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Cited by 196Genomics and Chromatin DynamicsChromatin Remodeling and CancerEpigenetics and DNA Methylation

Super-enhancers include classical enhancers and facilitators to fully activate gene expression

Open access
Blayney, Joseph W., Francis, Helena, Rampasekova, Alexandra, Camellato, Brendan, Mitchell, Leslie, Stolper, Rosa, Cornell, Lucy, Babbs, Christian +3 more·2023·Cell
DOI 10.1016/j.cell.2023.11.030PMC PMC10858684
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Abstract

Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.

AI snapshot

Written from the paper’s full text.

TL;DR

The mouse α-globin super-enhancer contains functionally distinct element types: classical enhancers R1 and R2, and facilitator elements R3, Rm, and R4 that lack intrinsic enhancer activity but are required for full target gene activation. Facilitators potentiate classical enhancer function, including co-activator recruitment, eRNA transcription, and enhancer-promoter interactions, with their potency depending more on position than sequence.

Key findings

  • The α-SE comprises two classical enhancers, R1 and R2, and three facilitators, R3, Rm, and R4. Facilitators have little or no intrinsic enhancer activity, but without them classical enhancers cannot fully upregulate α-globin.
  • In R2-only models, α-globin expression was only 10% in EB-derived erythroid cells versus a predicted 50%, and 15% in E12.5 fetal liver erythroid cells versus WT. R2-only mice were largely non-viable; only one homozygote survived, with anemia and severe splenomegaly, dying at 7 weeks.
  • R2 retained enhancer identity in R2-only cells—open chromatin and erythroid TF occupancy—but showed severely reduced Med1 and Brd4 recruitment, reduced Pol II at α-globin promoters, ~3-fold reduced R2 eRNA transcription, and reduced R2–promoter interaction frequency.
  • Rebuilding the SE showed R1 and R2 cooperate synergistically: each upregulated expression 100-fold alone versus 450-fold when combined. Reinserting Rm or R4 into R1R2-only increased expression by 50% and 80%, respectively, while R3 rescued only ~10% (not statistically significant). R4’s rescue was position-dependent: R2R4[R1] expressed only 12%, whereas R1R2R3[R4] increased expression by >85% compared with ~12% for R3 in its native position. The β-globin LCR HS1 element in the R4 position increased α-globin expression by 36%.

Methods

  • The authors synthesized 86 kb wild-type and variant α-globin alleles by de novo assembly, integrated them into mESCs by recombinase-mediated genomic replacement, and used CRISPR-Cas9 to generate hemizygous WT, Δα-SE, R2-only, R1-only, R1R2-only, and facilitator titration models. An R2-only mouse model was also generated.
  • Erythroid cells were produced by embryoid body-based in vitro differentiation of mESCs and erythroid purification; mouse analyses used embryonic days E9.5–E17.5 and E12.5 fetal liver erythroid cells. Assays included RT-qPCR, poly-A minus RNA-seq, ATAC-seq, ChIPmentation for histones, TFs, Med1, Brd4, TBP, and Pol II, tiled-C, FACS, and motif analysis.
  • Expression analysis used one-way ANOVA with Tukey multiple comparisons; at least three separately targeted clones were generated per mESC model, with PCR/Sanger verification and in silico screening for unintended TF motifs or accessibility changes.

Limitations

  • Stated by the paper: whether facilitators are common in multipartite enhancer clusters remains to be tested; genome-wide identification is limited by the lack of a distinguishing signature and the extensive genetic engineering required; the mechanism by which facilitators potentiate classical enhancers was not formally addressed.
  • Evident from the text: the functional dissection is centered on one locus, the mouse α-globin SE, and many rebuilt models rely on the in vitro EB-derived erythroid system rather than in vivo mice; some supporting comparisons use reanalysis of previously published datasets.

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

Integrity signals

Registry

Crossref · 10.1016/j.cell.2023.11.030

Cites

86 of 86 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

42 of 44 cited sources checked · 25 supported · 2 flagged · accuracy not yet measured

  • Premisecitation

    HS1 has no intrinsic enhancer activity in embryonic, fetal, or adult erythropoiesis.

    “when tested individually, HS1 has no intrinsic enhancer activity in embryonic, fetal, or adult erythropoiesis;45”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [45] Fraser P, Pruzina S, Antoniou M, and Grosveld F (1993). Each hypersensitive site of the human beta-globin locus control region confers a different developmental pattern of expression on the globin genes. Genes Dev. 7, 106– 113. 8422981 source

      The HS1 transgenic-line results (Fig. 3; page unavailable in our copy) report expression above the comparison lines in the embryonic yolk sac, fetal liver, and adult blood. The paper concludes that HS1 has very weak activity, not no activity; the Abstract likewise describes its activity as very low. (LLM, conf=0.95)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation

    Deletion of HS1 from the SE results in substantial sensitivity to position effects in transgenic mice.

