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Cited by 221Genomics and Chromatin DynamicsChromatin Remodeling and CancerChromosomal and Genetic Variations

Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo

Open access
Hanssen, Lars L. P., Kassouf, Mira T., Oudelaar, A. Marieke, Biggs, Daniel, Preece, Chris, Downes, Damien J., Gosden, Matthew, Sharpe, Jacqueline A. +4 more·2017·Nature Cell Biology
DOI 10.1038/ncb3573PMC PMC5540176
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Abstract

The genome is organized via CTCF–cohesin-binding sites, which partition chromosomes into 1–5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF–cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF–cohesin boundary extends the sub-TAD to adjacent upstream CTCF–cohesin-binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF–cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters.

AI snapshot

Written from the paper’s full text.

TL;DR

In vivo chromosome engineering of the mouse α-globin sub-TAD shows that convergent CTCF/cohesin boundaries are ubiquitously bound but erythroid-specifically interact, delimiting which promoters enhancers can access. Removing the upstream boundary extends enhancer contacts and ectopically activates flanking genes, including Polycomb-silenced Rhbdf1, without affecting α-globin expression.

Key findings

  • The ~80-kb α-globin sub-TAD within a ~1-Mb TAD is flanked by predominantly convergent CTCF/cohesin sites that are bound in erythroid and non-erythroid cells but interact strongly only during erythropoiesis, forming a self-interacting erythroid compartment.
  • Deleting both HS-38 and HS-39 (D3839) shifts the upstream boundary to HS-59, increases R1 enhancer interactions with Mpg, Rhbdf1, and Snrnp25, and up-regulates these genes: Mpg 12-fold, Snrnp25 6-fold, and Rhbdf1 ~600-fold; α- and θ-globin expression and haematological phenotype were not significantly changed.
  • Single deletions show non-equivalent CTCF sites: D38 up-regulates upstream genes less than D3839, with Rhbdf1 ten-fold less up-regulated than in D3839; D39 alone has no strong effect; D29 increases enhancer–promoter interactions but causes no detectable local gene-expression or histone-modification changes.
  • The boundary constrains enhancer access rather than simply blocking chromatin modification: in D3839 erythroid cells, Rhbdf1 gains H3K4me3, PolII, and chromatin accessibility while retaining H3K27me3 and Ezh2 binding.

Methods

  • Mouse chromosome engineering: TALEN and CRISPR-mediated small deletions of CTCF/cohesin sites HS-38, HS-39, and HS-29, singly and in combination, in C57BL/6J mice; primary Ter119+ erythroid cells from APH-treated spleens and ES-E14TG2a embryonic stem cells were analysed.
  • Next-generation Capture-C, ATAC-seq, RNA-seq/qPCR, and ChIP-seq for CTCF, H3K4me3, H3K27me3, Rad21, and Ezh2, plus CTCF motif and DNaseI footprint analysis.
  • Expression and Capture-C experiments used three biological replicates; ChIP experiments were performed at least in biological duplicate; differential analyses used DESeq2/DiffBind with FDR thresholds.

Limitations

  • Stated in the paper: no statistical method was used to predetermine sample size; Capture-C, gene expression, and ChIP-seq experiments were not randomised and investigators were not blinded; qPCR cannot detect fractional changes in α-globin expression; the molecular basis for differing CTCF-site activity is unknown; insulation may not be absolute, as human α-globin enhancers may influence NME4 ~400 kb away.
  • Evident from the text: the study is limited to one mouse locus and to erythroid and ES cell contexts, with no human or other tissue validation presented.

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

Integrity signals

Registry

Crossref · 10.1038/ncb3573

Cites

54 of 54 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 23 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    The α-globin genes and the closely linked widely expressed gene Nprl3 are expressed at high levels in erythroid cells.

    “The α-globin genes and the closely linked, widely expressed gene (Nprl3), which lie together within the H3K4me1-marked domain are expressed at high levels in erythroid cells (Fig 1C).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Mutation of the two CTCF core sequences resulted in a complete loss of CTCF binding at HS-38 and HS-39.

