Transcriptional enhancers and their communication with gene promoters

At least some pioneer factors, such as FOXA, OCT4 and SOX2/SOX11, initiate chromatin opening by direct displacement of histones.

“The mechanisms of pioneer factor action are still being established [37], but at least some of them (such as FOXA, OCT4 and SOX2/SOX11) have been shown to initiate chromatin opening by direct displacement of histones [38–41], whilst many others recruit chromatin remodelers [42–50].”

• cites [38] Iwafuchi-Doi M, Donahue G, Kakumanu A et al (2016) The pioneer transcription factor FoxA maintains an accessible nucleosome configuration at enhancers for tissue-specific gene activation. Mol Cell 62:79–91 sourceSupported
• cites [39] Echigoya K, Koyama M, Negishi L et al (2020) Nucleosome binding by the pioneer transcription factor OCT4. Sci Rep 10:11832 sourceDisputed

  The Introduction says OCT4 may help convert closed chromatin to open chromatin, but it does not attribute that opening to direct histone displacement. The Results section “Linker histone H1 competes with OCT4 for nucleosome binding” reports the opposite of the claimed mechanism for OCT4: adding OCT4 did not displace H1; adding H1 removed OCT4 from the nucleosome. The Discussion describes SOX2/SOX11 peeling DNA from histones, not displacing the histones. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [40] Dodonova SO, Zhu F, Dienemann C et al (2020) Nucleosome-bound SOX2 and SOX11 structures elucidate pioneer factor function. Nature 580:669–672 sourceNot checked — no full text of the source
• cites [41] Jacobs J, Atkins M, Davie K et al (2018) The transcription factor Grainy head primes epithelial enhancers for spatiotemporal activation by displacing nucleosomes. Nat Genet 50:1011–1020 sourcePartly supported

  The introductory passage (labelled Abstract in our copy; page unavailable) describes pioneer factors as transcription factors that can displace nucleosomes and names FOXA1 and SOX2 as examples. The Results section, “Functional Grainyhead motifs are embedded in a specific DNA context” (page unavailable), also associates pioneer-factor binding with nucleosome displacement. But the paper does not establish direct histone displacement for each named factor: it mentions OCT4 as a possible additional factor needed for SOX2-dependent enhancer activation and does not mention SOX11. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

The majority of active enhancers are themselves transcribed, producing enhancer RNAs in a bi-directional manner.

“The majority of active enhancers are themselves transcribed, producing enhancer RNAs (eRNAs) in a bi-directional manner [73–77].”

• cites [73] Core LJ, Martins AL, Danko CG et al (2014) Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers. Nat Genet 46:1311–1320 sourcePartly supported

  The Abstract says enhancer transcription is widespread and that eRNAs are produced bidirectionally. The Discussion also reports bidirectional initiation at thousands of enhancers. Neither passage establishes that a majority of active enhancers are transcribed; the nearly 80% figure in the Abstract concerns promoters, not enhancers. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [74] Andersson R, Gebhard C, Miguel-Escalada I et al (2014) An atlas of active enhancers across human cell types and tissues. Nature 507:455–461 sourceSupported
• cites [75] Lam MTY, Cho H, Lesch HP et al (2013) Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription. Nature 498:511–515 sourcePartly supported

  The passage reporting GRO-Seq results (section labeled Abstract; page unavailable) found eRNA initiation at 76% of Rev-Erb binding sites in enhancer-like regions and bidirectional transcription at 56% of those sites. This supports the pattern in the studied macrophage-associated sites, but not the broader claim that a majority of active enhancers generally are transcribed bidirectionally. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [76] De Santa F, Barozzi I, Mietton F et al (2010) A large fraction of extragenic RNA pol II transcription sites overlap enhancers. PLoS Biol 8:e1000384 sourceDisputed

  The Discussion says that non-transcribed enhancers greatly outnumber transcribed ones, contrary to the claim that a majority are transcribed. The Introduction also gives an example of unidirectional enhancer-region transcription; this paper does not establish bidirectional production as the general pattern. No page numbers are available in our copy. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [77] Kim T-K, Hemberg M, Gray JM et al (2010) Widespread transcription at neuronal activity-regulated enhancers. Nature 465:182–187 sourcePartly supported

  The Transcription at enhancers section reports that eRNAs are produced bi-directionally at neuronal activity-regulated enhancers. It does not support the claim that a majority of active enhancers are transcribed: the Methods Summary reports detectable eRNAs at 44% of extragenic enhancers, and the findings are limited to neuronal enhancers. Page numbers are not provided in our copy. (LLM, conf=0.89)

  Experimental — accuracy not yet measured

A significant minority of enhancers in human ESCs show pre-existing "permissive" promoter contacts prior to activation.