    “deletion of HS1 from the SE results in substantial sensitivity to position effects in transgenic mice;46”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [46] Milot E, Strouboulis J, Trimborn T, Wijgerde M, de Boer E, Langeveld A, Tan-Un K, Vergeer W, Yannoutsos N, Grosveld F, and Fraser P (1996). Heterochromatin Effects on the Frequency and Duration of LCR-Mediated Gene Transcription. Cell 87, 105– 114. 8858153 source
  • Premisecitation

    Previous studies deleting the entire β-globin super-enhancer showed that the β-globin genes remain accessible and marked by histone acetylation.

    “previous studies deleting the entire β-globin SE showed that the β-globin genes remain accessible and marked by histone acetylation;55–57”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [55] Bender MA, Bulger M, Close J, and Groudine M (2000). β-globin Gene Switching and DNase I Sensitivity of the Endogenous β-globin Locus in Mice Do Not Require the Locus Control Region. Mol. Cell 5, 387– 393. 10882079 source

      The Discussion reports that mice lacking the β-globin LCR (5′HS 1–6) retain a DNase I–sensitive, open β-globin locus, supporting the accessibility part of the claim. Our copy does not report that the β-globin genes remain marked by histone acetylation; its Discussion mentions histone acetylation only in relation to a different locus, IgH/c-myc. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [56] Epner E, Reik A, Cimbora D, Telling A, Bender MA, Fiering S, Enver T, Martin DI, Kennedy M, Keller G, and Groudine M (1998). The β-Globin LCR Is Not Necessary for an Open Chromatin Structure or Developmentally Regulated Transcription of the Native Mouse β-Globin Locus. Mol. Cell 2, 447– 455. 9809066 source

      The Results report that deleting the mouse β-globin LCR did not make the locus DNaseI-resistant, supporting the accessibility claim. Our copy does not report retained histone acetylation at the β-globin genes; its discussion of histone acetylation concerns an immunoglobulin LCR and linked c-myc genes. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [57] Schübeler D, Groudine M, and Bender MA (2001). The murine β-globin locus control region regulates the rate of transcription but not the hyperacetylation of histones at the active genes. Proc. Natl. Acad. Sci. USA 98, 11432– 11437. 11553791 PMC58747 source
  • Premisecitation

    The α-SE and the TAD in which it is contained have been extensively characterized.

    “The α-SE is an ideal genetic model for this study; the SE and the TAD in which it is contained have been extensively characterized,58”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [58] Oudelaar AM, Beagrie RA, Kassouf MT, and Higgs DR (2021). The mouse alpha-globin cluster: a paradigm for studying genome regulation and organization. Curr. Opin. Genet. Dev. 67, 18– 24. 33221670 PMC8100094 source
  • Premisecitation

    Sahu and colleagues used STARR-seq to show that four out of the five MYC super-enhancer constituents have no detectable enhancer activity in HepG2 cells.

    “Sahu and colleagues recently used a STARR-seq method to show that four out of the five MYC SE constituents have no detectable enhancer activity in HepG2 cells.59”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [59] Sahu B, Hartonen T, Pihlajamaa P, Wei B, Dave K, Zhu F, Kaasinen E, Lidschreiber K, Lidschreiber M, Daub CO, (2022). Sequence determinants of human gene regulatory elements. Nat. Genet. 54, 283– 294. 35190730 PMC8920891 source

      The Results (Fig. 4b caption; page unavailable) show one STARR-seq peak at a MYC super-enhancer in HepG2 cells, alongside chromatin-dependent enhancers that the Results describe as poorly detected by STARR-seq. Our copy does not state that the super-enhancer has five constituents or establish that exactly four lack detectable activity. It also treats chromatin-dependent enhancers as active through mechanisms the assay may not detect. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 30
  • May combine two claims. 23

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

    Study design and conductown data

    The two synthetic BAC constructs were sequence-verified using Illumina short-read sequencing.

