Genetic dissection of the α-globin super-enhancer in vivo
Open accessAbstract
Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.
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Written from the paper’s full text.
TL;DR
A functional dissection of the mouse α-globin super-enhancer shows that its five constituent elements act independently and additively, with no evidence of synergistic or higher-order super-enhancer properties. Only R1 and R2 behave as strong enhancers in vivo, while R3, R4 and Rm contribute little or nothing to α-globin expression.
Key findings
- In primary mouse erythroid cells, 15,849 DNase I hypersensitive sites were identified; after filtering, 1,963 putative enhancers were defined, and 95 regions (<7%) were classified as super-enhancers by Med1 occupancy. The α- and β-globin regulatory regions were the two highest-scoring erythroid super-enhancers.
- The mouse α-globin super-enhancer spans 24 kb and contains five enhancer-like elements. R1 and R2 were strong enhancers in vivo; R3, R4 and Rm had little or no discernible impact on α-globin RNA synthesis, despite chromatin signatures and positive transient enhancer assays.
- Deletion of R1 or R2 individually caused significant, reproducible reductions in α-globin RNA synthesis and increased reticulocyte counts, while R1/R2 double homozygotes died in utero at ~E14.5 and produced very little α-globin RNA, with remaining <10% of normal α-globin RNA levels likely transcribed under intact cis-elements.
- Chromatin structure and chromosome conformation were largely independent of the intact super-enhancer: no individual hypersensitive site depended on any other, and even in R1/R2−/− cells, hypersensitive sites at R3, R4 and Rm still formed; Capture-C showed little change in the tissue-specific chromatin compartment.
Methods
- Mouse models: seven homologous-recombination models were generated, deleting each of the five α-globin super-enhancer elements singly (R1, R2, R3, Rm, R4) plus two informative double knockouts (R2/R3 and R1/R2).
- Data types: primary mouse erythroid cells were profiled by DNase I hypersensitivity, ChIP-seq for chromatin marks and transcription factors, ATAC-seq, and Next Generation Capture-C; enhancer activity was tested in transient chick reporter assays and stably integrated mouse LacZ transgenic assays.
- Expression and phenotype: hematologic parameters, reticulocyte counts, total and nascent RNA (4-thiouridine labeling followed by NanoString) were measured in fetal liver erythroid cultures and adult mice; statistical analysis used Levene’s test followed by one-way ANOVA with Dunnett’s correction.
Limitations
- Stated: the authors did not assess possible changes in Med1 binding, activating chromatin marks, or eRNA production in the context of each enhancer knockout; they also state that findings cannot be extrapolated to all super-enhancers.
- Stated: it remains possible that the apparently functionless elements have weak enhancer function not revealed by current physiological assays, or roles at different developmental stages or in defining enhancer polarity, which were not tested.
- Evident: the study is restricted to the mouse α-globin locus in erythroid cells, and conclusions about super-enhancer generality rely on this single locus plus prior β-globin LCR work; the authors call for genetic dissection of other super-enhancers in other cell types.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
7 of 11 cited sources checked · 4 supported · 0 flagged · accuracy not yet measured
- Interpretationown data
Multiple enhancers may provide robustness in gene expression.
“From the data presented here and from others29, 30, it seems likely that multiple enhancers may provide robustness in gene expression.”
Quote in the paperSubstantial claim2 of 2 sources checked- cites [29] Frankel, N. et al. Phenotypic robustness conferred by apparently redundant transcriptional enhancers. Nature 466, 490–493 (2010). source
- cites [30] Perry, M.W., Boettiger, A.N., Bothma, J.P. & Levine, M. Shadow enhancers foster robustness of Drosophila gastrulation. Curr. Biol. 20, 1562–1567 (2010). source
- Interpretationcitation
The apparently functionless enhancer-like elements identified in this study reflect evolutionary turnover in cis-acting elements.
“We would suggest, however, that these elements reflect evolutionary turnover in cis-acting elements as seen in other studies32.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [32] Villar, D. et al. Enhancer evolution across 20 mammalian species. Cell 160, 554–566 (2015). source
- Rests on an assumption: It is unlikely that cis-acting elements emerge fully formed during evolution or that they disappear entirely when they lose function.
- Findingnone given
Knockouts of R3, R4 and Rm appear to have little, if any, effect on α-globin expression or erythropoiesis.
“Knockouts of R3, R4 and Rm appear to have little, if any, effect on α-globin expression or erythropoiesis.”
Quote in the paperSubstantial claim - Findingown data
Deletion of R4 has a minor impact on α-globin RNA synthesis.
“Deletion of R4 has a minor impact.”
Quote in the paperSubstantial claim - Findingown data
Deletions of R3 and Rm have no discernible impact on α-globin RNA synthesis.
“Deletions of R3 and Rm, however, have no discernible impact on α-globin RNA synthesis (Fig. 5b).”
Quote in the paperSubstantial claim
- Findingown data
The α- and β-globin gene regulatory regions are the two highest scoring super-enhancers in erythroid cells using Mediator-binding as the defining parameter.
“The previously reported α- and β-globin gene regulatory regions were the two highest scoring super-enhancers in erythroid cells using Mediator-binding as the defining parameter (Fig. 1c and Supplementary Table 1).”
Quote in the paperNot a substantial claim - Findingown data
Deletions of R1 and R2 have a significant and reproducible impact on the synthesis of α-globin RNA.
“Deletions of R1 and R2 have a significant and reproducible impact on the synthesis of α-globin RNA.”
Quote in the paperSubstantial claim - Findingown data
R1/R2-/- mice die in utero at approximately E14.5.
“These mice die in utero at ~E14.5”
Quote in the paperSubstantial claim - Findingown data
R1/R2-/- fetal liver cells produced very little α-globin RNA.
“Analysis of α:β globin RNA ratios showed that R1/R2-/- fetal liver cells produced very little α-globin RNA (Fig. 5).”
Quote in the paperSubstantial claim - Findingown data
Only two of the five α-globin super-enhancer elements behave as strong enhancers in vivo during embryonic, fetal, and adult erythropoiesis.
“We find that no single element is critical for globin gene expression and, although each element scores as an erythroid enhancer based on conventional chromatin signatures and enhancer assays, only two of the five elements behave as strong enhancers in vivo during embryonic, fetal, and adult erythropoiesis.”
Quote in the paperSubstantial claim - Findingown data
The two strong enhancers fall into a subgroup of individual erythroid enhancers bound by the greatest amount of Mediator and the highest numbers of erythroid master regulators.
“These two strong enhancers fall into a subgroup of individual erythroid enhancers which are bound by the greatest amount of Mediator and the highest numbers of erythroid master regulators.”
Quote in the paperSubstantial claim - Findingown data
Even in the most phenotypically severe R1/R2-/- model, hypersensitive sites at R3, R4 and Rm still form.
“Even in the most phenotypically severe R1/R2-/- model, hypersensitive sites at R3, R4 and Rm still form.”
Quote in the paperSubstantial claim - Findingown data
R1-/- and R2-/- mice have significantly increased reticulocyte counts.
“We evaluated this using the reticulocyte count and noted significantly increased counts in R1-/- and R2-/- mice (Fig. 4c and d).”
Quote in the paperSubstantial claim - Findingown data
No single element of the α-globin super-enhancer is critical for globin gene expression.
“We find that no single element is critical for globin gene expression and, although each element scores as an erythroid enhancer based on conventional chromatin signatures and enhancer assays, only two of the five elements behave as strong enhancers in vivo during embryonic, fetal, and adult erythropoiesis.”
Quote in the paperSubstantial claim - Findingown data
The contributions of R1 and R2 to transcription appear to be additive rather than clearly synergistic.
“Further, within the limits of the experimental method, the contributions of R1 and R2 to transcription appear to be additive rather than clearly synergistic.”
Quote in the paperSubstantial claim - Findingown data
Genome-wide Capture-C analysis showed that the only statistically significant changes in even the most phenotypically severe double knockouts were at the α-globin promoters.
“When we extended the assessment of chromosomal conformation genome-wide using next-generation Capture-C, the only statistically significant changes seen in even the most phenotypically severe double knockouts were those at the α-globin promoters (Supplementary Fig. 7).”
