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Cited by 420Genomics and Chromatin DynamicsChromatin Remodeling and CancerEpigenetics and DNA Methylation

Insulated Neighborhoods: Structural and Functional Units of Mammalian Gene Control

Open access
Hnisz, Denes, Day, Daniel S., Young, Richard A.·2016·Cell
DOI 10.1016/j.cell.2016.10.024PMC PMC5125522
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Abstract

Understanding how transcriptional enhancers control over 20,000 protein-coding genes to maintain cell-type-specific gene expression programs in all human cells is a fundamental challenge in regulatory biology. Recent studies suggest that gene regulatory elements and their target genes generally occur within insulated neighborhoods, which are chromosomal loop structures formed by the interaction of two DNA sites bound by the CTCF protein and occupied by the cohesin complex. Here, we review evidence that insulated neighborhoods provide for specific enhancer-gene interactions, are essential for both normal gene activation and repression, form a chromosome scaffold that is largely preserved throughout development, and are perturbed by genetic and epigenetic factors in disease. Insulated neighborhoods are a powerful paradigm for gene control that provides new insights into development and disease.

AI snapshot

Written from the paper’s full text.

TL;DR

This review argues that insulated neighborhoods—CTCF-CTCF loops co-bound by cohesin that contain at least one gene—are structural and functional units of mammalian gene control. It synthesizes evidence that these loops constrain enhancer-gene interactions, are largely preserved across cell types, and are disrupted in disease.

Key findings

  • In human ESCs, there are ~13,000 insulated neighborhoods, ranging from 25 kb to 940 kb and containing 1–10 genes; the median neighborhood is ~190 kb and contains 3 genes.
  • Most enhancer/promoter DNA loops occur within insulated neighborhood boundaries: ~97% in human ESCs and ~90% in T cell leukemia cells; 59% of human ESC neighborhoods have an insulation score of 100%.
  • Insulated neighborhoods are proposed as the mechanistic basis of TADs: at least 50% of TADs have TAD-spanning CTCF-CTCF loops, and 70% of loop domains and 54% of CTCF contact domains share boundaries with an insulated neighborhood in human lymphoblastoid cells.
  • Neighborhood boundaries are largely shared across cell types (~80% shared by any two cell types), while enhancer-gene interactions within them are cell-type specific; CTCF sites at neighborhood boundaries are evolutionarily conserved, and ~55% of CTCF binding sites do not appear to participate in neighborhood loops.

Methods

  • Narrative review synthesizing published studies on chromosome conformation, CTCF/cohesin binding, enhancer-promoter interactions, and disease-associated non-coding variation.
  • Evidence discussed includes Hi-C, ChIA-PET, 5C, CRISPR/Cas9 perturbation, DNA methylation editing, and cancer genomics studies; no new experimental data are reported.
  • Main analysis is conceptual synthesis, proposing insulated neighborhoods as a unifying model for enhancer-gene specificity, TAD structure, and gene dysregulation in disease.

Limitations

  • The paper states that loop dynamics and cell-to-cell heterogeneity are not understood; current assays generally study cell populations and are inadequate for single-cell dynamics.
  • The paper states that DNA interaction data are noisy and sparse, making it difficult to estimate how extensively neighborhoods are shared across cell types; mechanisms regulating loop anchors, how a loop insulates, and whether anchors vary in insulation strength remain unclear.
  • Evident from the text: as a review, it presents no primary data and does not describe a systematic search or meta-analysis, so its conclusions depend on the cited studies and their experimental/analytical assumptions.

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

Integrity signals

Registry

Crossref · 10.1016/j.cell.2016.10.024

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 155 cited sources checked · 0 supported · 0 flagged

  • Findingnone given

    In neighborhoods with two genes, the activity of the two is coherent in 60% (both are active or both are silent).

    “We estimate that in neighborhoods with two genes, the activity of the two is coherent in 60% (both are active or both are silent).”
    Quote in the paperSubstantial claim
  • Premisecitation

    Each chromosome contains thousands of DNA loops, formed by the interaction of two CTCF molecules bound to different sites, and reinforced by a cohesin molecule.

    “In the models that emerge from these data, each chromosome contains thousands of DNA loops, formed by the interaction of two CTCF molecules bound to different sites, and reinforced by a cohesin molecule (Figure 2A).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Chepelev et al., 2012 source

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  • Premisecitation

    TADs have similar boundaries in all human cell types examined.