    “Mutation of the two CTCF core sequences resulted in a complete loss of CTCF binding at these sites (Figs 3, 4A, and 5A)”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The small deletions generated by TALEN and CRISPR mutagenesis specifically disrupt the intended CTCF binding sites without affecting other genomic elements or causing off-target effects.
  • Findingown data

    Deletion of HS-39 alone did not have a strong effect on local gene expression.

    “However, deletion of HS-39 alone did not have a strong effect on local gene expression (Fig. 5E).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The small deletions generated by TALEN and CRISPR mutagenesis specifically disrupt the intended CTCF binding sites without affecting other genomic elements or causing off-target effects.
  • Findingown data

    The HS-29 CTCF site binds lower levels of CTCF and Cohesin than HS-38.

    “Notably, the HS-29 CTCF site binds lower levels of CTCF and Cohesin than HS-38”
    Quote in the paperSubstantial claim
  • Findingown data

    Expression of Mpg, Rhbdf1 and Snrnp25 is strongly up-regulated in D3839 mutant mice.

    “We found that expression of the three genes whose promoters are located in the genomic region that shows increased interactions with the R1 enhancer (Fig 4B), Mpg, Rhbdf1 and Snrnp25, is strongly up-regulated in D3839 mutant mice (Figs 5A, B, and C).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The small deletions generated by TALEN and CRISPR mutagenesis specifically disrupt the intended CTCF binding sites without affecting other genomic elements or causing off-target effects.
  • May combine two claims. 19
  • Classified as probably not a substantial claim. 25

  • May combine two claims.

    Findingown data

    The flanking CTCF/cohesin sites interact with each other only during erythropoiesis, defining a self-interacting erythroid compartment.

    “only interact during erythropoiesis, defining a self-interacting erythroid compartment”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Changes in H3K4me3 in the D3839 mutant are accompanied by higher levels of RNA Polymerase II recruitment and increased chromatin accessibility at the gene promoters.

    “Changes in H3K4me3 in the D3839 mutant are accompanied by higher levels of RNA Polymerase II (PolII) recruitment and increased chromatin accessibility (ATAC-seq) at the gene promoters.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The small deletions generated by TALEN and CRISPR mutagenesis specifically disrupt the intended CTCF binding sites without affecting other genomic elements or causing off-target effects.

  • May combine two claims.

    Findingown data

    Housekeeping genes Mpg and Snrnp25 are normally expressed in wild-type erythroid cells but in the absence of HS-38 and HS-39 their expression increases by 12- and 6-fold, respectively.

    “Housekeeping genes Mpg and Snrnp25 are normally expressed in wild-type erythroid cells but in the absence of HS-38 and HS-39 their expression increases by 12- and 6-fold, respectively.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The small deletions generated by TALEN and CRISPR mutagenesis specifically disrupt the intended CTCF binding sites without affecting other genomic elements or causing off-target effects.

  • May combine two claims.

    Findingown data

    The expression of Mpg and Rhbdf1 lying outside the sub-TAD is unchanged and repressed in erythroid cells respectively.

    “whereas the expression of Mpg and Rhbdf1 lying outside the sub-TAD is unchanged and repressed in erythroid cells respectively (Fig. 1C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The Il9r gene, whose promoter is located within the chromatin region in which interactions between flanking CTCF/cohesin domains are retained in the D3839 mutant, remained inactive and insulated from the influence of the α-globin enhancers.

    “By contrast, the Il9r gene, whose promoter is located within the chromatin region in which interactions between flanking CTCF/cohesin domains are retained in the D3839 mutant (Fig. 4A), remained inactive (Fig. 5B) and insulated from the influence of the α-globin enhancers.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The small deletions generated by TALEN and CRISPR mutagenesis specifically disrupt the intended CTCF binding sites without affecting other genomic elements or causing off-target effects.

  • May combine two claims.

    Findingown data

    In ES cells, α-globin is not expressed whereas transcription of flanking genes (Snrnp25, Mpg, Rhbdf1 and Nprl3) occurs in the absence of erythroid-specific enhancer activity.