Quote in the paperSubstantial claim - Findingown data
Chromatin conformation analysis in knockout models demonstrates very little change in the compartment between wild type and any single knockout.
“Assessing this in fetal liver culture erythroid cells from the knockout models demonstrates very little change in the compartment between the wild type and any single knockout (Fig. 6b).”
Quote in the paperSubstantial claim - Findingown data
No single knockout of an α-globin super-enhancer element reduced the hemoglobin concentration in adult mice below the normal range.
“No single knockout reduced the hemoglobin concentration in adult mice below the normal range (Fig. 4b)”
Quote in the paperSubstantial claim - Findingown data
The constituents of the α-globin super-enhancer show considerable variation in Med1 and transcription factor binding.
“Thus, the constituents of the α-globin super-enhancer show considerable variation in Med1 and transcription factor binding, raising the possibility that they may vary in their contribution to α-globin gene regulation.”
Quote in the paperSubstantial claim - Findingown data
The entire α-globin cluster is flanked by two pairs of CTCF binding sites.
“The entire α-globin cluster is flanked by two pairs of CTCF binding sites (Fig. 1f), a common observation for super-enhancers15.”
Quote in the paperNot a substantial claim - Findingown data
Enhancer elements bound by a high number of transcription factors are enriched in erythroid super-enhancers.
“Consistent with the recent identification of transcription factor “hotspots” as key components of adipogenic super-enhancers 6, enhancer elements bound by a high number of transcription factors are enriched in erythroid super-enhancers.”
Quote in the paperSubstantial claim - Findingown data
When tested at the α-globin locus, clustering of enhancers in super-enhancers does not appear to lead to any unexplained emergent properties.
“When tested at the α-globin locus, clustering of enhancers in super-enhancers does not appear to lead to any unexplained emergent properties.”
Quote in the paperSubstantial claim - Findingown data
Only R1 and R2 score positively in an enhancer assay in which the sequences are incorporated into chromatin.
“Of interest only R1 and R2 score positively in an enhancer assay in which the sequences are incorporated into chromatin.”
Quote in the paperSubstantial claim - Findingown data
Med1 binding varied considerably between the five constituent enhancers of the α-globin super-enhancer.
“When individual enhancers were ranked for Med1 occupancy without stitching, Med1 binding varied considerably between the five constituent enhancers of the α-globin super-enhancer (Fig. 2b).”
Quote in the paperSubstantial claim - Findingown data
The paper's data do not support the suggestion that super-enhancers have a singular composite function.
“Our data do not support the suggestion that super-enhancers have a singular composite function”
Quote in the paperSubstantial claim - Findingown data
Cluster 2, containing enhancers binding all four erythroid transcription factors, was significantly enriched in erythroid super-enhancers, being present in over 30% of erythroid super-enhancers.
“Cluster 2, containing enhancers binding all four erythroid transcription factors, was significantly enriched, being present in over 30% of erythroid super-enhancers.”
Quote in the paperNot a substantial claim - Findingown data
An increase in the number of constituent elements correlated to higher levels of Med1.
“An increase in the number of constituent elements correlated to higher levels of Med1 (Fig. 2a).”
Quote in the paperNot a substantial claim - Interpretationown data
Distal strong enhancers R1 and R2 potentiate the interactions between the elements R3, Rm and R4 and the α-globin promoters.
“These data suggest that distal strong enhancers R1 and R2 potentiate the interactions between the elements (R3, Rm and R4) and the α-globin promoters.”
Quote in the paperSubstantial claim - Interpretationown data
The regulatory effect of the α-globin super-enhancer depends mainly on two elements, R1 and R2; the other three elements contribute very little, if at all, to the activity of the super-enhancer in vivo.
“Thus, the regulatory effect of the α-globin super-enhancer depends mainly on two elements, R1 and R2; the other three elements contribute very little, if at all, to the activity of the super-enhancer in vivo.”
Quote in the paperSubstantial claim - Interpretationown data
The intact α-globin super-enhancer is not required for the formation or maintenance of the chromatin compartment.
“The intact super-enhancer is therefore not required for the formation or maintenance of the chromatin compartment.”
Quote in the paperSubstantial claim - Interpretationown data
Each constituent enhancer of the α-globin super-enhancer appears to act independently and in an additive fashion with respect to hematologic phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects.
“we demonstrate that each constituent enhancer appears to act independently and in an additive fashion with respect to hematologic phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects.”
Quote in the paperSubstantial claim - Premisecitation
The neomycin resistance cassette affects local gene expression.
“mice bearing the floxed neomycin resistance gene were crossed with GATA1-Cre expressing mice to excise the neomycin resistance cassette in all tissues40 as this has previously been shown to affect local gene expression41.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [41] Russell, E.S. & Berstein, S.E. in Biology of the Laboratory Mouse 2nd edn. (ed. Green, E.L.) Chapter 17 (Dover Publications, 1966).
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- Interpretationown data
The effects of non-integrated constructs in transient assays reflect the numbers and types of transcription factor binding sites present in each element.
“Consistent with our findings in the PAM analysis the effects of these non-integrated constructs in transient assays reflect the numbers and types of transcription factor binding sites present in each element.”
Quote in the paperNot a substantial claim - Interpretationown data
Removal of either strong enhancer R1 or R2 at the α-globin locus produces stressed erythropoiesis to ensure normal hemoglobin production.
“Removal of either strong enhancer (R1 or R2) at the α-globin locus produces stressed erythropoiesis to ensure normal hemoglobin production.”
Quote in the paperSubstantial claim - Interpretationown data
The study, together with previous work at the β-globin LCR, does not support further sub-classification of enhancers beyond the simple designation of strong to weak elements.
“Our study, together with previous work at the β-globin LCR, does not support further sub-classification of enhancers beyond the simple designation of strong to weak elements.”
Quote in the paperSubstantial claim - Interpretationown data
Strong enhancers augment the interactions between their chromatin environment and cognate promoters.
“but rather that strong enhancers simply augment the interactions between their chromatin environment and cognate promoters.”
Quote in the paperSubstantial claim - Interpretationreasoning
The augmentation of interactions between strong enhancers and cognate promoters generates a compartment of interaction between the chromatin surrounding the enhancer and the promoter.
“This generates a compartment of interaction between the chromatin surrounding the enhancer and the promoter”
Quote in the paperSubstantial claim - Interpretationown data
The super-enhancer associated with the globin genes may be more simply described as a group of conventional enhancers including at least one strong enhancer, rather than as a new discrete entity with properties greater than the sum of its parts.
“Thus the super-enhancer associated with the globin genes may be more simply described as a group of conventional enhancers including at least one strong enhancer, rather than as a new discrete entity with properties greater than the sum of its parts.”
Quote in the paperSubstantial claim - Interpretationnone given
Enhancer assays do not reliably describe super-enhancer elements.
“Enhancer assays do not reliably describe super-enhancer elements”
Quote in the paperSubstantial claim - Interpretationreasoning
The apparently functionless elements identified in this study could represent evolving enhancers.
“the apparently functionless elements we identify could therefore represent evolving enhancers.”
Quote in the paperNot a substantial claim- Rests on an assumption: It is unlikely that cis-acting elements emerge fully formed during evolution or that they disappear entirely when they lose function.
- Interpretationreasoning
The compartment of interaction between the chromatin surrounding the enhancer and the promoter results in an environment that might favor the evolution of other enhancers.
“which results in an environment that might favor the evolution of other enhancers.”
Quote in the paperNot a substantial claim
- Classified as probably not a substantial claim. 15
- May combine two claims. 15
- The quote only partly states this claim. 1
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationPrevious less extensive studies have shown that the individual regulatory elements of the β-globin LCR behave in a similar independent and additive manner.
“Previous less extensive studies have also shown that the individual regulatory elements of the β-globin LCR, which we show here also corresponds to an erythroid super-enhancer, behave in a similar independent and additive manner28.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [28] Bender, M.A. et al. The hypersensitive sites of the murine β-globin locus control region act independently to affect nuclear localization and transcriptional elongation. Blood 119, 3820–3827 (2012). source
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenSample sizes were chosen with a view to detecting a 25% reduction in haemoglobin.
