-specific.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [53] Hu, S. et al. DNA methylation presents distinct binding sites for human transcription factors. eLife 2, e00726 (2013). This study identifies the transcription factors that preferentially bind to methylated DNA using a protein microarray-based approach and verified that endogenous KLF4 binds to methylated DNA in human ES cells. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Some transcription factors identified by protein microarray bind methylated DNA in vivo and regulate gene expression.

    “A subsequent validation assay showed that some of these TFs indeed bind to methylated DNA in vivo and regulate gene expression53.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [53] Hu, S. et al. DNA methylation presents distinct binding sites for human transcription factors. eLife 2, e00726 (2013). This study identifies the transcription factors that preferentially bind to methylated DNA using a protein microarray-based approach and verified that endogenous KLF4 binds to methylated DNA in human ES cells. source

      Not checked in the free run.

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

    Premisecitation

    An arginine and glutamate pair in KLF4 recognizes the mCpG site.

    “A separate study suggested that an arginine and glutamate pair in KLF4 recognizes the mCpG site68.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [68] Liu, Y. et al. Structural basis for Klf4 recognition of methylated DNA. Nucleic Acids Res. 42, 4859–4867 (2014). This study determined the crystal structure of the KLF4-methylated DNA complex and provided the structural basis for mCpG–TF interactions. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    KLF4 recognizes the non-methylated motif 5′-TTTACGCC and specifically recognizes 5′-TCCmCGCCC only when the CpG is methylated.

    “although KLF4 recognizes a non-methylated canonical motif of 5′-TTTACGCC, it has been demonstrated that KLF4 specifically recognizes a 5′-TCCmCGCCC motif only when the CpG is methylated53.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [53] Hu, S. et al. DNA methylation presents distinct binding sites for human transcription factors. eLife 2, e00726 (2013). This study identifies the transcription factors that preferentially bind to methylated DNA using a protein microarray-based approach and verified that endogenous KLF4 binds to methylated DNA in human ES cells. source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductreasoning

    ChIP-BS-seq requires prior knowledge of methyl-CpG-binding proteins and antibodies directed against the proteins of interest.

    “it requires prior knowledge of mCpG-binding proteins and the availability of antibodies that are directed against the proteins of interest.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    When the paternal imprinting control region is methylated, CTCF cannot bind, allowing distal enhancers to activate downstream genes.

    “when the paternal ICR is methylated, CTCF cannot bind to the ICR, thus allowing the activation of downstream genes by distal enhancers.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [81] Bell, A. C. & Felsenfeld, G. Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405, 482–485 (2000). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Those positively correlated methylation regions are enriched in DNase I hypersensitive sites and are conserved across evolution.

    “These methylation regions are overrepresented in DHSs and are evolutionarily conserved”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [101] Wan, J. et al. Characterization of tissue-specific differential DNA methylation suggests distinct modes of positive and negative gene expression regulation. BMC Genomics 16, 49 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Insertion of a CTCF-binding motif into a genomic locus in mouse embryonic stem cells induces CTCF binding and reduces local methylation.

    “Insertion of the binding site induced CTCF binding and resulted in a reduced methylation level in local genomic regions.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [78] Stadler, M. B. et al. DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480, 490–495 (2011). This paper demonstrates that some proteins, such as CTCF and REST, can reduce DNA methylation levels at the genomic regions near their binding regions. source

      Not checked in the free run.

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

    Premisecitation

    Methylation in promoters is often considered the hallmark for gene repression.

    “Methylation in promoters is often considered the hallmark for gene repression99.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [99] Baylin, S. B. DNA methylation and gene silencing in cancer. Nat. Clin. Pract. Oncol. 2, S4–S11 (2005). source

      Not checked in the free run.

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

    Interpretationnone given

    Binding to an mCpG site is only one way a pioneer transcription factor can access condensed chromatin; other pioneer factors might use alternative routes such as binding partial motifs.