    “A variant of the eSwAP-In method24 was used to produce the two constructs, which were sequence-verified using illumina short-read sequencing (Figure S1A).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [24] Mitchell LA, McCulloch LH, Pinglay S, Berger H, Bosco N, Brosh R, Bulajić M, Huang E, Hogan MS, Martin JA, (2021). De novo assembly and delivery to mouse cells of a 101 kb functional human gene. Genetics 218, iyab038. 33742653 PMC8128383 source

      The paper’s “Structural and sequence verification of assembled DNA” section says that pLM750 underwent PacBio sequence verification, not Illumina short-read sequencing. It describes several assembled plasmids but does not report Illumina verification of two constructs. (LLM, conf=0.84)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

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

    Study design and conductcitation

    Consensus peak files were generated from multiple biological replicates using bedtools intersect.

    “Generation of consensus peak files from multiple biological replicates was performed using bedtools intersect”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [81] Quinlan AR, and Hall IM (2010). BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841– 842. 20110278 PMC2832824 source

  • May combine two claims.

    Premisecitation

    The β-globin LCR includes six regulatory elements (HS1-6) and has been classified as a super-enhancer.

    “The β-globin LCR includes six regulatory elements (HS1–6) and has been classified as a super-enhancer.17”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] Hay D, Hughes JR, Babbs C, Davies JOJ, Graham BJ, Hanssen L, Kassouf MT, Marieke Oudelaar AM, Sharpe JA, Suciu MC, (2016). Genetic dissection of the α-globin super-enhancer in vivo. Nat. Genet. 48, 895– 903. 27376235 PMC5058437 source

      The Results and Discussion (page numbers unavailable) classify the β-globin regulatory region/LCR as an erythroid super-enhancer. Our copy does not specify that it contains six regulatory elements named HS1–6, so that part of the claim is unsupported. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Previous dissection of the endogenous α-SE suggested it is a cluster of five independent elements: two classical enhancers (R1 and R2) capable of significantly upregulating gene expression, and three inactive elements (R3, Rm, and R4) with little/no ability to activate transcription in all previously defined assays of enhancer function.

    “Previous dissection of the endogenous α-SE, removing each of its constituents individually or in selective pairs, suggested that it is a cluster of five independent elements: two classical enhancers (R1 and R2) capable of significantly upregulating gene expression, and three inactive elements (R3, Rm, and R4)”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [17] Hay D, Hughes JR, Babbs C, Davies JOJ, Graham BJ, Hanssen L, Kassouf MT, Marieke Oudelaar AM, Sharpe JA, Suciu MC, (2016). Genetic dissection of the α-globin super-enhancer in vivo. Nat. Genet. 48, 895– 903. 27376235 PMC5058437 source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [23] Hughes Jim R., Cheng Jan-Fang, Ventress Nicki, Prabhakar Shyam, Clark Kevin, Anguita Eduardo, Marco De Gobbi, Pieter de Jong, Eddy Rubin, and Douglas R. Higgs. Proc Natl Acad Sci U S A. 2005 Jul 12; 102(28): 9830– 9835. 15998734 PMC1174996

      Not checked in the free run.

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

    Premisecitation

    A number of groups have dissected super-enhancers, yielding various conclusions, from additive to super-additive, redundant, synergistic, and hierarchical cooperation.

    “A number of groups have dissected SEs, yielding various conclusions, from additive16 to super-additive,22 redundant,19 synergistic,21 and hierarchical20 cooperation.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [16] Bender MA, Ragoczy T, Lee J, Byron R, Telling A, Dean A, and Groudine M (2012). The hypersensitive sites of the murine β-globin locus control region act independently to affect nuclear localization and transcriptional elongation. Blood 119, 3820– 3827. 22378846 PMC3335386 source
    • cites [22] Thomas HF, Kotova E, Jayaram S, Pilz A, Romeike M, Lackner A, Penz T, Bock C, Leeb M, Halbritter F, (2021). Temporal dissection of an enhancer cluster reveals distinct temporal and functional contributions of individual elements. Mol. Cell 81, 969– 982.e13. 33482114 source
    • cites [19] Hörnblad A, Bastide S, Langenfeld K, Langa F, and Spitz F (2021). Dissection of the Fgf8 regulatory landscape by in vivo CRISPR-editing reveals extensive intra- and inter-enhancer redundancy. Nat. Commun. 12, 439. 33469032 PMC7815712 source

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

    Study design and conductcitation

    Tiled-C was conducted as previously described.

    “Tiled-C, a high-resolution Chromosome Conformation Capture (3C) method that produces contact matrices of selected regions of interest was conducted as previously described.41”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [41] Oudelaar AM, Beagrie RA, Gosden M, de Ornellas S, Georgiades E, Kerry J, Hidalgo D, Carrelha J, Shivalingam A, El-Sagheer AH, (2020). Dynamics of the 4D Genome during in Vivo Lineage Specification and Differentiation. Nat. Commun. 11, 2722. 32483172 PMC7264236 source

  • May combine two claims.