“Sample sized were chosen with a view to detecting a 25% reduction in haemoglobin; smaller confirmatory datasets were obtained where the analysis of phenotype had already been reported (as for R2 and R3 deletions14, 19.”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites [14] Kowalczyk, M.S. et al. Intragenic enhancers act as alternative promoters. Mol. Cell 45, 447–458 (2012). source
- cites [19] Anguita, E. et al. Deletion of the mouse α-globin regulatory element (HS −26) has an unexpectedly mild phenotype. Blood 100, 3450–3456 (2002). source
The Cytologic and hematologic analysis section describes how haemoglobin was measured, and the Results report haemoglobin findings, but neither states how sample sizes were chosen. Searches for a 25% reduction, sample-size planning, and a power calculation found no passage addressing the claimed detection target. The paper’s finding of essentially normal haemoglobin in untreated knockout mice does not establish what reduction the citing authors planned to detect. (LLM, conf=0.76)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).
PremisecitationTranscription factor hotspots, such as R1 and R2, create a permissive chromatin environment for the emergence of new transcription factor binding regions.
“Indeed, it has been proposed that transcription factor hotspots, such as R1 and R2, create a permissive chromatin environment for the emergence of new transcription factor binding regions6.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [6] Siersbæk, R. et al. Transcription factor cooperativity in early adipogenic hotspots and super-enhancers. Cell Rep. 7, 1443–1455 (2014). source
The Discussion (page unavailable) says transcription factors at hotspots cooperate in chromatin remodeling and establish an active epigenomic signature, supporting the idea of a permissive chromatin environment. Our copy does not identify hotspots R1 and R2 or say that hotspots enable new transcription factor binding regions to emerge. (LLM, conf=0.82)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductcitationHematologic phenotypes were examined at more than 7 weeks of age to avoid inter-individual variation.
“Hematologic phenotypes of a balanced mix of male and female mice for all models were examined at more than 7 weeks of age to avoid inter-individual variation42.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [42] Kent, W.J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002). source
The 'Tracks Based on Homology with Other Species' section discusses mouse genome sequence alignments, not hematologic testing in mice. Searches for hematologic phenotypes, the seven-week age threshold, and inter-individual variation found no passage addressing the claim in our copy. (LLM, conf=0.76)
Experimental — accuracy not yet measured
May combine two claims.
Findingown dataIn R1/R2-/- cells, interactions between the α-globin promoters and the regions around the R1 and R2 enhancers are markedly reduced but remain above baseline levels found in ES cells.
“Although the interactions between the α-globin promoters and the regions around the R1 and R2 enhancers are markedly reduced in R1/R2-/- cells, they remain above the baseline levels found in ES cells (Supplementary Fig. 6.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe mouse α-globin super-enhancer spans 24 kb and contains five enhancer-like elements, four of which lie in introns of the adjacent widely-expressed gene Nprl3.
“The mouse α-globin super-enhancer identified using these methods spans 24 kb and contains five enhancer-like elements, four of which lie in the introns of an adjacent, widely-expressed gene (Nprl3) (Fig. 1f).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe presence of no individual hypersensitive site within the α-globin super-enhancer critically depends on any other; they appear to form independently.
“We show that the presence of no individual hypersensitive site critically depends on any other: they appear to form independently.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIn R2/R3-/- cells, the promoters' interactions with R1 are diminished but those with Rm and R4 are virtually unchanged compared to wild type.
“In R2/R3-/-,cells, the promoters’ interactions with R1 are diminished but those with Rm and R4 are virtually unchanged compared to wild type.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataR1 and R2 are distinguished by binding the greatest number of cell-specific transcription factors and the greatest amount of Mediator, and they are also associated with the most extensively modified chromatin.
“Further sub-classification of all erythroid enhancers shows that R1 and R2 are distinguished by binding the greatest number of cell-specific transcription factors and the greatest amount of Mediator, and they are also associated with the most extensively modified chromatin.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataSuper-enhancers are enriched for composite enhancers; the greater the number of constituent elements, the more likely the region will be classified as a super-enhancer.
“Analysis of the composition of regular enhancers and super-enhancers showed that, as expected, super-enhancers are enriched for composite enhancers (Fig. 1d); the greater the number of constituent elements, the more likely the region will be classified as a super-enhancer (Supplementary Fig. 1g).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
FindingcitationThe β-globin LCR corresponds to an erythroid super-enhancer.
“Previous less extensive studies have also shown that the individual regulatory elements of the β-globin LCR, which we show here also corresponds to an erythroid super-enhancer, behave in a similar independent and additive manner28.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe four evolutionarily conserved α-globin regulatory elements R1-4 fell into the class of highly Med1 bound regions when analysed as single elements, whereas Rm fell below the proposed criterion.
“Using the same methodology as described above to determine a cut-off, the four evolutionarily conserved α-globin regulatory elements (R1-4)16 still fell into the class of highly Med1 bound regions when analysed as single elements, whereas Rm (a species-specific cis-element) fell below the proposed criterion (Fig. 2b).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based). The quote only partly states this claim.
Findingnone givenEnhancer-like elements that score positively on indirect assays have no discernible function in their natural chromosomal environment.
“One puzzle emerging from this study is why enhancer-like elements which clearly score positively on indirect assays have no discernible function in their natural chromosomal environment.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataDespite their chromatin signatures, R3, R4 and Rm would not be classified as enhancers by the standard mouse transgenic assay; only those two elements with the most extensive transcription factor binding profile as determined by PAM analysis scored positively.
“Thus, despite their chromatin signatures, these three elements would not be classified as enhancers by this standard assay; only those two elements with the most extensive transcription factor binding profile as determined by PAM analysis scored positively in the assay.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataRemoval of two elements does not appear to abrogate the formation of other enhancer elements, showing that the cluster does not act as an interdependent holo-complex.
“Similarly, removal of two elements does not appear to abrogate the formation of other enhancer elements showing that the cluster does not act as an interdependent holo-complex.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataEach element within the α-globin super-enhancer cluster appears to act independently rather than co-operatively, with removal of any one element having little or no effect on the function of the others.
“we have found that each element within the cluster appears to act independently rather than co-operatively, with removal of any one element having little or no effect on the function of the others.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataR1 and R2 are strong enhancers in their natural chromosomal environment, whereas R3, Rm and R4 are either very weak enhancers or are not enhancers at all, despite their chromatin marks.
“Whereas R1 and R2 are strong enhancers in their natural chromosomal environment, R3, Rm and R4 are either very weak enhancers or are not enhancers at all, despite their chromatin marks.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataAlthough all components of the α-globin cluster would be classified as enhancers, they are not functionally equivalent.
“Importantly we have shown that although all components of the cluster would be classified as enhancers they are not functionally equivalent.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataThe α-globin super-enhancer does not act as a multi-component structure; removal of any single element cannot abolish its function.
“Importantly, it can be seen that the super-enhancer does not act as a multi-component structure; removal of any single element cannot abolish its function.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
Study design and conductreasoningSouthern blotting was used to identify clones in which the second enhancer had been targeted in cis, and deleted in isolation, maintaining the coding sequence of Nprl3.
“Southern blotting was used to identify clones in which the second enhancer had been targeted in cis, and deleted in isolation, maintaining the coding sequence of Nprl3.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataMotif analysis results were used to validate the PAM clustering analysis.
“The results were inspected, and used to validate the PAM clustering analysis.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll mouse work was performed in accordance with UK Home Office regulations under approved project licences.
“All mouse work was performed in accordance with UK Home Office regulations under approved project licences.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Study design and conductnone givenSouthern blotting and sequencing were used to confirm the correct deletion.
“Southern blotting and sequencing were used to confirm the correct deletion.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll mice were generated on the same complex background.
“All mice were generated on the same complex background.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenCorrectly targeted clones were identified by Southern blotting and karyotyped prior to blastocyst injection and chimera generation.