    “Yet, binding to an mCpG site is just one approach for a pioneer TF to access condensed chromatin. Other pioneer TFs might have alternative approaches such as binding to partial motifs.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Although only about 25% of known transcription factor binding motifs contain at least one CpG site, transcription factors without CpGs in their motifs could also be influenced by DNA methylation through indirect crosstalk mechanisms.

    “Although only approximately 25% of known TF binding motifs contain at least one CpG site98, through such indirect crosstalk mechanisms, the binding of TFs without CpGs in their binding motifs could also be influenced by DNA methylation.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [98] Blattler, A. & Farnham, P. J. Cross-talk between site-specific transcription factors and DNA methylation states. J. Biol. Chem. 288, 34287–34294 (2013). source

      Not checked in the free run.

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

    Premisecitation

    The human genome is mainly arranged as heterochromatin, where DNA and histones are highly condensed and genes are inactive, or euchromatin, where DNA is largely accessible to transcription factors and genes can be activated.

    “One is heterochromatin (or condensed chromatin), in which DNA sequences and histones are highly condensed, and genes in these regions are inactive. The other form is euchromatin (or open chromatin), in which DNA sequences are largely accessible to TFs, and genes in these regions can be activated102,103.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [102] Kornberg, R. D. Chromatin structure: a repeating unit of histones and DNA. Science 184, 868–871 (1974). source

      Not checked in the free run.

    • cites [103] Mikkelsen, T. S. et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560 (2007). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Novel NRF1 binding sites in triple-knockout cells correlate with novel DNase I hypersensitivity sites and exhibit predominantly low methylation levels.

    “These binding sites often correlated with novel DHSs in the TKO cells, which exhibited predominantly low methylation levels due to their generation in cells lacking DNMTs.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [97] Domcke, S. et al. Competition between DNA methylation and transcription factors determines binding of NRF1. Nature 528, 575–579 (2015). This paper shows that the removal of DNA methylation would create novel binding sites for NRF1 and thus affect the NRF1–DNA interactions in vivo , whereas other studies showed that DNA methylation could affect TF–DNA interactions in vitro. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Deletion of CTCF motifs in a reporter construct leads to hypermethylation and decreased NRF1 binding, suggesting that NRF1 binding in vivo depends on both methylation levels and co-occurring transcription factors such as CTCF.

    “Deletion of CTCF motifs within the construct led to hypermethylation and thus decreased NRF1 binding, suggesting that NRF1 binding in vivo depends on both methylation levels and co-occurring TFs, such as CTCF97.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [97] Domcke, S. et al. Competition between DNA methylation and transcription factors determines binding of NRF1. Nature 528, 575–579 (2015). This paper shows that the removal of DNA methylation would create novel binding sites for NRF1 and thus affect the NRF1–DNA interactions in vivo , whereas other studies showed that DNA methylation could affect TF–DNA interactions in vitro. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Novel NRF1 binding sites were hypermethylated in wild-type cells, suggesting that removal of DNA methylation in triple-knockout cells generated new NRF1 binding sites.

    “novel NRF1 binding sites were hypermethylated in the wild-type cell line97, suggesting that the removal of DNA methylation in TKO cells generated new binding sites for NRF1.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [97] Domcke, S. et al. Competition between DNA methylation and transcription factors determines binding of NRF1. Nature 528, 575–579 (2015). This paper shows that the removal of DNA methylation would create novel binding sites for NRF1 and thus affect the NRF1–DNA interactions in vivo , whereas other studies showed that DNA methylation could affect TF–DNA interactions in vitro. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    CTCF changes local methylation status, and its binding is also methylation sensitive.

    “although CTCF is known to change the local methylation status78, it has been shown that the binding of CTCF is also methylation sensitive96.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [78] Stadler, M. B. et al. DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480, 490–495 (2011). This paper demonstrates that some proteins, such as CTCF and REST, can reduce DNA methylation levels at the genomic regions near their binding regions. source

      Not checked in the free run.