    “These TADs, which have similar boundaries in all human cell types examined, have been proposed to constrain enhancer-gene interactions because most DNA contacts occur within the TADs (Dixon et al., 2015; Dixon et al., 2012).”
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  • Premisecitation

    Insulators are regulatory elements that can block the ability of an enhancer to activate a gene when located between them.

    “Another potential solution to the enhancer-gene specificity conundrum lies in insulators, which are regulatory elements that can block the ability of an enhancer to activate a gene when located between them (Chung et al., 1993; Geyer and Corces, 1992; Kellum and Schedl, 1991; Udvardy et al., 1985).”
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    • cites Geyer and Corces, 1992

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    • cites Kellum and Schedl, 1991

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  • Premisecitation

    Gene regulatory elements and their target genes generally occur within insulated neighborhoods, which are chromosomal loop structures formed by the interaction of two DNA sites bound by CTCF and occupied by the cohesin complex.

    “Recent studies suggest that gene regulatory elements and their target genes generally occur within insulated neighborhoods, which are chromosomal loop structures formed by the interaction of two DNA sites bound by the CTCF protein and occupied by the cohesin complex.”
    • Also stated in: § Preamble · plain
      “A core principle that has emerged from these studies is that genes and their regulatory elements typically occur together within specific DNA loop structures, which we have called “insulated neighborhoods”.”
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    Premisecitation

    In human embryonic stem cells, there are ~13,000 insulated neighborhoods, which range from 25 kb to 940 kb in size, and contain from 1-10 genes.

    “In human embryonic stem cells (ESCs), there are ~13,000 insulated neighborhoods, which range from 25 kb to 940 kb in size, and contain from 1 – 10 genes (Figure 2B) (Dowen et al., 2014; Ji et al., 2016).”
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    Premisecitation

    The majority of disease-associated non-coding variation occurs in the vicinity of enhancers and thus likely impacts these enhancers' target genes.

    “This mystery, which we will call the enhancer-gene specificity conundrum, is important to solve because the majority of disease-associated non-coding variation occurs in the vicinity of enhancers and thus likely impacts these enhancers’ target genes (Ernst et al., 2011; Farh et al., 2015; Hnisz et al., 2013; Maurano et al., 2012).”
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    Premisecitation

    Human germline variation is rare in CTCF binding sites at insulated neighborhood boundaries and few GWAS variants occur in these sites.

    “Human germline variation is rare in CTCF binding sites at insulated neighborhood boundaries and few GWAS variants occur in these sites (Ji et al., 2016).”
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    • cites Ji et al., 2016

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    Premisecitation

    Deletion of a CTCF loop anchor site of the miR-290-295 neighborhood reduced expression of the miRNA precursor and activated an adjacent gene outside the neighborhood, concomitant with looping of the super-enhancer to this outside gene.

    “For example, the miR-290–295 miRNA gene cluster, which plays important roles in ES cell pluripotency, occurs within an insulated neighborhood together with a super-enhancer; when a CTCF loop anchor site of this neighborhood was deleted, there was a reduction in expression of the miRNA precursor and activation of an adjacent gene outside of the neighborhood concomitant with looping of the super-enh”
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    Premisecitation

    At least 50% of TADs have TAD-spanning CTCF-CTCF loops and are thus insulated neighborhoods.

    “ChIA-PET data revealed that at least 50% of TADs have TAD-spanning CTCF-CTCF loops (Ji et al., 2016) and are thus insulated neighborhoods.”
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    Premisecitation

    CTCF-CTCF looping has been implicated in bringing DNA segments into spatial proximity and in constraining the off-target effects of the recombinase.

    “CTCF-CTCF looping has been implicated in bringing these segments into spatial proximity and also in constraining the off-target effects of the recombinase (Dong et al., 2015; Hu et al., 2015).”
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    Premisecitation

    The median insulated neighborhood is ~190 kb and contains 3 genes.

    “The median insulated neighborhood is ~190kb and contains 3 genes.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Dowen et al., 2014 source

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    • cites Ji et al., 2016

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    Premisecitation

    Some TADs appear to be a single insulated neighborhood, while others consist of multiple nested or multiple independent insulated neighborhoods.

    “Some TADs appear to be a single insulated neighborhood, while others consist of multiple nested or multiple independent insulated neighborhoods (Figure 4B–D).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Ji et al., 2016

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    Premisecitation

    Different cell types share very similar insulated neighborhood boundaries, but the enhancer-gene interactions that occur within these neighborhoods are cell-type specific because enhancer activity is cell-type specific.