    “In ES cells, α-globin is not expressed whereas transcription of flanking genes (Snrnp25, Mpg, Rhbdf1 and Nprl3) occurs in the absence of the erythroid-specific enhancer activity (Figs 1B and C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Despite near-identical CTCF/Cohesin binding landscape across the α-globin sub-TAD in erythroid and non-erythroid cells, interactions between flanking CTCF/Cohesin clusters are significantly increased in erythroid cells.

    “despite the near-identical CTCF/Cohesin binding landscape across the α-globin sub-TAD in erythroid and non-erythroid cells (Fig. 1B), we observed significantly increased interactions between flanking CTCF/Cohesin clusters in erythroid cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Rhbdf1 is marked by high levels of H3K27me3 and completely silenced in erythroid cells.

    “one of these genes (Rhbdf1) is marked by high levels of the Polycomb-mediated repressive mark (H3K27me3) and completely silenced in erythroid cells (Figs 1B and C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    Only HS-38 is conserved across mammals including human and bound by higher levels of CTCF/cohesin.

    “only HS-38 is conserved across mammals including human and bound by higher levels of CTCF/cohesin”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Interaction profiles from the Rhbdf1 and Mpg promoters show a strong increase of interactions with the R1 and R2 enhancers and the α-globin genes, while losing interactions with the downstream genomic region flanking the cluster.

    “the interaction profiles obtained from the Rhbdf1 and Mpg promoters which show a strong increase of interactions with the R1 and R2 enhancers and the α-globin genes, while losing interactions with the downstream genomic region flanking the cluster (Fig. 4C).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The small deletions generated by TALEN and CRISPR mutagenesis specifically disrupt the intended CTCF binding sites without affecting other genomic elements or causing off-target effects.

  • May combine two claims.

    Findingown data

    The normally silent Rhbdf1 promoter acquired high levels of H3K4me3 in D3839 erythroid cells, consistent with its ectopic expression in these mutant cells.

    “it was of interest that the normally silent Rhbdf1 promoter acquired high levels of H3K4me3 in D3839 erythroid cells, consistent with its ectopic expression in these mutant cells (Figs 6A and B).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The small deletions generated by TALEN and CRISPR mutagenesis specifically disrupt the intended CTCF binding sites without affecting other genomic elements or causing off-target effects.

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

    Findingcitation

    Motifs up- and downstream of the CTCF core motif identified from 6000 randomly selected 20 bp flanking sequences were similar to those previously identified.

    “Motifs up- and downstream of the core motif were identified from 6000 randomly selected 20 bp sequences of up- and downstream flanks and were similar to those previously identified51.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [51] Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013). source

      Not checked in the free run.

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

    Findingown data

    The motif with the highest score in de novo motif discovery from ter119+ CTCF peaks matched the previously published consensus CTCF core binding motif.

    “The motif with the highest score matched the previously published consensus CTCF core binding motif.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Effective chromatin boundaries are formed by two directly adjacent divergent CTCF binding sites.

    “It has been suggested that effective chromatin boundaries are formed by two directly adjacent divergent CTCF binding sites30.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] Gómez-Marín, C. et al. Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders. Proc. Natl Acad. Sci. USA 112, 7542–7547 (2015). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S1 (asserted). May combine two claims.

    Interpretationreasoning

    These CTCF/cohesin sites may act individually or together as an insulator, shielding these upstream genes from enhancer activity.

    “suggesting that they may act individually or together as an insulator, shielding these upstream genes from enhancer activity.”
    Quote in the paperNot a substantial claim

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

    Study design and conductreasoning

    Minor changes in α-globin gene expression may not have been detected by qPCR, which cannot detect fractional changes in expression.

    “However, minor changes in α-globin gene expression may not have been detected by qPCR, which cannot detect fractional changes in expression.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    The mouse α-globin cluster is located in close proximity to a cluster of 10 widely expressed genes near the boundary of a ~1Mb TAD as defined by Dixon et al.

    “The mouse α-globin cluster is located in close proximity to a cluster of 10 widely expressed genes near the boundary of a ~1Mb TAD as defined by Dixon et al (Fig. 1A)12.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Dixon, J. R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    CTCF/Cohesin sites dispersed within TADs are thought to contribute to the formation of smaller self-interacting domains termed sub-TADs, contact domains, and insulated neighborhoods.