“Nonetheless, a significant minority of enhancers in human ESCs also show pre-existing “permissive” promoter contacts prior to activation [141, 394].”

• cites [141] Freire-Pritchett P, Schoenfelder S, Várnai C et al (2017) Global reorganisation of regulatory units upon lineage commitment of human embryonic stem cells. Elife 6:e21926 sourceSupported
• cites [394] Rubin AJ, Barajas BC, Furlan-Magaril M et al (2017) Lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation. Nat Genet 49:1522–1528 sourceDisputed

  The Abstract says the pre-established enhancer–promoter contacts studied were present in epidermal progenitor cells but not in embryonic stem cells. Its analysis of hESC promoter-contact data likewise reports reduced contact signal relative to keratinocytes. The paper places these pre-established contacts after pluripotency, not in hESCs. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

In the classic scenario, the boundary is presented by CTCF binding to its sequence motifs located in a divergent orientation at either end of the loop.

“In the classic scenario, this boundary is presented by CTCF binding to its sequence motifs located in a divergent orientation at either end of the loop [277, 438–440]”

• cites [277] Rao SSP, Huntley MH, Durand NC et al (2014) A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159:1665–1680 sourceDisputed

  The Results section reports that 92% of CTCF motif pairs at the examined loop loci are convergent, not divergent, and that convergent pairs are overwhelmingly more frequent than divergent pairs. The Discussion likewise says that motifs at loop anchors are convergent in more than 90% of cases. Page numbers were not supplied in our copy. (LLM, conf=0.95)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [438] Guo Y, Monahan K, Wu H et al (2012) CTCF/cohesin-mediated DNA looping is required for protocadherin α promoter choice. Proc Natl Acad Sci USA 109:21081–21086 sourceNot checked — no full text of the source
• cites [439] Guo Y, Xu Q, Canzio D et al (2015) CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162:900–910 sourcePartly supported

  In Results, under “Directional CTCF Binding in Genome-wide DNA Looping Specificity” (page unavailable), the paper reports reverse-forward CTCF-binding-site pairs at boundaries between neighboring domains, consistent with divergent sites at a boundary. But it reports that most CTCF-mediated interactions within loops involve forward-reverse, convergent site pairs. It therefore does not support describing the sites at either end of a loop as divergent. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [440] Nanni L, Ceri S, Logie C (2020) Spatial patterns of CTCF sites define the anatomy of TADs and their boundaries. Genome Biol 21:197 sourcePartly supported

  The Results section reports that divergent CTCF-site patterns are enriched at TAD boundaries and suggests they confer boundary function (page not provided). But the paper distinguishes boundaries from loop ends: its Results section says loop bases involve pairs of convergent CTCF sites. It therefore does not support placing divergent motifs at either end of a loop. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

In Drosophila, Zelda is critical for lowering the nucleosome barrier at enhancers for genes driving zygotic gene activation.

“In Drosophila, the Zinc Finger Early Drosophila Activator (Zelda) is critical for lowering the nucleosome barrier at enhancers for genes driving zygotic gene activation [33–35].”

• cites [33] Liang H-L, Nien C-Y, Liu H-Y et al (2008) The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila. Nature 456:400–403 sourcePartly supported

  The paper’s Abstract reports that Zelda binds regulatory TAGteam sites and plays a key role in activating the early zygotic genome. It does not say that Zelda lowers a nucleosome barrier at enhancers; the passages retrieved for nucleosome occupancy and chromatin accessibility do not establish that mechanism. No page numbers are available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [34] Harrison MM, Li X-Y, Kaplan T et al (2011) Zelda binding in the early Drosophila melanogaster embryo marks regions subsequently activated at the maternal-to-zygotic transition. PLoS Genet 7:e1002266 sourcePartly supported