    • cites [96] Maurano, M. T. et al. Role of DNA methylation in modulating transcription factor occupancy. Cell Rep. 12, 1184–1195 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    CTCF binding in an exon creates a roadblock for RNA polymerase II elongation and promotes inclusion of that exon.

    “In one study, the authors observed that the binding of CTCF in an exon region created a roadblock for RNA polymerase II elongation and thus promoted the inclusion of the exon112.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [112] Shukla, S. et al. CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing. Nature 479, 74–79 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    KLF4 is an mCpG-binding protein and has been shown to be a pioneer factor.

    “The mCpG-binding protein KLF4 was also shown to be a pioneer factor105,111.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [105] Iwafuchi-Doi, M. & Zaret, K. S. Pioneer transcription factors in cell reprogramming. Genes Dev. 28, 2679–2692 (2014). source

      Not checked in the free run.

    • cites [111] Buganim, Y., Faddah, D. A. & Jaenisch, R. Mechanisms and models of somatic cell reprogramming. Nat. Rev. Genet. 14, 427–439 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Pioneer transcription factors are a distinct subset that drive chromatin changes.

    “Pioneer TFs are a unique subset of TFs that drive these chromatin changes.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [105] Iwafuchi-Doi, M. & Zaret, K. S. Pioneer transcription factors in cell reprogramming. Genes Dev. 28, 2679–2692 (2014). source

      Not checked in the free run.

    • cites [106] Zaret, K. S. & Carroll, J. S. Pioneer transcription factors: establishing competence for gene expression. Genes Dev. 25, 2227–2241 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Chromatin organization is dynamic, and chromatin types can switch during development or differentiation.

    “Chromatin organization is dynamic, and the different types of chromatin can change from one form to another during development or differentiation104”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [104] Ho, L. & Crabtree, G. R. Chromatin remodelling during development. Nature 463, 474–484 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    DNMT3A and DNMT3B interact with NR6A1, and this interaction induces methylation of the OCT4 promoter carrying the NR6A1 binding site.

    “Sato et al.86 demonstrated that DNMT3A and DNMT3B interact with an orphan nuclear receptor, NR6A1 (nuclear receptor subfamily 6 group A member 1), and that this interaction induced the methylation of the OCT4 (also known as OCT3 and POU5F1) promoter carrying the NR6A1 binding site.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [86] Sato, N., Kondo, M. & Arai, K. The orphan nuclear receptor GCNF recruits DNA methyltransferase for Oct-3/4 silencing. Biochem. Biophys. Res. Commun. 344, 845–851 (2006). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Differences in chromatin states create or eliminate transcription factor binding sites and lead to differential transcription factor binding.

    “Differences in chromatin states will either create or eliminate TF binding sites and thus lead to differential TF binding.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Pioneer transcription factors can bind directly to heterochromatic DNA and recruit other factors to convert it to euchromatin and start transcription.

    “A typical characteristic of pioneer TFs is their ability to bind directly to heterochromatic DNA and recruit other factors to change the status to euchromatin to initiate transcription105,106.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [105] Iwafuchi-Doi, M. & Zaret, K. S. Pioneer transcription factors in cell reprogramming. Genes Dev. 28, 2679–2692 (2014). source

      Not checked in the free run.

    • cites [106] Zaret, K. S. & Carroll, J. S. Pioneer transcription factors: establishing competence for gene expression. Genes Dev. 25, 2227–2241 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Regions linked to alternative splicing were enriched for CTCF motifs, but they also contained other enriched motifs.

    “Although CTCF motifs were significantly enriched in differentially methylated regions associated with alternative splicing, other motifs were also enriched”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [115] Wan, J. et al. Integrative analysis of tissue-specific methylation and alternative splicing identifies conserved transcription factor binding motifs. Nucleic Acids Res. 41, 8503–8514 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    IDH mutant gliomas show hypermethylation at CTCF binding sites, which reduces CTCF binding.