    “These CTCF/Cohesin sites are thought to contribute to the formation of smaller self-interacting domains that have been termed sub-TADs18, contact domains19, and insulated neighborhoods11,20.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [18] Phillips-Cremins, J. E. et al. Architectural protein subclasses shape 3D organization of genomes during lineage commitment. Cell 153, 1281–1295 (2013). source

      Not checked in the free run.

    • cites [19] Rao, S. S. P. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014). source

      Not checked in the free run.

    • cites [11] Dowen, J. M. et al. Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes. Cell 159, 374–387 (2014). source

      Not checked in the free run.

    • cites [20] Hnisz, D., Day, D. S. & Young, R. A. Insulated neighborhoods: structural and functional units of mammalian gene control. Cell 167, 1188–1200 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Enhancer/promoter interactions may be promiscuous rather than specific and such interactions are normally constrained, in some way, by CTCF/cohesin binding sites.

    “This suggests that enhancer/promoter interactions may be promiscuous rather than specific and that such interactions are normally constrained, in some way, by CTCF/cohesin binding sites.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Sub-TADs often appear in a cell specific manner and may be identified via an increased density of chromatin interactions and specific histone modifications in a specific cell type.

    “sub-TADs (40kb to 3Mb, median size of 185kb19) often appear in a cell specific manner and, as in the case of the 80kb sub-TAD corresponding to the H3K4me1 and histone acetylation-marked α-globin domain, may be identified via an increased density of chromatin interactions (as seen by Capture–C, Fig. 2A) and specific histone modifications in a specific cell type.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [19] Rao, S. S. P. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Recent studies have shown a strong preference for interactions to occur between CTCF sites lying in a convergent orientation with respect to each other.

    “recent studies have shown a strong preference for interactions to occur between CTCF sites lying in a convergent orientation with respect to each other21–24.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [21] Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015). source

      Not checked in the free run.

    • cites [22] Sanborn, A. L. et al. Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes. Proc. Natl Acad. Sci. USA 112, E6456–E6465 (2015). source

      Not checked in the free run.

    • cites [23] de Wit, E. et al. CTCF binding polarity determines chromatin looping. Mol. Cell 60, 676–684 (2015). source

      Not checked in the free run.

    • cites [24] Rudan, M. V. et al. Comparative Hi-C reveals that CTCF underlies evolution of chromosomal domain architecture. Cell Rep. 10, 1297–1309 (2015). source

      Not checked in the free run.

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

    Study design and conductown data

    The investigators were not blinded to allocation during Capture-C, gene expression and ChIP-seq experiments and outcome assessments.

    “Experiments for Capture-C, gene expression and ChIP-seq analysis were not randomised and the investigators were not blinded to allocation during these experiments and outcome assessments.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All Capture-C experiments were performed on three biological replicates with similar results.

    “All Capture-C experiments were performed on three biological replicates with similar results.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Differentially enriched regions between wild-type and CTCF binding site mutant mice were identified using two biological duplicate datasets and independent peak calls with a false discovery rate of 0.05.

    “Two biological duplicate datasets and independent peak calls of CTCF, H3K4me3 and H3K27me3 were used to identify differentially enriched regions with a false discovery rate (FDR) of 0.05.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Purity of the isolated erythroid cells was routinely verified by FACS.

    “Purity of the isolated erythroid cells was routinely verified by FACS.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The standard deviation of 250bp bins was calculated in R and visualised to illustrate the reproducibility of the chromatin interaction analysis.

    “The standard deviation of 250bp bins was calculated in R and visualised to illustrate the reproducibility of this chromatin interaction analysis.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Mice were given neutral identifiers and analysed by research technicians unaware of mouse genotype during outcome assessment.

    “Mice were given neutral identifiers and analysed by research technicians unaware of mouse genotype during outcome assessment.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The E14TG2a cell line has been extensively authenticated by blastocyst injection.

    “The E14TG2a line was a kind gift from Andrew Smith and was tested negative for mycoplasma and has been extensively authenticated by blastocyst injection.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The E14TG2a cell line was tested negative for mycoplasma.