“Correctly targeted clones were identified by Southern blotting, and were karyotyped prior to blastocyst injection and chimera generation.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
PremisecitationThe study of mammalian enhancers is hampered by the observation that these elements are defined by criteria only partially or indirectly related to their role in vivo.
“The study of mammalian enhancers is hampered by the observation that these elements are defined by criteria only partially or indirectly related to their role in vivo9,10.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [9] Heintzman, N.D. et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311–318 (2007). source
- cites [10] Kvon, E.Z. Using transgenic reporter assays to functionally characterize enhancers in animals. Genomics 106, 185–192 (2015). source
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenReticulocyte counts were made by two independent assessors blinded to genotype.
“Reticulocyte preparations were made using Brilliant Cresyl Blue staining, and counts were made by two independent assessors blinded to genotype.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductown dataThe PAM clustering of annotated enhancers was validated using silhouette plots, and the best clustering was 16 clusters.
“The clustering results were visually inspected, and validated in silhouette plots, and the best clustering (fewest outliers, high average silhouette value for all clusters) was seen when our annotated enhancers were divided to 16 clusters.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
It is unlikely that cis-acting elements emerge fully formed during evolution or that they disappear entirely when they lose function.
“It is unlikely that cis-acting elements emerge fully formed during evolution or that they disappear entirely when they lose function”
The paper relies on this without support.
The residual loxP site and multiple cloning site footprint left by the recombination do not affect local gene expression.
“This recombination leaves only a single residual loxP site and multiple cloning site footprint from the targeting vector.”
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?
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)”
From Super-enhancers include classical enhancers and facilitators to fully activate gene expression (2023) · cites this paper as [17]
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 measuredThe α-SE is an ideal model for detailed genetic analysis, as its perturbation has no effect on cell identity or differentiation.
“The α-SE is an ideal model for detailed genetic analysis, as its perturbation has no effect on cell identity or differentiation.17”
From Super-enhancers include classical enhancers and facilitators to fully activate gene expression (2023) · cites this paper as [17]
The Abstract (page unavailable) describes the α-globin super-enhancer as the subject of a detailed genetic dissection, supporting its use as a model. But the Results (“No single element is critical for globin gene expression,” page unavailable) report ineffective erythropoiesis after some deletions, and the paper does not establish that perturbation has no effect on cell identity or differentiation. (LLM, conf=0.84)
Experimental — accuracy not yet measuredFacilitators, as described here, may be relatively common elements in multipartite enhancers.
“Review of diverse, albeit incomplete, previous analyses of SEs16,17,19,20,22 suggest that facilitators, as described here, may be relatively common elements in multipartite enhancers.”
From Super-enhancers include classical enhancers and facilitators to fully activate gene expression (2023) · cites this paper as [17]
The Abstract and Discussion (page unavailable) describe a multipartite α-globin enhancer cluster containing strong and weak or apparently inactive elements. They do not identify any element as a facilitator or establish that facilitators are relatively common; the Discussion says the apparently inactive elements have no discernible function in their natural chromosomal environment. (LLM, conf=0.76)
Experimental — accuracy not yet measuredThe 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”
From Super-enhancers include classical enhancers and facilitators to fully activate gene expression (2023) · cites this paper as [17]
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 measuredThe mouse α-globin SE is made up of five constituent elements (R1, R2, R3, Rm, and R4).
“The mouse α-globin SE (α-SE) is made up of five constituent elements (R1, R2, R3, Rm, and R4)”
From Super-enhancers include classical enhancers and facilitators to fully activate gene expression (2023) · cites this paper as [17]
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”
From Super-enhancers include classical enhancers and facilitators to fully activate gene expression (2023) · cites this paper as [17]
Transcriptional effects of enhancer deletions within extended super-enhancer loci are consistent with generally additive effects of individual enhancers.
“the transcriptional effects of enhancer deletions within extended super-enhancer loci are also consistent with the generally additive effects of individual enhancers [517, 518].”
From Transcriptional enhancers and their communication with gene promoters (2021) · cites this paper as [517]
Targeting disruption of specific enhancer elements has demonstrated that many long-range regulatory elements additively enhance transcription.
“Furthermore, targeting disruption of specific enhancer elements has demonstrated that many of these long range regulatory elements additively enhance transcription (Hay et al., 2016) (Figure 4A).”
From Unraveling Hematopoiesis through the Lens of Genomics (2020) · cites this paper as Hay et al., 2016
The α-globin genes are regulated by four conserved erythroid-specific enhancers (R1-R4) and a mouse specific element (Rm) located 14-38 kb upstream of the promoters.
“We have shown that the α-globin genes are regulated by four conserved erythroid-specific enhancers (R1-R4) and a mouse specific element (Rm) located 14-38 kb upstream of the promoters15–17.”
From Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo (2017) · cites this paper as [15]
Four of these enhancers (R1, R2, R3 and Rm) lie within the introns of an adjacent widely expressed gene (Nprl3).
“Four of these enhancers (R1, R2, R3 and Rm) lie within the introns of an adjacent widely expressed gene (Nprl3)15.”
From Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo (2017) · cites this paper as [15]
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Self-citation
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Research works printed but not linked: 1
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A Phase Separation Model for Transcriptional Control
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Transcriptional Addiction in Cancer
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“…2016; Kieffer-Kwon et al., 2013) and can function as independent or interdependent components of these large transcription-regulating complexes to drive high-level expression of their associated genes (Hah et al., 2015; Hay et al., 2016; Hnisz et al., 2015; Jiang et al., 2016; Shin et al., 2016).”
Ever-Changing Landscapes: Transcriptional Enhancers in Development and Evolution
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“Many demonstrations of the modular and additive behavior of enhancers exist, both for elements controlling the same locus across different tissues and for simultaneously active individual elements within a super-enhancer (for example, Hay et al., 2016; Maeda and Karch, 2011; Visel et al., 2009).”
BackgroundStructural variation in the 3D genome
Spielmann, M., Lupiáñez, D. G., & Mundlos, S. (2018) · Nature Reviews Genetics · doi:10.1038/s41576-018-0007-0 · Cited by 719 · see citing works
Enhancer redundancy provides phenotypic robustness in mammalian development
Osterwalder, M., Barozzi, I., Tissières, V., Fukuda-Yuzawa, Y., Mannion, B. J., Afzal, S. Y., Lee, E. A., Zhu, Y., Plajzer-Frick, I., Pickle, C. S., et al. (2018) · Nature · doi:10.1038/nature25461 · Cited by 642 · see citing works
“Founder (F0) mice were genotyped using PCR with High Fidelity Platinum Taq Polymerase (Thermo Fisher Scientific) to identify those with the desired non-homologous end joining (NHEJ)-generated deletion breakpoints (see Extended Data Figs 1a–j, 5a–c, 7a, d and Supplementary Table 3 for genotyping strategy, primer sequen…”
MethodInfluentialSelf-citationMultiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells
Xie, S., Duan, J., Li, B., Zhou, P., & Hon, G. C. (2017) · Molecular Cell · doi:10.1016/j.molcel.2017.03.007 · Cited by 334 · see citing works
“These results are consistent with recent reports (Hay et al., 2016; Hnisz mbination with sgRNAs targeting PIM1 SE2 (right).”
ResultThe AP-1 transcriptional complex: Local switch or remote command?
Bejjani, F., Evanno, E., Zibara, K., Piechaczyk, M., & Jariel‐Encontre, I. (2019) · Biochimica et Biophysica Acta (BBA) - Reviews on Cancer · doi:10.1016/j.bbcan.2019.04.003 · Cited by 355 · see citing works
Super-Enhancer-Driven Transcriptional Dependencies in Cancer
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Regulation of disease-associated gene expression in the 3D genome
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“Examples of genes that depend on multiple enhancers for expression in a single tissue include the α and βglobin genes 31,33 , Wap (encoding whey acidic protein) 30 and Aicda (encod ing activationinduced cytidine deaminase) 34 .”
The relationship between genome structure and function
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Self-citationDeciphering the multi-scale, quantitative cis-regulatory code
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“OCT4 peaks were annotated as putative promoters or putative distal regulatory elements in a manner similar to that described previously (Hay et al., 2016), using the relative and absolute coverage of H3K4me3 (a promoter-associated modification; Yue et al.”