    Premisecitation

    The α-SE is activated exclusively during terminal erythroid differentiation, meaning its manipulation has no effect on cell fate or in non-erythroid cells.

    “and the SE is activated exclusively during terminal erythroid differentiation, meaning its manipulation has no effect on cell fate or in non-erythroid cells.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [58] Oudelaar AM, Beagrie RA, Kassouf MT, and Higgs DR (2021). The mouse alpha-globin cluster: a paradigm for studying genome regulation and organization. Curr. Opin. Genet. Dev. 67, 18– 24. 33221670 PMC8100094 source

      The Introduction (page unavailable) says perturbing α-globin regulatory elements can leave cell lineage, differentiation, and maturation unchanged. But the section on α-globin activation during erythropoiesis says the enhancers become accessible in very early erythroid progenitors, not exclusively during terminal differentiation. The paper does not establish that manipulating them has no effect in non-erythroid cells. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

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

    Study design and conductcitation

    R2[R4], R1R2R3[R4] and R1R2HS1[R4] donors were screened with Sasquatch and JASPAR to ensure no novel accessibility sites or motifs were predicted at the newly created junctions.

    “R2[R4], R1R2R3[R4] and R1R2HS1[R4] donors were screened with Sasquatch 43and JASPAR42,43 to ensure no novel accessibility sites or motifs were predicted at the newly created junctions,43,44 prior to synthesis and transfection.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [43] Fornes O, Castro-Mondragon JA, Khan A, van der Lee R, Zhang X, Richmond PA, Modi BP, Correard S, Gheorghe M, Baranašić D, (2020). JASPAR 2020: update of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 48, D87– D92. 31701148 PMC7145627 source
    • cites [42] Castro-Mondragon JA, Riudavets-Puig R, Rauluseviciute I, Lemma RB, Turchi L, Blanc-Mathieu R, Lucas J, Boddie P, Khan A, Manosalva Pérez N, (2022). JASPAR 2022: the 9th release of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 50, D165– D173. 34850907 PMC8728201 source
    • cites [44] Schwessinger R, Suciu MC, McGowan SJ, Telenius J, Taylor S, Higgs DR, and Hughes JR (2017). Sasquatch: predicting the impact of regulatory SNPs on transcription factor binding from cell- and tissue-specific DNase footprints. Genome Res. 27, 1730– 1742. 28904015 PMC5630036 source

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

    Premisecitation

    A number of studies have suggested that enhancer clusters, including super-enhancers, may act cooperatively to form foci containing high concentrations of tissue-specific transcription factors, co-activators such as the Mediator complex, and Pol II.

    “A number of studies have suggested that enhancer clusters, including SEs, may act cooperatively to form foci containing high concentrations of tissue-specific TFs, co-activators such as the Mediator complex, and Pol II.11”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [11] Grosveld F, van Staalduinen J, and Stadhouders R (2021). Transcriptional Regulation by (Super)Enhancers: From Discovery to Mechanisms. Annu. Rev. Genom. Hum. Genet. 22, 127– 146. 33951408 source

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

    Study design and conductcitation

    ChIPmentation experiments were performed as previously described with few modifications.

    “ChIPmentation experiments were performed as previously described,76 with few modifications.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [76] Schmidl C, Rendeiro AF, Sheffield NC, and Bock C (2015). ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors. Nat. Methods 12, 963– 965. 26280331 PMC4589892 source

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

    Premisecitation

    Current theories propose liquid-liquid phase separation and some form of transcription factor trapping or engagement of transcription factors in multivalent interactions independent of assembly into phase-separated liquid-like droplets.

    “current theories propose liquid-liquid phase separation18,64–66 and some form of TF trapping22,67 or engagement of TFs in multivalent interactions independent of assembly into phase-separated liquid-like droplets.68”
    Quote in the paperNot a substantial claim7 of 7 sources checked
    • cites [18] Hnisz D, Schuijers J, Lin CY, Weintraub AS, Abraham BJ, Lee TI, Bradner JE, and Young RA (2015). Convergence of Developmental and Oncogenic Signaling Pathways at Transcriptional Super-Enhancers. Mol. Cell 58, 362– 370. 25801169 PMC4402134 source