    “While different cell types share very similar insulated neighborhood boundaries, the enhancer-gene interactions that occur within these neighborhoods are cell-type specific because enhancer activity is cell-type specific (Figure 3) (Ji et al., 2016; Smith et al., 2016).”
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    Premisecitation

    Silent proto-oncogenes typically occur within insulated neighborhoods and genetic modification of the neighborhood loop anchors can cause activation of these oncogenes.

    “Silent proto-oncogenes typically occur within insulated neighborhoods and genetic modification of the neighborhood loop anchors can cause activation of these oncogenes (Flavahan et al., 2016; Hnisz et al., 2016).”
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    • cites Flavahan et al., 2016 source

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    Interpretationcitation

    CTCF contact domains are either insulated neighborhoods or clusters of insulated neighborhoods.

    “These features suggest that CTCF contact domains are either insulated neighborhoods or clusters of insulated neighborhoods.”
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    Premisecitation

    The anchor sites of CTCF contact domains are bound by CTCF and cohesin, and the CTCF anchors of ~90% of the CTCF contact domains occur in a convergent orientation.

    “The anchor sites of CTCF contact domains are bound by CTCF and cohesin, and the CTCF anchors of ~90% of the CTCF contact domains occur in a convergent orientation (Tang et al., 2015).”
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    Premisecitation

    70% of loop domains, and 54% CTCF contact domains have the same boundaries as an insulated neighborhood in human lymphoblastoid cells.

    “For example, 70% of loop domains, and 54% CTCF contact domains have the same boundaries as an insulated neighborhood in human lymphoblastoid cells (Rao et al., 2014; Tang et al., 2015).”
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    Premisecitation

    In vertebrates, the beta globin locus contains a cluster of fetal and adult globin genes, and the developmental control of these genes is exerted by an upstream regulatory element called the locus control region (LCR).

    “In vertebrates, the beta globin locus contains a cluster of fetal and adult globin genes, and the developmental control of these genes is exerted by an upstream regulatory element called the locus control region (LCR) (Figure 6D).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
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    Premisecitation

    ~5,000 chromatin loops whose boundaries are occupied by CTCF and cohesin in multiple human cell types were identified and termed loop domains.

    “Another study used high-resolution hi-C technology to identify ~5,000 chromatin loops whose boundaries are occupied by CTCF and cohesin in multiple human cell types; these were termed “loop domains” (Rao et al., 2014).”
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    Premisecitation

    Hereditary mutations that invert or delete a TAD boundary at the EPHA4 locus have been linked to limb malformations in humans, and this TAD has a TAD-spanning CTCF-CTCF loop in ES cells, indicating that it is an insulated neighborhood.

    “Hereditary mutations that invert or delete a TAD boundary at the EPHA4 locus have recently been linked to limb malformations in humans (Lupianez et al., 2015), and this TAD has a TAD-spanning CTCF-CTCF loop in ES cells, indicating that it is an insulated neighborhood.”
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    Premisecitation

    In fetal brain cells, CTCF binding to one of the beta-globin loop anchor regions is absent and CTCF-CTCF loop formation is not detected.

    “In fetal brain cells, CTCF binding to one of the beta-globin loop anchor regions is absent and CTCF-CTCF loop formation is not detected (Splinter et al., 2006; Tolhuis et al., 2002), which suggests that tissue-specific CTCF-CTCF loops participate in developmental gene control.”
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    Premisecitation

    CTCF sites located within a Hox gene cluster play a critical role in proper expression of Hox genes; some of these critical CTCF sites form insulated neighborhood anchors in ES cells.

    “Positional information in the developing embryo depends on the precise expression of Homeobox (Hox) genes, and CTCF sites located within a Hox gene cluster play a critical role in proper expression of Hox genes (Narendra et al., 2015); some of these critical CTCF sites form insulated neighborhood anchors in ES cells.”
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    • cites Narendra et al., 2015 source

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    Premisecitation

    The CTCF DNA binding motif is asymmetric and directional, and the CTCF anchors of >90% of loop domains occur in a convergent orientation.

    “This and other studies have noted that the DNA binding motif of CTCF is asymmetric and thus directional, and the CTCF anchors of >90% the loop domains occur in a convergent orientation (de Wit et al., 2015; Gomez-Marin et al., 2015; Guo et al., 2015; Ji et al., 2016; Vietri Rudan et al., 2015).”
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    Premisecitation

    Paternal allele-specific DNA methylation of a CTCF site in the H19 promoter region abrogates CTCF binding, causing differential CTCF-CTCF loop formation while silencing H19 expression.