MethodSuper-Enhancer-Mediated RNA Processing Revealed by Integrative MicroRNA Network Analysis
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“…recent reports have explored the contribution of the constituent enhancers of SEs to expression of mRNA transcripts, with varying results (Hay et al., 2016; Hnisz et al., 2015; Shin et al., 2016); it remains unclear whether multiple SE constituents have redundant or non-redundant…”
Dissecting super-enhancer hierarchy based on chromatin interactions
Huang, J., Li, K., Cai, W., Liu, X., Zhang, Y., Orkin, S. H., Xu, J., & Yuan, G.-C. (2018) · Nature Communications · doi:10.1038/s41467-018-03279-9 · Cited by 241 · see citing works
“In some studies, the results are consistent with a model whereby SEs are composed of a hierarchy of both essential and dispensable constituent enhancers to coordinate gene transcription (Hay et al., 2016; Hnisz et al., 2015; Huang et al., 2016; H. Y. Shin et al., 2016).”
BackgroundResultEnhancer redundancy in development and disease
Kvon, E. Z., Waymack, R., Gad, M., & Wunderlich, Z. (2021) · Nature Reviews Genetics · doi:10.1038/s41576-020-00311-x · Cited by 226 · see citing works
Transposable elements as a potent source of diverse cis -regulatory sequences in mammalian genomes
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Evaluating Enhancer Function and Transcription
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Functional Dissection of the Enhancer Repertoire in Human Embryonic Stem Cells
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“Four of these enhancers (R1, R2, R3 and Rm) lie within the introns of an adjacent widely expressed gene (Nprl3)15.”
Self-citationCatarino, R. R., & Stark, A. (2018) · Genes & Development · doi:10.1101/gad.310367.117 · Cited by 205 · see citing works
“…deletion of individual constituent enhancers within SEs revealed that enhancer activity is mostly dependent on a few constituents that activate transcription predominately additively (Hnisz et al. 2015; Hay et al. 2016; Moorthy et al. 2017; Xie et al. 2017; for discussion, see Dukler et al. 2016).”
BackgroundSuper-enhancers include classical enhancers and facilitators to fully activate gene expression
Blayney, J. W., Francis, H., Rampasekova, A., Camellato, B., Mitchell, L., Stolper, R., Cornell, L., Babbs, C., Boeke, J. D., Higgs, D. R., et al. (2023) · Cell · doi:10.1016/j.cell.2023.11.030 · Cited by 196 · see citing works
“16,17 As we have shown here, this level of dissection is insufficient to unequivocally conclude whether a cluster combines additively or non-additively.”
BackgroundInfluentialSelf-citationSingle-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains
Oudelaar, A. M., Davies, J. O. J., Hanssen, L. L. P., Telenius, J. M., Schwessinger, R., Liu, Y., Brown, J. M., Downes, D. J., Chiariello, A. M., Bianco, S., et al. (2018) · Nature Genetics · doi:10.1038/s41588-018-0253-2 · Cited by 196 · see citing works
Epigenetics of Atherosclerosis: Emerging Mechanisms and Methods
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Moorthy, S. D., Davidson, S., Shchuka, V. M., Singh, G., Malek-Gilani, N., Langroudi, L., Martchenko, A., So, V., Macpherson, N. N., & Mitchell, J. A. (2016) · Genome Research · doi:10.1101/gr.210930.116 · Cited by 190 · see citing works
“Furthermore, Hay et al. (2016) showed by deletions within the Hba super-enhancer that each constituent enhancer acts independently in an additive manner to regulate expression of Hba genes in vivo.”
Fuentes, D., Swigut, T., & Wysocka, J. (2018) · eLife · doi:10.7554/elife.35989 · Cited by 191 · see citing works
Kron, K. J., Murison, A. J., Zhou, S., Huang, V., Yamaguchi, T. N., Shiah, Y.-J., Fraser, M. E., van der Kwast, T., Boutros, P. C., Bristow, R. G., et al. (2017) · Nature Genetics · doi:10.1038/ng.3930 · Cited by 184 · see citing works
Polymer Simulations of Heteromorphic Chromatin Predict the 3D Folding of Complex Genomic Loci
Buckle, A., Brackley, C. A., Boyle, S., Marenduzzo, D., & Gilbert, N. (2018) · Molecular Cell · doi:10.1016/j.molcel.2018.09.016 · Cited by 172 · see citing works
Defining genome architecture at base-pair resolution
Peng, H., Badat, M., Hanssen, L. L. P., Hentges, L. D., Crump, N. T., Downes, D. J., Jeziorska, D. M., Oudelaar, A. M., Schweßinger, R., Taylor, S., et al. (2021) · Nature · doi:10.1038/s41586-021-03639-4 · Cited by 176 · see citing works
Self-citationLoss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder
Long, H. K., Osterwalder, M., Welsh, I. C., Hansen, K. L., Davies, J. O. J., Liu, Y. E., Koska, M., Adams, A. T., Aho, R., Arora, N., et al. (2020) · Cell stem cell · doi:10.1016/j.stem.2020.09.001 · Cited by 160 · see citing works
Self-citationEnhancer Logic and Mechanics in Development and Disease
Rickels, R. A., & Shilatifard, A. (2018) · Trends in Cell Biology · doi:10.1016/j.tcb.2018.04.003 · Cited by 165 · see citing works
Uyehara, C. M., & Apostolou, E. (2023) · Cell Reports · doi:10.1016/j.celrep.2023.112068 · Cited by 143 · see citing works
Thandapani, P. (2019) · Pharmacology & Therapeutics · doi:10.1016/j.pharmthera.2019.02.014 · Cited by 163 · see citing works
Unraveling Hematopoiesis through the Lens of Genomics
Liggett, L. A., & Sankaran, V. G. (2020) · Cell · doi:10.1016/j.cell.2020.08.030 · Cited by 163 · see citing works
“targeting disruption of specific enhancer elements has demonstrated that many of these long range regulatory elements additively enhance transcription (Hay et al., 2016) (Figure 4A).”
Berthelot, C., Villar, D., Horvath, J. E., Odom, D. T., & Flicek, P. (2017) · Nature Ecology & Evolution · doi:10.1038/s41559-017-0377-2 · Cited by 159 · see citing works
Generating specificity in genome regulation through transcription factor sensitivity to chromatin
Isbel, L., Grand, R. S., & Schübeler, D. (2022) · Nature Reviews Genetics · doi:10.1038/s41576-022-00512-6 · Cited by 149 · see citing works
Enhancer grammar in development, evolution, and disease: dependencies and interplay
Jindal, G. A., & Farley, E. K. (2021) · Developmental Cell · doi:10.1016/j.devcel.2021.02.016 · Cited by 147 · see citing works
“Other aspects of transcriptional regulation that may affect enhancer grammar include enhancer-enhancer interactions, additive or synergistic activity of enhancers, and transvection (Fukaya and Levine, 2017; Hay et al., 2016; Maekawa et al., 1989).”
BackgroundSuper-enhancers in transcriptional regulation and genome organization
Wang, X., Cairns, M. J., & Yan, J. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz1038 · Cited by 154 · see citing works
Reconstruction of the Global Neural Crest Gene Regulatory Network In Vivo
Williams, R. M., Candido-Ferreira, I., Repapi, E., Gavriouchkina, D., Senanayake, U., Ling, I. T. C., Telenius, J., Taylor, S., Hughes, J., & Sauka-Spengler, T. (2019) · Developmental Cell · doi:10.1016/j.devcel.2019.10.003 · Cited by 122 · see citing works
Self-citationPeng, L., Jiang, B., Yuan, X., Qiu, Y., Peng, J., Huang, Y., Zhang, C., Zhang, Y., Lin, Z., Li, J., et al. (2018) · Cancer Research · doi:10.1158/0008-5472.can-18-0367 · Cited by 141 · see citing works
Genome-wide mapping of autonomous promoter activity in human cells
van Arensbergen, J., FitzPatrick, V. D., de Haas, M., Pagie, L., Sluimer, J., Bussemaker, H. J., & van Steensel, B. (2017) · Nature Biotechnology · doi:10.1038/nbt.3754 · Cited by 140 · see citing works
An encyclopedia of enhancer-gene regulatory interactions in the human genome
Gschwind, A. R., Mualim, K. S., Karbalayghareh, A., Sheth, M. U., Dey, K. K., Jagoda, E., Nurtdinov, R. N., Xi, W., Tan, A. S., Jones, H., et al. (2023) · doi:10.1101/2023.11.09.563812 · Cited by 119 · see citing works
The three-dimensional genome: regulating gene expression during pluripotency and development
Andrey, G., & Mundlos, S. (2017) · Development · doi:10.1242/dev.148304 · Cited by 129 · see citing works
“…vivo studies of the α-globin gene locus, which is controlled by a cluster of enhancer elements, also referred to as a super enhancer, have shown that each enhancer seems to act independently and in an additive fashion without clear evidence of synergistic or higher-order effects (Hay et al., 2016).”