    “The E14TG2a line was a kind gift from Andrew Smith and was tested negative for mycoplasma and has been extensively authenticated by blastocyst injection.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Upon activation of the α-globin enhancers, selective convergent CTCF/Cohesin binding sites act as boundary elements and create an erythroid-specific chromatin structure, delimiting enhancer interactions and consequently ensuring an erythroid-specific transcriptional program.

    “The above results indicate that upon activation of the α-globin enhancers, selective convergent CTCF/Cohesin binding sites act as boundary elements and create an erythroid-specific chromatin structure, delimiting enhancer interactions and consequently ensuring an erythroid-specific transcriptional program (Fig. 6E).”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    All gene expression experiments (both RT-qPCR and RNA-seq) were performed on three biological replicates with similar results.

    “All gene expression experiments (both RT-qPCR and RNA-seq) were performed on three biological replicates with similar results (standard deviation (SD) is shown for all measurements).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Genetic perturbation of CTCF binding has been shown to result in misregulation of gene expression in various cell lines and cancer.

    “While genetic perturbation of CTCF binding has been shown to result in misregulation of gene expression in various cell lines and cancer9–11,28”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [9] Flavahan, W. A. et al. Insulator dysfunction and oncogene activation in IDH mutant gliomas. Nature 529, 110–114 (2016). source

      Not checked in the free run.

    • cites [10] Hnisz, D. et al. Activation of proto-oncogenes by disruption of chromosome neighborhoods. Science 351, 1454–1458 (2016). source

      Not checked in the free run.

    • cites [11] Dowen, J. M. et al. Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes. Cell 159, 374–387 (2014). source

      Not checked in the free run.

    • cites [28] Narendra, V. et al. CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation. Science 347, 1017–1021 (2015). source

      Not checked in the free run.

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

    Study design and conductnone given

    The E14TG2a cell line is not found in the database of commonly misidentified cell lines maintained by ICLAC and NCBI Biosample.

    “This cell line is not found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Experiments to determine haematological parameters were blinded.

    “Experiments to determine haematological parameters were blinded.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    No samples or animals were excluded from the analysis.

    “No samples or animals were excluded from the analysis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Not all CTCF/Cohesin sites subserve the same functions in the sub-TAD: HS-38 acts as a strong boundary element, HS-39 as a weaker element and HS-29 has no apparent insulator activity.

    “Importantly, we have shown that not all CTCF/Cohesin sites subserve the same functions in the sub-TAD: HS-38 acts as a strong boundary element, HS-39 as a weaker element and HS-29 has no apparent insulator activity.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    No statistical method was used to predetermine sample size.

    “No statistical method was used to predetermine sample size.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    More widespread and bidirectional enhancer interactions appeared and were associated with the up-regulation of three genes; in one case (Rhbdf1) overcoming Polycomb-mediated repression.

    “More widespread and bidirectional enhancer interactions appeared and were associated with the up-regulation of three genes; in one case (Rhbdf1) overcoming Polycomb-mediated repression.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Statistical analysis was performed using multiple unpaired, two-tailed t-tests and corrected for multiple comparisons using the Holm-Sidak method where appropriate.

    “Statistical analysis was performed using multiple unpaired, two-tailed t-tests and corrected for multiple comparisons using the Holm-Sidak method where appropriate.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All ChIP experiments newly generated for this study were performed at least in biological duplicate with similar results.

    “All ChIP experiments newly generated for this study were performed at least in biological duplicate with similar results.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Experiments for Capture-C, gene expression and ChIP-seq analysis were not randomised.

    “Experiments for Capture-C, gene expression and ChIP-seq analysis were not randomised and the investigators were not blinded to allocation during these experiments and outcome assessments.”
    Quote in the paperNot a substantial claim
41 of 41

  • The paper relies on this without support.

    The α-globin regulatory elements normally act solely on promoters downstream of the enhancers.

    “Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers”

  • The paper relies on this without support.

    The small deletions generated by TALEN and CRISPR mutagenesis specifically disrupt the intended CTCF binding sites without affecting other genomic elements or causing off-target effects.