    “IDH mutant gliomas have been shown to exhibit hypermethylation at CTCF binding sites, which leads to a reduction in CTCF binding”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [117] Flavahan, W. A. et al. Insulator dysfunction and oncogene activation in IDH mutant gliomas. Nature (2015). source

      Not checked in the free run.

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

    Premisecitation

    Abnormal DNA methylation has been linked to many human diseases, among them several cancers.

    “Many studies have shown that aberrant DNA methylation is associated with various human diseases, including some types of cancer19–21.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [19] Jones, P. A. & Baylin, S. B. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. 3, 415–428 (2002). source

      Not checked in the free run.

    • cites [20] Jones, P. A. & Baylin, S. B. The epigenomics of cancer. Cell 128, 683–692 (2007). source

      Not checked in the free run.

    • cites [21] Jones, P. A. & Laird, P. W. Cancer epigenetics comes of age. Nat. Genet. 21, 163–167 (1999). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In honeybee brain methylation data, histone genes with introns had high methylation, while those without introns had none.

    “A comparison of methylation levels between queen and worker bees revealed that intron-containing histone genes were highly methylated, whereas intronless histone genes were not methylated”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [114] Lyko, F. et al. The honey bee epigenomes: differential methylation of brain DNA in queens and workers. PLoS Biol. 8, e1000506 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Loss of CTCF in topologically associated domains removes the domain boundary and causes aberrant gene activation.

    “loss of CTCF in topologically associated domains removed the domain boundary and caused aberrant gene activation117.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [117] Flavahan, W. A. et al. Insulator dysfunction and oncogene activation in IDH mutant gliomas. Nature (2015). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    IDH1 and IDH2 encode isocitrate dehydrogenases, and mutations in these genes are among the most frequent in diffuse gliomas.

    “The IDH genes (IDH1 and IDH2) encode isocitrate dehydrogenases, and mutations in these genes are among the most frequent found in diffuse gliomas118,119.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [118] Parsons, D. W. et al. An integrated genomic analysis of human glioblastoma multiforme. Science 321, 1807–1812 (2008). source

      Not checked in the free run.

    • cites [119] Yan, H. et al. IDH1 and IDH2 mutations in gliomas. N. Engl. J. Med. 360, 765–773 (2009). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationnone given

    This new concept does not refute the conventional view that most transcription factors do not interact with methylated DNA.

    “Of note, this new concept does not refute the conventional view that most TFs do not interact with methylated DNA.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Most transcription factors do not interact with methylated DNA.

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

    Interpretationnone given

    The concept may apply only to a subset of transcription factors and a subset of their binding sites.

    “Here, we have focused on this exciting and novel concept with a full awareness that it may apply only to a subset of TFs and to a subset of their binding sites.”
    Quote in the paperNot a substantial claim
42 of 42

  • The paper relies on this without support.

    NRF1 and TAF1 do not interact with methylated DNA.

    “we selected two TFs (nuclear respiratory factor 1 (NRF1) and transcription initiation factor TFIID subunit 1 (TAF1)), which are known not to interact with methylated DNA (based on our current knowledge), as negative controls”

  • The paper relies on this without support.

    DNA in heterochromatin is often methylated and tightly wrapped around nucleosomes, making it inaccessible to most transcription factors.

    “As DNA in heterochromatin is wrapped tightly around the nucleosomes and is often methylated, it is inaccessible to most TFs; pioneer TFs must possess special features to enable protein–DNA interactions.”

  • The paper relies on this without support.

    Splicing occurs co-transcriptionally.

    “However, it is now well-established that splicing occurs co-transcriptionally, which means that DNA modification could influence RNA splicing.”

  • The paper relies on this without support.

    The role of MBD proteins as non-sequence-specific methylation readers has been fairly well studied.

    “Although the role of MBD proteins, as non-sequence-specific methylation readers, has been fairly well-studied, the biological functions of an emerging class of sequence-specific methylation readers and/or effectors remain elusive.”

  • The paper relies on this without support.

    The biological functions of sequence-specific methylation readers and/or effectors remain elusive.