BackgroundWill, A. J., Cova, G., Osterwalder, M., Chan, W.-L., Wittler, L., Brieske, N., Heinrich, V., de Villartay, J., Vingron, M., Klopocki, E., et al. (2017) · Nature Genetics · doi:10.1038/ng.3939 · Cited by 126 · see citing works
Thurner, M., van de Bunt, M., Torres, J., Mahajan, A., Nylander, V., Bennett, A. J., Gaulton, K. J., Barrett, A., Burrows, C., Bell, C. G., et al. (2018) · eLife · doi:10.7554/elife.31977 · Cited by 124 · see citing works
Novo, C. L., Javierre, B.-M., Cairns, J., Segonds-Pichon, A., Wingett, S. W., Freire-Pritchett, P., Furlan-Magaril, M., Schoenfelder, S., Fraser, P., & Rugg-Gunn, P. J. (2018) · Cell Reports · doi:10.1016/j.celrep.2018.02.040 · Cited by 119 · see citing works
“Equally, questions have been raised as to whether SEs should be considered as separate entities from clusters of canonical enhancers or locus control regions (Moorthy et al., 2017; Bojcsuk et al., 2017; Hay et al., 2016; Pott and Lieb, 2015), and thus, it is fundamental to investigate the properties of SEs.”
BackgroundDynamics of the 4D Genome during in Vivo Lineage Specification and Differentiation
Oudelaar, A. M., Beagrie, R. A., Gosden, M., de Ornellas, S., Georgiades, E., Kerry, J., Hidalgo, D., Carrelha, J., Shivalingam, A., El-Sagheer, A. H., et al. (2020) · Nature Communications · doi:10.1038/s41467-020-16598-7 · Cited by 119 · see citing works
Self-citationNested epistasis enhancer networks for robust genome regulation
Lin, X., Liu, Y., Liu, S., Zhu, X., Wu, L., Zhu, Y., Zhao, D., Xu, X., Chemparathy, A. G., Wang, H., et al. (2022) · Science · doi:10.1126/science.abk3512 · Cited by 111 · see citing works
Evidence for additive and synergistic action of mammalian enhancers during cell fate determination
Choi, J., Lysakovskaia, K., Stik, G., Demel, C., Söding, J., Tian, T. V., Graf, T., & Cramer, P. (2021) · eLife · doi:10.7554/elife.65381 · Cited by 117 · see citing works
“In contrast to our time-resolved study, previous studies investigated enhancer cooperation in steady state or at an end point, and at selected genes, rather than genome-wide (Hay et al., 2016; Osterwalder et al., 2018; Stine et al., 2011).”
BackgroundResultTranscriptional Regulation by (Super)Enhancers: From Discovery to Mechanisms
Grosveld, F., van Staalduinen, J., & Stadhouders, R. (2021) · Annual Review of Genomics and Human Genetics · doi:10.1146/annurev-genom-122220-093818 · Cited by 115 · see citing works
Carleton, J. B., Berrett, K. C., & Gertz, J. (2017) · Cell Systems · doi:10.1016/j.cels.2017.08.011 · Cited by 113 · see citing works
“In dissection of the murine alpha globin enhancer (Hay et al., 2016), as well as enhancers near the c-fos gene (Joo et al., 2016), similar seemingly non-functional regulatory elements were identified, suggesting that these sites may be a pervasive feature of mammalian genomes.”
BackgroundGillespie, M. A., Palii, C. G., Sanchez-Taltavull, D., Shannon, P., Longabaugh, W. J. R., Downes, D. J., Sivaraman, K., Espinoza, H. M., Hughes, J. R., Price, N. D., et al. (2020) · Molecular Cell · doi:10.1016/j.molcel.2020.03.031 · Cited by 110 · see citing works
Self-citationThomas, H. F., Kotova, E., Jayaram, S., Pilz, A., Romeike, M., Lackner, A., Penz, T., Bock, C., Leeb, M., Halbritter, F., et al. (2021) · Molecular Cell · doi:10.1016/j.molcel.2020.12.047 · Cited by 98 · see citing works
Transcription imparts architecture, function and logic to enhancer units
Tippens, N. D., Liang, J. H., Leung, A. K.-Y., Wierbowski, S. D., Ozer, A., Booth, J. G., Lis, J. T., & Yu, H. (2020) · Nature Genetics · doi:10.1038/s41588-020-0686-2 · Cited by 101 · see citing works
Targeting Super-Enhancers for Disease Treatment and Diagnosis.
Shin, H. Y. (2018) · Europe PMC (PubMed Central) · doi:10.14348/molcells.2018.2297 · Cited by 97 · see citing works
“However, individual constituent super-enhancers in erythroid cells showed independent functions without any hierarchy, indicating that the constituent enhancers function differently in different cell types or under different environments (Hay et al., 2016).”
BackgroundTesting the super-enhancer concept
Blobel, G. A., Higgs, D. R., Mitchell, J. A., Notani, D., & Young, R. A. (2021) · Nature Reviews Genetics · doi:10.1038/s41576-021-00398-w · Cited by 94 · see citing works
Self-citationKLF1 drives the expression of fetal hemoglobin in British HPFH
Wienert, B., Martyn, G. E., Kurita, R., Nakamura, Y., Quinlan, K. G. R., & Crossley, M. (2017) · Blood · doi:10.1182/blood-2017-02-767400 · Cited by 94 · see citing works
Li, P., Mitra, S., Spolski, R. J., Oh, J., Liao, W., Tang, Z., Mo, F., Li, X., West, E. E., Gromer, D. J., et al. (2017) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1714019114 · Cited by 94 · see citing works
Self-citationOudelaar, A. M., Harrold, C. L., Hanssen, L. L. P., Telenius, J. M., Higgs, D. R., & Hughes, J. R. (2019) · Nature Communications · doi:10.1038/s41467-019-13404-x · Cited by 80 · see citing works
“The α-globin genes and their five enhancer elements, which fulfil the criteria for a super-enhancer (Hay et al., 2016), are located in a small TAD which is activated during erythroid differentiation (Brown et al., 2018).”
BackgroundSelf-citationOncogenic super-enhancer formation in tumorigenesis and its molecular mechanisms
Jia, Q., Chen, S., Tan, Y., Li, Y., & Tang, F. (2020) · Experimental & Molecular Medicine · doi:10.1038/s12276-020-0428-7 · Cited by 90 · see citing works
Transcription factor competition at the γ-globin promoters controls hemoglobin switching
Liu, N., Xu, S., Yao, Q., Zhu, Q., Yan, K., Hsu, J. Y., Sakon, P., Pinello, L., Yuan, G., Bauer, D. E., et al. (2021) · Nature Genetics · doi:10.1038/s41588-021-00798-y · Cited by 86 · see citing works
Ober-Reynolds, B., Wang, C., Ko, J. M., Rios, E. J., Aasi, S. Z., Davis, M. M., Oro, A. E., & Greenleaf, W. J. (2023) · Nature Genetics · doi:10.1038/s41588-023-01445-4 · Cited by 83 · see citing works
“These findings support an additive, modular model of enhancer activity—a model substantiated by genetic perturbation studies of individual enhancers for alpha-globin 37 and Myc 38 , studies of enhancers involved in limb development 39 and genomic-scale measures of enhancer activity 40,41 .”