      The Results section (‘Signaling modules at super-enhancers’; page unavailable) reports that multiple transcription factors bind the same super-enhancers, and the Discussion describes physical and functional interactions among enhancer constituents. These findings support clustered TF engagement, but the paper does not establish multivalent TF interactions or address whether they occur independently of phase-separated droplets. Citation 68 is attached to that latter proposal, not to the sentence’s separate phase-separation or TF-trapping proposals. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [64] Boija A, Klein IA, Sabari BR, Dall’Agnese A, Coffey EL, Zamudio AV, Li CH, Shrinivas K, Manteiga JC, Hannett NM, (2018). Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains. Cell 175, 1842– 1855.e16. 30449618 PMC6295254 source
    • cites [65] Sabari BR, Dall’Agnese A, Boija A, Klein IA, Coffey EL, Shrinivas K, Abraham BJ, Hannett NM, Zamudio AV, Manteiga JC, (2018). Coactivator condensation at super-enhancers links phase separation and gene control. Science 361, eaar3958. 29930091 PMC6092193 source
    • cites [66] Gurumurthy A, Shen Y, Gunn EM, and Bungert J (2019). Phase Separation and Transcription Regulation: Are Super-Enhancers and Locus Control Regions Primary Sites of Transcription Complex Assembly? Bio-essays 41, 1800164. PMC6484441 30500078 source
    • cites [22] Thomas HF, Kotova E, Jayaram S, Pilz A, Romeike M, Lackner A, Penz T, Bock C, Leeb M, Halbritter F, (2021). Temporal dissection of an enhancer cluster reveals distinct temporal and functional contributions of individual elements. Mol. Cell 81, 969– 982.e13. 33482114 source

      The citation is attached to the proposed engagement of transcription factors in multivalent interactions without liquid-like droplets. The Discussion (p. 13) mentions phase separation as one proposed mechanism, while p. 15 speculates that a combination of transcription factors and co-activators could contribute to Pol II accumulation. Neither passage addresses multivalent transcription-factor interactions independent of droplet assembly (searched those terms and a paraphrase). (LLM, conf=0.73)

      Experimental — accuracy not yet measured
    • cites [67] Sigova AA, Abraham BJ, Ji X, Molinie B, Hannett NM, Guo YE, Jangi M, Giallourakis CC, Sharp PA, and Young RA (2015). Transcription factor trapping by RNA in gene regulatory elements. Science 350, 978– 981. 26516199 PMC4720525 source
    • cites [68] Trojanowski J, Frank L, Rademacher A, Mücke N, Grigaitis P, and Rippe K (2022). Transcription activation is enhanced by multivalent interactions independent of phase separation. Mol. Cell 82, 1878– 1893.e10. 35537448 source

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

    Premisecitation

    Numerous publications have demonstrated that super-enhancers activate high levels of gene expression with a tendency to regulate lineage-specific genes.

    “Numerous publications have demonstrated that SEs activate high levels of gene expression with a tendency to regulate lineage-specific genes.4,11,14,18”
    Quote in the paperNot a substantial claim4 of 4 sources checked
    • cites [4] Whyte WA, Orlando DA, Hnisz D, Abraham BJ, Lin CY, Kagey MH, Rahl PB, Lee TI, and Young RA (2013). Master Transcription Factors and Mediator Establish Super-Enhancers at Key Cell Identity Genes. Cell 153, 307– 319. 23582322 PMC3653129 source
    • cites [11] Grosveld F, van Staalduinen J, and Stadhouders R (2021). Transcriptional Regulation by (Super)Enhancers: From Discovery to Mechanisms. Annu. Rev. Genom. Hum. Genet. 22, 127– 146. 33951408 source
    • cites [14] Wang X, Cairns MJ, and Yan J (2019). Super-enhancers in transcriptional regulation and genome organization. Nucleic Acids Res. 47, 11481– 11496. 31724731 PMC7145697 source
    • cites [18] Hnisz D, Schuijers J, Lin CY, Weintraub AS, Abraham BJ, Lee TI, Bradner JE, and Young RA (2015). Convergence of Developmental and Oncogenic Signaling Pathways at Transcriptional Super-Enhancers. Mol. Cell 58, 362– 370. 25801169 PMC4402134 source

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

    Premisecitation

    A recent study in Drosophila has identified elements that are not enhancers but are thought to facilitate interactions between regulatory elements by tethering them together.

    “A recent study in Drosophila has identified what may be similar elements, which are not enhancers but are thought to facilitate interactions between regulatory elements by tethering them together.63”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [63] Levo M, Raimundo J, Bing XY, Sisco Z, Batut PJ, Ryabichko S, Gregor T, and Levine MS (2022). Transcriptional coupling of distant regulatory genes in living embryos. Nature 605, 754– 760. 35508662 PMC9886134 source

  • May combine two claims.