    “we used TALEN26 and CRISPR-mediated mutagenesis27 to generate mice with small deletions in the binding sequences of these three CTCF/cohesin sites, singly and in combination”

  • The paper relies on this without support.

    The observed changes in gene expression in D3839 are caused by the altered enhancer-promoter interactions rather than by other consequences of CTCF site deletion.

    “Thus, the elimination of CTCF binding in the D3839 mutant is associated with an entirely new set of contacts between the α-globin enhancers and the Rhbdf1 and Mpg genes.”
3 of 3

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?

  • Changes in CTCF binding profiles seem to play an important role in the reconfiguration of the 3D genome during cell differentiation.

    “Changes in CTCF binding profiles seem to play an important role in the reconfiguration of the 3D genome during cell differentiation [95,96].”

    From Enhancer Function in the 3D Genome (2023) · cites this paper as [96]

    The Discussion (page unavailable in our copy) says CTCF/cohesin sites help create an erythroid-specific chromatin structure, supporting a role for CTCF in cell-type-specific 3D architecture. But it also says the relevant CTCF and cohesin binding is present in embryonic stem cells even though interactions are weaker or absent there. The paper therefore does not establish that changes in CTCF binding profiles drive reconfiguration during differentiation. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Incorporating specific chromatin modifications and boundary elements at distant regulatory sites has created a more complete picture of the underlying regulation at the α-globin locus.

    “Incorporating specific chromatin modifications and boundary elements at these distant regulatory sites has created a more complete picture of the underlying regulation at this locus (Brown et al., 2018; Hanssen et al., 2017; Oudelaar et al., 2017).”

    From Unraveling Hematopoiesis through the Lens of Genomics (2020) · cites this paper as Hanssen et al., 2017

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Authors
Hanssen, L. L. P., Kassouf, M. T., Oudelaar, A. M., Biggs, D., Preece, C., Downes, D. J., Gosden, M., Sharpe, J. A., Sloane-Stanley, J. A., Hughes, J. R., Davies, B., & Higgs, D. R.
Affiliations
  • Centre for Human Genetics (GB)
  • MRC Weatherall Institute of Molecular Medicine (GB)
Corresponding author
Higgs, Douglas R.
ORCID
Venue
Nature Cell Biology
Publisher
Springer Science and Business Media LLC
Published
2017-07-24
Volume
19
Issue
8
Pages
952-961
ISSN
  • 1465-7392 (ISSN-L)
  • 1476-4679
DOI
10.1038/ncb3573
PMC
PMC5540176
OpenAlex
W2736857446
Semantic Scholar
904bc3df54f089e1833cb8c233ebc13e7fd2a2b9
Cited by
219
References
56
Citations by year
2026: 15 · 2025: 11 · 2024: 21 · 2023: 17 · 2022: 26 · 2021: 35 · 2020: 37 · 2019: 18 · 2018: 32 · 2017: 6
FWCI
10.61
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Cell Biology (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
Funders
  • Wellcome Trust (203141/Z/16/Z, 083323/Z/07/Z, 203141, 099684/Z/12/Z)
  • Medical Research Council (MC_UU_00016/4, MC_UU_00016/1, MR/N00969X/1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Chromosomal and Genetic Variations
Keywords
topologically associating domains, chromatin structure, enhancer-promoter interactions, alpha-globin gene cluster, erythropoiesis, 3D genome organization, chromatin looping, enhancer accessibility
MeSH terms
CCCTC-Binding Factor, Cohesins, Animals, Binding Sites, Blood Group Antigens, Cell Line, Chromatin, Chromosomal Proteins, Non-Histone, Enhancer Elements, Genetic, Female, Multigene Family, Genotype, Hematopoietic Stem Cells, Male, Mice, Inbred C57BL, Mutation, Phenotype, Promoter Regions, Genetic, Protein Binding, Repressor Proteins, Transfection, Gene Expression Regulation, Developmental, Cell Cycle Proteins, Erythroid Cells, Chromatin Assembly and Disassembly, Embryonic Stem Cells, alpha-Globins

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 2026-10-10.

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

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.

What counts as a claim ›How we measure confidence ›How we check this ›

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