    “Paternal allele-specific DNA methylation of a CTCF site in the H19 promoter region abrogates CTCF binding, thus causing differential CTCF-CTCF loop formation while silencing H19 expression (Bell and Felsenfeld, 2000; Hark et al., 2000; Kanduri et al., 2000; Szabo et al., 2000).”
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    Premisecitation

    DNA hypermethylation occurs in some cancer cells, and tumor-specific DNA methylation has been implicated in the disruption of CTCF binding, alteration of chromosome structure and dysregulation of oncogene expression.

    “DNA hypermethylation occurs in some cancer cells, and tumor-specific DNA methylation has recently been implicated in the disruption of CTCF binding, alteration of chromosome structure and dysregulation of oncogene expression (Flavahan et al., 2016).”
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    Premisecitation

    Individuals who lose these allele-specific insulated neighborhoods develop Beckwith-Wiedemann syndrome or Silver-Russell syndrome.

    “Individuals who lose these allele-specific insulated neighborhoods develop Beckwith-Wiedemann syndrome (when both alleles have the paternal type of insulated neighborhood; Figure 6B) or Silver-Russell syndrome (when both alleles have the maternal type of insulated neighborhood; Figure 6C) (Nativio et al., 2011).”
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    Premisecitation

    Sub-TADs were found to be constitutively present in multiple cell types and cell type-specific enhancer-promoter contacts occurred within sub-TAD boundaries.

    “Sub-TADs were found to be constitutively present in multiple cell types and cell type-specific enhancer-promoter contacts occurred within sub-TAD boundaries (Phillips-Cremins et al., 2013).”
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    Premisecitation

    CTCF binding and CTCF-CTCF loop formation can be cell type-specific.

    “there is some evidence that CTCF binding and CTCF-CTCF loop formation can be cell type-specific (Narendra et al., 2015; Splinter et al., 2006; Tolhuis et al., 2002; Wang et al., 2012).”
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    Premisecitation

    A genetic variant associated with obesity and previously assigned to the FTO gene has no impact on FTO, but affects the IRX3 and IRX5 genes.

    “For example, a recent study revealed that a genetic variant associated with obesity and previously assigned to the FTO gene in fact has no impact on FTO, but affects the IRX3 and IRX5 genes (Claussnitzer et al., 2016).”
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    Premisecitation

    Condensin is known to inhibit transvection in Drosophila polytene chromosomes, where it presumably prevents interactions between two alleles.

    “Condensin is known to inhibit transvection in Drosophila polytene chromosomes, where it presumably prevents interactions between two alleles (Hartl et al., 2008).”
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    Premiseown data

    Examination of insulated neighborhood structures reveals that CAMK1D is located in the same neighborhood as the Type 2 diabetes variant, whereas CDC123 is not.

    “Examination of insulated neighborhood structures reveals that CAMK1D is located in the same neighborhood as the variant, whereas CDC123 is not (Figure 7C).”
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    Premisecitation

    The obesity-associated variant is located in an intronic enhancer within FTO, and both IRX3 and IRX5 are located in the same insulated neighborhood as the variant.

    “Although the variant is located in an intronic enhancer within FTO, both IRX3 and IRX5 are located in the same insulated neighborhood as the variant (Figure 7B).”
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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Additional structuring of the neighborhood, such as condensing the looped chromatin into a compact ball, would reduce the opportunity to interact with other neighborhoods.

    “Additional structuring of the neighborhood, such as condensing the looped chromatin into a compact ball, would reduce the opportunity to interact with other neighborhoods.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: An element in a 2-D chromatin loop should be able to contact elements in other loops.

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

    Premisecitation

    In Drosophila, a number of proteins have been identified that influence CTCF binding and insulator function.

    “In Drosophila, a number of proteins have been identified that influence CTCF binding and insulator function (Phillips-Cremins and Corces, 2013)”
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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Differences in insulation strength at CTCF anchors might occur and perhaps correlate with certain genomic features.

    “Early efforts to quantify insulation on a genome-wide scale suggest that differences in insulation strength at CTCF anchors might occur, and perhaps correlate with certain genomic features (Phillips-Cremins and Corces, 2013).”
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    Premisecitation

    Tens of thousands of non-coding genetic variants have been linked with various human diseases and traits in genome-wide association studies (GWAS), and the majority of these variants occur in enhancers.