    Premisecitation

    Hnisz and colleagues previously showed that the E8 enhancer within Pri-miR-290-295 has very little enhancer activity measured by luciferase assay; nevertheless, deletion of E8 caused a large decrease in Pri-miR-290-295 expression.

    “Similarly, Hnisz and colleagues previously showed that the E8 enhancer within the Pri-miR-290–295 has very little enhancer activity measured by luciferase assay; nevertheless, deletion of E8 caused a large decrease in Pri-miR-290–295 expression.18”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Hnisz D, Schuijers J, Lin CY, Weintraub AS, Abraham BJ, Lee TI, Bradner JE, and Young RA (2015). Convergence of Developmental and Oncogenic Signaling Pathways at Transcriptional Super-Enhancers. Mol. Cell 58, 362– 370. 25801169 PMC4402134 source

      The Results section, “Perturbation of super-enhancer constituents” (page unavailable), reports that deleting miR-290-295 E8 caused a substantial reduction in gene expression—more than 50% versus wild type. The Results section, “Enhancer activity of super-enhancer constituents” (page unavailable), describes luciferase assays but does not state that E8 specifically had very little activity. That part of the attributed claim is unsupported by our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Previous dissection of the α-SE suggested that its five constituents combine additively as independent elements.

    “Previous dissection of the α-SE suggested that its five constituents combine additively as independent elements,17”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] Hay D, Hughes JR, Babbs C, Davies JOJ, Graham BJ, Hanssen L, Kassouf MT, Marieke Oudelaar AM, Sharpe JA, Suciu MC, (2016). Genetic dissection of the α-globin super-enhancer in vivo. Nat. Genet. 48, 895– 903. 27376235 PMC5058437 source

  • May combine two claims.

    Findingown data

    In the absence of other α-SE elements, R2 retains its identity as an enhancer, recruiting transcription factors and creating a region of open chromatin.

    “We conclude that in the absence of other elements in the α-SE, R2 retains its identity as an enhancer, recruiting transcription factors and creating a region of open chromatin.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only one surviving R2-only homozygote was obtained; it had anemia and severe splenomegaly and died prematurely at 7 weeks.

    “We obtained only one surviving homozygote with anemia and severe splenomegaly, which died prematurely at 7 weeks.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Individual deletion of R1 or R2 significantly reduces α-globin expression.

    “Individual deletion of R1 (R2R3RmR4) or R2 (R1R3RmR4) significantly reduced α-globin expression”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Deleting R3 or Rm alone has no discernible effect on α-globin expression.

    “deleting R3 (R1R2RmR4) or Rm (R1R2R3R4) alone had no discernible effect on gene expression”
    Quote in the paperSubstantial claim

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

    Findingown data

    Wild-type and R2-only fetal liver cells are composed of approximately 95% CD71+/Ter119+ erythroid cells.

    “FACS analysis following staining for the CD71 and Ter119 cell surface markers in E12.5 FL cells, revealed that WT and R2-only FL are composed of ~95% CD71+/Ter119+ erythroid cells”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    R4's sequence is not sufficient to rescue expression; when placed at the R1 position, R2R4[R1] cells expressed only 12% α-globin, comparable to R2-only.

    “EB-derived R2R4[R1] erythroid cells only expressed 12% α-globin, a level comparable to that in R2-only erythroid cells, suggesting that R4’s rescue capacity is not exclusively based on its sequence”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    R2 by itself exhibits very low levels of co-activator recruitment, reduced interactions with its target genes' promoters, and lower levels of eRNA transcription.

    “exhibiting very low levels of co-activator recruitment, reduced interactions with its target genes’ promoters, and lower levels of eRNA transcription.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    R1 and R2 contained the highest density of TF motifs and the most complex DNase foot-printing signals among the five α-SE elements.

    “Unsurprisingly, R1 and R2 contained the highest density of TF motifs and the most complex DNase foot-printing signals”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    R1 and R2 cooperate synergistically, each upregulating gene expression 100-fold alone versus 450-fold when combined.

    “R1 and R2 cooperate synergistically, each upregulating gene expression 100-fold alone versus 450-fold when combined”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Rm and R4 function in a context-dependent manner, each partially redundant to the other.

    “Rm and R4 function in a context-dependent manner, each partially redundant to the other.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    The erythroid populations derived from WT, R2-only heterozygotes, and R2-only homozygotes are not affected by the R2-only α-globin genotype and are comparable.