Current challenges in understanding the role of enhancers in disease
Zaugg, J. B., Sahlén, P., Andersson, R., Alberich-Jordà, M., de Laat, W. L., Deplancke, B., Ferrer, J. P., Mandrup, S., Natoli, G., Plewczyński, D., et al. (2022) · Nature Structural & Molecular Biology · doi:10.1038/s41594-022-00896-3 · Cited by 77 · see citing works
“As a consequence of such regulatory complexity, many enhancers might not, individually, reveal a strong phenotype when disrupted in their endogenous context 23,56 , while still possessing endogenous enhancer activity.”
BackgroundA suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types
Ben‐Simon, Y., Hooper, M. J., Narayan, S., Daigle, T. L., Dwivedi, D., Way, S. W., Oster, A., Stafford, D. A., Mich, J. K., Taormina, M. J., et al. (2025) · Cell · doi:10.1016/j.cell.2025.05.002 · Cited by 81 · see citing works
“61,62 To diversify the use of our enhancer-based genetic toolkit, we replaced SYFP2 with a Cre recombinase, which would enable expression of a variety of tools from existing or newly generated Cre-dependent AAVs or mouse transgenes.”
Transcriptional enhancers and their communication with gene promoters
Ray-Jones, H., & Spivakov, M. (2021) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-021-03903-w · Cited by 81 · see citing works
“Likewise, the transcriptional effects of enhancer deletions within extended super-enhancer loci are also consistent with the generally additive effects of individual enhancers [517, 518].”
BackgroundKrüppel-like factors compete for promoters and enhancers to fine-tune transcription
Ilsley, M. D., Gillinder, K. R., Magor, G. W., Huang, S., Bailey, T. L., Crossley, M., & Perkins, A. C. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx441 · Cited by 81 · see citing works
Mammalian Transcription Factor Networks: Recent Advances in Interrogating Biological Complexity
Wilkinson, A. C., Nakauchi, H., & Göttgens, B. (2017) · Cell Systems · doi:10.1016/j.cels.2017.07.004 · Cited by 76 · see citing works
“However, the functional relevance of this 9 classification is still unclear (Hay et al., 2016).”
BackgroundIs a super-enhancer greater than the sum of its parts?
Dukler, N., Gulko, B., Huang, Y.-F., & Siepel, A. (2017) · Nature Genetics · doi:10.1038/ng.3759 · Cited by 79 · see citing works
Enhancer and super‐enhancer: Positive regulators in gene transcription
Peng, Y., & Zhang, Y. (2018) · Animal Models and Experimental Medicine · doi:10.1002/ame2.12032 · Cited by 74 · see citing works
Anania, C., Acemel, R. D., Jedamzick, J., Bolondi, A., Cova, G., Brieske, N., Kühn, R., Wittler, L., Real, F. M., & Lupiáñez, D. G. (2022) · Nature Genetics · doi:10.1038/s41588-022-01117-9 · Cited by 73 · see citing works
Enhancers and chromatin structures: regulatory hubs in gene expression and diseases
Hu, Z., & Tee, W. (2017) · Bioscience Reports · doi:10.1042/bsr20160183 · Cited by 78 · see citing works
Pijuan-Sala, B., Wilson, N. K., Xia, J., Hou, X., Hannah, R., Kinston, S., Calero‐Nieto, F. J., Poirion, O., Preißl, S., Liu, F., et al. (2020) · Nature Cell Biology · doi:10.1038/s41556-020-0489-9 · Cited by 71 · see citing works
LDB1-mediated enhancer looping can be established independent of mediator and cohesin
Krivega, I. V., & Dean, A. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx433 · Cited by 73 · see citing works
Sancisi, V., Manzotti, G., Gugnoni, M., Rossi, T., Gandolfi, G., Gobbi, G., Torricelli, F., Catellani, F., do Valle, Í. F., Remondini, D., et al. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx802 · Cited by 74 · see citing works
Perenthaler, E., Yousefi, S., Niggl, E., & Barakat, T. S. (2019) · Frontiers in Cellular Neuroscience · doi:10.3389/fncel.2019.00352 · Cited by 71 · see citing works
“On the other hand, an increasing number of studies have challenged this view and consider super enhancers as a collection of normal enhancers that together do not have a larger activity than the sum of the individual parts (Hay et al., 2016; Shin et al., 2016).”
BackgroundBrown, J. M., Roberts, N. A., Graham, B., Waithe, D., Lagerholm, C., Telenius, J. M., De Ornellas, S., Oudelaar, A. M., Scott, C., Szczerbal, I., et al. (2018) · Nature Communications · doi:10.1038/s41467-018-06248-4 · Cited by 72 · see citing works
Benton, M. L., Talipineni, S. C., Kostka, D., & Capra, J. A. (2019) · BMC Genomics · doi:10.1186/s12864-019-5779-x · Cited by 68 · see citing works
“We also consider a method that incorporates DNase I hypersensitive sites (DHSs) with histone modifications to generate the “ DNasePlusHistone ” enhancer set, which is composed of DHSs where the ratio of H3K4me1 to H3K4me3 is less than one [36].”
MethodVisualizing transcription: key to understanding gene expression dynamics
Brouwer, I., & Lenstra, T. L. (2019) · Current Opinion in Chemical Biology · doi:10.1016/j.cbpa.2019.05.031 · Cited by 71 · see citing works
Santos, M. D., Backer, S., Aurade, F., Wong, M. M.-K., Wurmser, M., Pierre, R., Vives, F. L., Cruzeiro, M. D., Schmitt, A., Concordet, J., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-28666-1 · Cited by 71 · see citing works
Synthetic regulatory genomics uncovers enhancer context dependence at the Sox2 locus
Brosh, R., Coelho, C., Ribeiro-dos-Santos, A. M., Ellis, G., Hogan, M. S., Ashe, H. J., Somogyi, N., Ordóñez, R., Luther, R. D., Huang, E., et al. (2023) · Molecular Cell · doi:10.1016/j.molcel.2023.02.027 · Cited by 67 · see citing works
Enhancers: bridging the gap between gene control and human disease
Karnuta, J. M., & Scacheri, P. C. (2018) · Human Molecular Genetics · doi:10.1093/hmg/ddy167 · Cited by 68 · see citing works
“There appears to be no common regulatory modality; some act additively like those at the a-globin locus (28), and others work synergistically, like those at the mammary Wap locus (29).”
BackgroundStudying Transcriptional Enhancers: The Founder Fallacy, Validation Creep, and Other Biases
Halfon, M. S. (2018) · Trends in Genetics · doi:10.1016/j.tig.2018.11.004 · Cited by 66 · see citing works
Matthews, B. J., & Waxman, D. J. (2018) · eLife · doi:10.7554/elife.34077 · Cited by 65 · see citing works
Mechanisms of Enhancer-Promoter Interactions in Higher Eukaryotes
Georgiev, P. G., & Kyrchanova, O. (2021) · International Journal of Molecular Sciences · doi:10.3390/ijms22020671 · Cited by 68 · see citing works
Transcription factors: building hubs in the 3D space
Giammartino, D. C. D., Polyzos, A., & Apostolou, E. (2020) · Cell Cycle · doi:10.1080/15384101.2020.1805238 · Cited by 66 · see citing works
Genetic Dissection of a Super Enhancer Controlling the Nppa-Nppb Cluster in the Heart
Man, J. C. K., van Duijvenboden, K., Krijger, P. H. L., Hooijkaas, I. B., van der Made, I., Vries, C. D. G.-D., Wakker, V., Creemers, E. E., de Laat, W. L., Boukens, B. J., et al. (2020) · Circulation Research · doi:10.1161/circresaha.120.317045 · Cited by 65 · see citing works
Vos, E. S. M., Valdes‐Quezada, C., Huang, Y., Allahyar, A., Verstegen, M. J. A. M., Felder, A.-K., van der Vegt, F., Uijttewaal, E. C. H., Krijger, P. H. L., & de Laat, W. L. (2021) · Molecular Cell · doi:10.1016/j.molcel.2021.06.008 · Cited by 61 · see citing works
Chromatin priming of genes in development: Concepts, mechanisms and consequences
Bonifer, C., & Cockerill, P. N. (2017) · Experimental Hematology · doi:10.1016/j.exphem.2017.01.003 · Cited by 64 · see citing works
Capture-C: a modular and flexible approach for high-resolution chromosome conformation capture
Downes, D. J., Smith, A., Karpińska, M. A., Velychko, T., Rue-Albrecht, K., Sims, D., Milne, T. A., Davies, J. O. J., Oudelaar, A. M., & Hughes, J. R. (2022) · Nature Protocols · doi:10.1038/s41596-021-00651-w · Cited by 59 · see citing works
Self-citationPeng, X. L., So, K. K. H., He, L., Zhao, Y. F., Zhou, J., Li, Y., Yao, M., Xu, B., Zhang, S., Yao, H., et al. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx488 · Cited by 61 · see citing works
Raffield, L. M., Ulirsch, J. C., Naik, R. P. P., Lessard, S., Handsaker, R. E., Jain, D., Kang, H. M., Pankratz, N., Auer, P. L., Bao, E. L., et al. (2018) · PLoS Genetics · doi:10.1371/journal.pgen.1007293 · Cited by 63 · see citing works
Spatial Organization of Chromatin: Emergence of Chromatin Structure During Development
Ghosh, R. P., & Meyer, B. J. (2021) · Annual Review of Cell and Developmental Biology · doi:10.1146/annurev-cellbio-032321-035734 · Cited by 56 · see citing works
“Finally, emergent properties of liquid condensates formed at SE clusters are difficult to reconcile with the additive rather than collective impact of enhancers in SE clusters, as seen in case of globin genes (Hay et al. 2016).”