    “Tens of thousands of non-coding genetic variants have been linked with various human diseases and traits in genome-wide association studies (GWAS), and the majority of these variants occur in enhancers (Ernst et al., 2011; Farh et al., 2015; Hnisz et al., 2013; Maurano et al., 2012).”
    Quote in the paperSubstantial claim0 of 3 sources checked
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    Interpretationreasoning

    Features such as multiple CTCF binding sites and nesting appear to produce a higher insulation score for enhancers and genes within the neighborhood and may represent a safeguard against perturbation.

    “These features appear to produce a higher “insulation score” for enhancers and genes within the neighborhood and may thus represent a safeguard against perturbation.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Insulated neighborhoods can have boundaries with multiple CTCF binding sites and can be nested within larger insulated neighborhoods.

  • May combine two claims.

    Study design and conductreasoning

    The experimental approaches used thus far to identify CTCF loops generally depend on the study of populations of cells, and thus the present data are inadequate to address questions of dynamics.

    “The experimental approaches used thus far to identify CTCF loops generally depend on the study of populations of cells, and thus the present data are inadequate to address questions of dynamics.”
    Quote in the paperNot a substantial claim
34 of 34

  • The paper relies on this without support.

    The existing ChIA-PET data is not saturating.

    “Because the existing ChIA-PET data is not saturating, this is a minimal estimate; it is possible that the majority of TADs have TAD-spanning CTCF-CTCF loops.”

  • The paper relies on this without support.

    Proximity-based assignment of target genes to non-coding genetic variants has proven, in some cases, to be inaccurate.

    “The identification of the target genes of these variants is challenging because proximity-based assignment has proven, in some cases, to be inaccurate.”

  • The paper relies on this without support.

    Enhancers and their associated factors can regulate expression of genes located far upstream or downstream by looping to the promoters of these genes.

    “Enhancers and their associated factors can regulate expression of genes located far upstream or downstream by looping to the promoters of these genes, so the features that cause enhancers to regulate only specific genes, generally on their own chromosomes, has been something of a mystery for several decades (Figure 1A).”

  • The paper relies on this without support.

    Insulated neighborhoods can have boundaries with multiple CTCF binding sites and can be nested within larger insulated neighborhoods.

    “Insulated neighborhoods can have boundaries with multiple CTCF binding sites and can be nested within larger insulated neighborhoods.”

  • The paper relies on this without support.

    An element in a 2-D chromatin loop should be able to contact elements in other loops.

    “An element in a 2-D chromatin loop should be able to contact elements in other loops.”
5 of 5

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?

  • Enhancers are punctate relative to broader chromatin domains, but their in vivo functionality depends on chromatin context and the trans milieu.

    “Enhancers are ‘punctate’ relative to broader chromatin domains (e.g. chromosomes, topologically associating domains [G] (TADs) and sub-compartments of TADs87), but their in vivo functionality is dependent on both the chromatin context in which they reside88 and on the trans milieu (e.g. cell-type-specific TFs)89.”

    From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [87]

  • 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.”

    From Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo (2017) · cites this paper as [20]

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References 107 · Cited by 420

Authors
Hnisz, D., Day, D. S., & Young, R. A.
Affiliations
Whitehead Institute for Biomedical Research (US)
Corresponding author
  • Hnisz, Denes
  • Day, Daniel S.
  • Young, Richard A.
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
2016-11
Volume
167
Issue
5
Pages
1188-1200
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/j.cell.2016.10.024
PMC
PMC5125522
OpenAlex
W2556041461
Cited by
420
References
107
Citations by year
2026: 14 · 2025: 20 · 2024: 32 · 2023: 38 · 2022: 37 · 2021: 61 · 2020: 62 · 2019: 44 · 2018: 56 · 2017: 53 · 2016: 1 · 2015: 1
FWCI
4.65
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-nd)
  • PMC
Funders
National Human Genome Research Institute (R01HG002668)
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 › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
Keywords
CTCF, cohesin, chromatin looping, enhancers, enhancer-gene interactions, cell type-specific gene expression, gene expression regulation, epigenetic regulation, chromatin structure, developmental gene regulation, disease
MeSH terms
CCCTC-Binding Factor, Animals, Chromosomes, Enhancer Elements, Genetic, Gene Expression Regulation, Humans, Mammals, Repressor Proteins, Insulator Elements

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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