    “this showed that the erythroid populations are not affected by the R2-only α-globin genotype and that the populations are comparable”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    In the R2-only model, the structural integrity of the locus was checked with linked-read library preparation followed by Illumina sequencing.

    “In the case of the R2-only model, the structural integrity of the locus was checked with linked-read library preparation (10x Genomics) followed by illumina sequencing”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Only RNA samples with an RNA integrity score of at least 8 were taken forwards for subsequent analysis.

    “Only samples with an RNA integrity score of at least 8 were taken forwards for subsequent analysis.”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    Super-enhancer cooperation may be variable, with subsets of super-enhancers combining additively and others non-additively.

    “Similar to the variation in TF cooperation and sequence grammar manifested within individual enhancers, SE cooperation may well be variable, with subsets of SEs combining additively and others non-additively.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Erythropoietic cells/compartments were isolated for analysis, blinded to the genotype.

    “Erythropoietic cells/compartments were then isolated for analysis, blinded to the genotype.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    To avoid interaction bias between regions within and outside of the tiled region, all data mapping to the tiled region was extracted and the remaining data discarded from subsequent analysis steps.

    “To avoid interaction bias between regions within and outside of the tiled region, all data mapping to the tiled region was extracted and the remaining data discarded from subsequent analysis steps.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    In their native context, Rm and R4, but not R3, appear to be at least partially redundant in their ability to facilitate the function of classical enhancers R1 and R2, with R4 having a stronger effect than Rm.

    “Therefore, in their native context, Rm and R4, but not R3, appear to be at least partially redundant in their ability to facilitate the function of the classical enhancers R1 and R2, with R4 having a stronger effect than Rm.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    PCR duplicates were removed from SAM files using SAMtools.

    “Resultant SAM files were filtered, sorted, and PCR duplicates removed, using SAMtools (samtools view, sort, and rmdup, respectively).”
    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

    All mouse work was performed in accordance with UK Home Office regulations, under the appropriate animal licenses.

    “All mouse work was performed in accordance with UK Home office regulations, under the appropriate animal licenses.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    R2-only BAC-integrated mESCs were karyotyped before microinjection.

    “R2-only BAC-integrated mESCs were karyotyped, microinjected into C57BL/6 blastocysts and implanted into pseudopregnant C57BL/6 females.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Reverse transcriptase negative controls were included to confirm that DNase treatment was complete.

    “Reverse transcriptase (RT-) enzyme negative controls were included to confirm that DNase treatment was complete.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Super-enhancers can act as cohorts of functionally distinct elements, including classical enhancers responsible for activating a target gene's expression, and facilitators, which in some way augment activator function.

    “We present evidence that SEs can act as cohorts of functionally distinct elements, including classical enhancers, responsible for activating a target gene’s expression, and facilitators, which in some way augment activator function.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: It remains unclear whether there is a functional distinction separating super-enhancers from clusters of regular enhancer elements.

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Clones were screened for successful enhancer deletion by PCR, Sanger sequencing, and ATAC-seq.

    “Clones were screened for successful enhancer deletion by the appropriate PCR strategy; this entailed PCR amplification using primers flanking each deleted element (sequences in Table S3), such that a successfully deleted allele would produce a smaller product than a WT allele. Clones were then screened by Sanger sequencing and ATAC-seq.”
    Quote in the paperSubstantial claim

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

    Interpretationnone given

    The hierarchy of facilitators is encoded in their positions more than their sequences.

    “it is interesting that the hierarchy of facilitators is encoded in their positions more than their sequences.”
    Quote in the paperNot a substantial claim

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

    Premisereasoning

    All of these proposals require recruitment of a critical mass of transcription factors within a given three-dimensional space.

    “All of these proposals require recruitment of a critical mass of TFs within a given three-dimensional space.”
    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

    Guide RNAs were designed using CRISPOR and BreakingCas, and candidates with the fewest predicted off-targets were selected and screened for effectiveness using an in vitro surveyor assay.

    “Guide RNAs were designed using the CRISPOR and BreakingCas online gRNA design tools (refer to KRT). Candidates with the fewest predicted off-targets were selected and further screened for their effectiveness, using an in vitro surveyor assay”
    Quote in the paperNot a substantial claim

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

    Premisenone given

    Other super-enhancer dissection studies have been confounded by disruption of clusters that regulate pleiotropic transcription factors or co-factors that influence cell fate, rendering it impossible to control whether wild-type and manipulated models are equivalent in their developmental stage and cell type.