    “Although the role of MBD proteins, as non-sequence-specific methylation readers, has been fairly well-studied, the biological functions of an emerging class of sequence-specific methylation readers and/or effectors remain elusive.”

  • The paper relies on this without support.

    Methylation readers typically bind both methylated and unmethylated sites.

    “A methylation reader usually interacts with both methylated sites and unmethylated sites.”

  • The paper relies on this without support.

    Most transcription factors do not interact with methylated DNA.

    “Of note, this new concept does not refute the conventional view that most TFs do not interact with methylated DNA.”

  • The paper relies on this without support.

    The mechanism by which altered epigenetic state causes disease remains elusive.

    “However, the mechanism by which the altered epigenetic state causes disease remains elusive.”
8 of 8

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?

  • H3K27ac is associated with the active state of both enhancers and promoters.

    “In addition, acetylation of lysine 27 of histone 3 (H3K27ac) is associated with the active state of both elements.”

    From Hepatocyte Nuclear Factor 4‐Alpha Is Essential for the Active Epigenetic State at Enhancers in Mouse Liver (2019) · cites this paper as [13]

    The Cis-regulatory elements section (page unavailable) describes H3K27ac as an active mark in enhancers, supporting the enhancer part of the claim. It does not link H3K27ac to active promoters, so the claim about both elements is only partially supported. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Numerous transcription factors can directly sense cytosine methylation through changes in their binding affinity and specificity.

    “In addition, studies suggest that numerous transcription factors (TFs) can also directly sense the state of cytosine methylation through the modulation of their binding affinity and specificity (26).”

    From Dynamic DNA methylation: In the right place at the right time (2018) · cites this paper as [26]

  • High-throughput assays for transcription factor-DNA interaction have identified many transcription factors whose binding specificity is altered by 5mC in recognition sequences.

    “The development of high-throughput assays for TF-DNA interaction has identified many transcription factors with binding specificity altered by the presence of 5mC in recognition sequences (26).”

    From Dynamic DNA methylation: In the right place at the right time (2018) · cites this paper as [26]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Kaiso studies are cited with the wrong reference numbers

    Minor

    The later passage assigns Kaiso's in-vivo binding studies to references 58-60, whereas the earlier passage identifies those studies as references 61-63.

  • The cited IDR reference describes a different method

    Minor

    The paper cites reference 66 for the Irreproducible Discovery Rate method, but its reference list identifies 66 as a paper on RNA-seq isoform modeling.

2 of 2

Cites

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Cited by

Sorted by citations

Related

Authors
Zhu, H., Wang, G., & Qian, J.
Affiliations
Johns Hopkins University (US)
Corresponding author
  • Zhu, Heng
  • Qian, Jiang
ORCID
Venue
Nature Reviews Genetics
Publisher
Springer Science and Business Media LLC
Published
2016-08-01
Volume
17
Issue
9
Pages
551-565
ISSN
  • 1471-0056 (ISSN-L)
  • 1471-0064
DOI
10.1038/nrg.2016.83
PMC
PMC5559737
OpenAlex
W2494121918
Semantic Scholar
6dee1bc645a95ab7b9afe8a303d0b916cc74f4b3
Cited by
649
References
146
Citations by year
2026: 35 · 2025: 46 · 2024: 81 · 2023: 53 · 2022: 107 · 2021: 102 · 2020: 74 · 2019: 62 · 2018: 44 · 2017: 38 · 2016: 7
FWCI
4.55
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Reviews Genetics (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Eye Institute (R01EY024580, R01EY023188)
  • National Institute of General Medical Sciences (R01GM111514)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cancer-related gene regulation
Keywords
DNA methylation, transcription factors, methyl-CpG binding domain, protein-DNA interactions, methylated DNA, differentially methylated sites, aberrant DNA methylation, epigenetic regulation, cancer epigenetics, human development
MeSH terms
DNA, DNA-Binding Proteins, Humans, Transcription Factors, Transcription, Genetic, DNA Methylation

About these checks

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 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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