BackgroundDecreased STARD10 Expression Is Associated with Defective Insulin Secretion in Humans and Mice
Carrat, G., Hu, M., Nguyen‐Tu, M., CHABOSSEAU, P. L., Gaulton, K. J., van de Bunt, M., Siddiq, A., Falchi, M., Thurner, M., Canouil, M., et al. (2017) · The American Journal of Human Genetics · doi:10.1016/j.ajhg.2017.01.011 · Cited by 62 · see citing works
“Raw FASTQ reads were processed with a departmental/in-house pipeline 42 and on the DNase and ChIP pipeline website (Web Resources).”
MethodSET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expressionPreprint
Cao, K., Collings, C. K., Marshall, S. A., Morgan, M. A., Rendleman, E. J., Wang, L., Sze, C. C., Sun, T., Bartom, E. T., & Shilatifard, A. (2017) · Genes & Development · doi:10.1101/gad.294744.116 · Cited by 61 · see citing works
“The redundancy observed for E1 and E2 is different from α-globin enhancers, which act in an additivemanner (Hay et al. 2016), suggesting the locus specificity of enhancer commissioning (Smith and Shilatifard 2014).”
BackgroundA model of active transcription hubs that unifies the roles of active promoters and enhancers
Zhu, I., Song, W., Ovcharenko, I., & Landsman, D. (2021) · Nucleic Acids Research · doi:10.1093/nar/gkab235 · Cited by 55 · see citing works
Influences of rare copy-number variation on human complex traits
Hujoel, M. L. A., Sherman, M. A., Barton, A. R., Mukamel, R. E., Sankaran, V. G., Terao, C. C., & Loh, P. (2022) · Cell · doi:10.1016/j.cell.2022.09.028 · Cited by 56 · see citing works
Regulating specificity in enhancer–promoter communication
Galouzis, C. C., & Furlong, E. E. M. (2022) · Current Opinion in Cell Biology · doi:10.1016/j.ceb.2022.01.010 · Cited by 60 · see citing works
Taylor, T., Sikorska, N., Shchuka, V. M., Chahar, S., Ji, C., Macpherson, N. N., Moorthy, S. D., de Kort, M. A. C., Mullany, S., Khader, N., et al. (2022) · Genes & Development · doi:10.1101/gad.349489.122 · Cited by 45 · see citing works
“…enhancers is markedly de-bated, with previous genetic dissections uncovering context-dependent transcriptional effects that may be functionally redundant or additive, but not synergistic (Hay et al. 2016; Moorthy et al. 2017; Saravanan et al. 2020; for discussion, see Blobel et al. 2021).”
Reactivation of a developmentally silenced embryonic globin gene
King, A. J., Songdej, D., Downes, D. J., Beagrie, R. A., Liu, S., Buckley, M., Peng, H., Suciu, M., Oudelaar, A. M., Hanssen, L. L. P., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-24402-3 · Cited by 53 · see citing works
Self-citationEnhancer cooperativity can compensate for loss of activity over large genomic distances
Antonović, M., Feng, S., Haslhofer, F., Huber, M., Gallardo, M. G., Loubiere, V., Vanina, D., Pitasi, M., Stark, A., & Buecker, C. (2024) · Molecular Cell · doi:10.1016/j.molcel.2024.11.008 · Cited by 50 · see citing works
Super-enhancers are transcriptionally more active and cell type-specific than stretch enhancers
Khan, A., Mathelier, A., & Zhang, X. (2018) · Epigenetics · doi:10.1080/15592294.2018.1514231 · Cited by 56 · see citing works
“Some studies have showed synergy and hierarchy between the individual enhancers within the clusters [23,43,44], but this synergy is less obvious for some developmentally regulated super-enhancers [45].”
Background
- Authors
- Hay, D., Hughes, J. R., Babbs, C., Davies, J. O. J., Graham, B. J., Hanssen, L. L. P., Kassouf, M. T., Oudelaar, A. M., Sharpe, J. A., Suciu, M. C., Telenius, J., Williams, R., Rode, C., Li, P.-S., Pennacchio, L. A., Sloane-Stanley, J. A., Ayyub, H., Butler, S., Sauka-Spengler, T., Gibbons, R. J., Smith, A. J. H., Wood, W. G., & Higgs, D. R.
- Venue
- Nature Genetics
- Published
- 2016-07-04
- Volume
- 48
- Issue
- 8
- Pages
- 895-903
- ISSN
- 1061-4036 (ISSN-L)
- 1546-1718
- PMC
- PMC5058437
- OpenAlex
- W2464000940
- Cited by
- 385
- References
- 42
- Citations by year
- 2026: 20 · 2025: 37 · 2024: 36 · 2023: 36 · 2022: 44 · 2021: 46 · 2020: 40 · 2019: 28 · 2018: 41 · 2017: 41 · 2016: 16
- FWCI
- 18.99
- Type
- journal article
- Language
- en
- Open access
- green
- www.osti.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Genetics (publishedVersion)
- PubMed (publishedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- PubMed Central (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Research Explorer (The University of Manchester) (submittedVersion)
- Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, other-oa)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Funders
- U.S. Department of Energy (-AC02-05CH11231, 05CH11231, DE AC02 05CH11231, DE-AC02-)
- Wellcome Trust (106130/Z/14/Z)
- National Institute for Health and Care Research
- National Institutes of Health (DE-AC02–05CH11231, R01-HG003988, U54 HG006997)
- Medical Research Council (MC_PC_15069, MR/N00969X/1, DE-AC02-05CH11231)
- Biomedical Research Council
- National Human Genome Research Institute (DE-AC02-05CH11231, U54HG006997, R01-HG003988)
- National Institute of Dental and Craniofacial Research (U01 DE020060, DE-AC02-05CH11231)
- Lawrence Berkeley National Laboratory (AC02‐05CH11231, DE-AC02-05CH11231, U54HG006997, R01HG003988, 05CH11231)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Health Sciences › Medicine › Genetics › Hemoglobinopathies and Related Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
- Keywords
- super-enhancers, enhancer deletion, erythroid cells, hematological phenotype, chromatin structure, chromosome conformation, Mediator complex, transcriptional regulation, gene expression
- MeSH terms
- Animals, Chromatin, Embryo, Mammalian, Enhancer Elements, Genetic, Gene Expression Regulation, Transcription Factors, Transcription, Genetic, Mice, Knockout, Erythroid Cells, Mice, 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.
Cited sources are checked by gpt-6-sol.
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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