    “Other SE dissection studies have been confounded by disruption of clusters that regulate pleiotropic transcription factors or co-factors that influence cell fate, rendering it impossible to control whether WT and manipulated models are equivalent in their developmental stage and cell type.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The donor vector sequence was verified using Plasmidsaurus.

    “The donor vector sequence was also verified using Plasmidsaurus.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The findings demonstrate that super-enhancers can constitute complex cohesive networks of regulatory elements, displaying simultaneous additive, redundant, and synergistic cooperation.

    “In summary, our findings demonstrate that SEs can constitute complex cohesive networks of regulatory elements, displaying simultaneous additive, redundant, and synergistic cooperation.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Indexed ATAC samples were assessed by TapeStation using a High Sensitivity D1000 ScreenTape.

    “Indexed ATAC samples were assessed by tape station, using a High Sensitivity (HS) D1000 ScreenTape (Agilent).”
    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

    Population purity and selection efficiency were determined by flow cytometry.

    “Population purity and selection efficiency were determined by flow cytometry.”
    Quote in the paperNot a substantial claim
46 of 46

  • The paper relies on this without support.

    It remains unclear whether there is a functional distinction separating super-enhancers from clusters of regular enhancer elements.

    “Despite this, it remains unclear whether there is a functional distinction separating SEs from clusters of regular enhancer elements.”

  • The paper relies on this without support.

    Observations on gene regulation made at the globin gene clusters illustrate general principles.

    “The fact that observations on gene regulation made at the globin gene clusters have always illustrated general principles is encouraging.”

  • The paper relies on this without support.

    The ROSE algorithm provides a clear bioinformatic definition of super-enhancers and the field has widely adopted the super-enhancer nomenclature.

    “To simplify our analyses, we focused on studying the functional characteristics of super-enhancers, selecting this particular class due to their clear bioinformatic definition (using the ROSE algorithm) and the fact that the field has widely adopted the “super-enhancer” nomenclature.”

  • The paper relies on this without support.

    Many enhancer clusters satisfy the criteria of multiple classes.

    “Many enhancer clusters satisfy the criteria of multiple classes.”
4 of 4

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?

  • The paper's observations expand the list of recently discovered long-range regulatory mechanisms that work by potentiating enhancer activity, such as facilitation and range extension.

    “Instead, our observations expand the list of recently discovered long-range regulatory mechanisms that work by potentiating enhancer activity, such as facilitation (60) and range extension (61).”

    From Synergy between regulatory elements can render cohesin dispensable for distal enhancer function (2026) · cites this paper as [60]

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.

  • Conflicting order of BAC integration and allele deletion

    Minor

    The Results say the synthetic alleles were integrated into cells in which one α-globin copy had already been deleted. The Methods say BAC targeting came first and the resulting cell lines were then made hemizygous by deleting an α-globin allele.

1 of 1

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Authors
Blayney, J. W., Francis, H., Rampasekova, A., Camellato, B., Mitchell, L., Stolper, R., Cornell, L., Babbs, C., Boeke, J. D., Higgs, D. R., & Kassouf, M.
Affiliations
  • John Radcliffe Hospital (GB)
  • NYU Langone Health (US)
Corresponding author
  • Boeke, Jef D.
  • Higgs, Douglas R.
  • Kassouf, Mira
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
2023-12
Volume
186
Issue
26
Pages
5826-5839.e18
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/j.cell.2023.11.030
PMC
PMC10858684
OpenAlex
W4389729890
Semantic Scholar
cfab22ef29ee3f1314123fde2b0e948b27abc190
Cited by
196
References
86
Citations by year
2026: 57 · 2025: 93 · 2024: 46
FWCI
17.19
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Funders
  • Wellcome Trust (109097/Z/15/Z, 215111/Z/18/Z, 219979/Z/19/Z, 222843/Z/21/Z)
  • Hansjörg Wyss Institute for Biologically Inspired Engineering, Harvard University
  • Memphis Research Consortium
  • Medical Research Council (MR/T014067/1)
  • National Institutes of Health
  • Chinese Academy of Medical Sciences
  • National Human Genome Research Institute (RM1HG009491)
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
super-enhancers, enhancers, Mediator complex, enhancer RNA, enhancer-promoter interactions, transcriptional activation, gene expression regulation
MeSH terms
Super Enhancers, Enhancer Elements, Genetic, Gene Expression Regulation, Promoter Regions, Genetic, Transcription Factors, Transcription, Genetic, 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 2026-10-27.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-27.

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Statistics and arithmetic are recomputed, not judged.

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