  The Discussion (page unavailable) says Zelda is important at promoters and enhancers and proposes that it promotes zygotic genome activation by controlling chromatin accessibility. But it does not establish that Zelda lowers a nucleosome barrier; it explicitly allows that Zelda may maintain chromatin that is already accessible rather than actively open it. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [35] Schulz KN, Bondra ER, Moshe A et al (2015) Zelda is differentially required for chromatin accessibility, transcription factor binding, and gene expression in the early Drosophila embryo. Genome Res 25:1715–1726 sourceSupported

A key function of sequence-specific transcription factors is the recruitment of workhorse cofactors that facilitate enzymatic chromatin remodelling, histone modification and act as scaffolds for recruitment of additional factors.

“Mechanistically, a key function of sequence-specific TFs is the recruitment of “workhorse” cofactors that facilitate enzymatic chromatin remodelling, histone modification and act as scaffolds for recruitment of additional factors.”

• cites [56] Zabidi MA, Stark A (2016) Regulatory enhancer-core-promoter communication via transcription factors and cofactors. Trends Genet 32:801–814 sourcePartly supported

  The section on differential activity of trans factors (page unavailable) says sequence-specific TFs recruit cofactors, including the SWI/SNF complex and p300, and identifies Mof as an acetyltransferase. The section on activating micro-environments describes cofactor activity leading to recruitment of BRD4, but does not establish the claimed scaffold role for recruiting additional factors. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [57] Winkler DD, Luger K (2011) The histone chaperone FACT: structural insights and mechanisms for nucleosome reorganization. J Biol Chem 286:18369–18374 sourcePartly supported

  The introduction and “FACT Function and Architecture” section describe FACT as a histone chaperone that reorganizes nucleosomes during transcription. The “Modifying” FACT Function section says histone modifications can recruit chromatin-altering machinery. The copy does not establish the claim’s central mechanism—that sequence-specific transcription factors recruit cofactors—or describe those cofactors as scaffolds for additional factors. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

P300 and CBP serve as scaffolds that connect RNA polymerase II and RNAP-associated general transcription factors to chromatin.

“P300 and CBP also serve as scaffolds that connect RNA polymerase II (RNAP) and RNAP-associated “general transcription factors” to the chromatin [64, 65].”

• cites [64] Chan HM, La Thangue NB (2001) p300/CBP proteins: HATs for transcriptional bridges and scaffolds. J Cell Sci 114:2363–2373 sourceNot checked — no full text of the source
• cites [65] Kim TK, Kim TH, Maniatis T (1998) Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-beta enhanceosome in vitro. Proc Natl Acad Sci USA 95:12191–12196 sourcePartly supported

  The Results and Discussion (page unavailable) show that interactions between CBP and the IFN-β enhanceosome help recruit an RNA polymerase II holoenzyme containing general transcription factors to a promoter. The paper does not establish the same role for p300 or show that either protein acts as a scaffold connecting those factors to chromatin; its experiments used an immobilized DNA template in vitro. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Degrading Mediator and BRD4 has shown that these proteins are critical for gene expression.

“Recent experiments degrading Mediator and BRD4 have shown that these proteins are critical for gene expression [69].”

• cites [69] Crump NT, Ballabio E, Godfrey L et al (2021) BET inhibition disrupts transcription but retains enhancer–promoter contact. Nat Commun 12:223 sourcePartly supported

  The Discussion (page unavailable) says BRD4 and Mediator play a key role in the transcription of many genes. The Introduction describes this paper’s use of a BRD4-degrading compound, but its experiments reduced Mediator binding rather than degrading Mediator; it mentions MED14 degradation only in a separate study of enhancer–promoter contact. Thus the gene-expression conclusion is supported, but the description of the experiments is not fully supported. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Non-coding enhancer deletions have been linked to severe human disease phenotypes such as beta thalassaemia and polydactyly.

“It soon became clear that enhancers also played vital roles in humans, with non-coding enhancer deletions starting to be linked to severe disease phenotypes such as beta thalassaemia and polydactyly [25–28].”

• cites [25] Kioussis D, Vanin E, deLange T et al (1983) Beta-globin gene inactivation by DNA translocation in gamma beta-thalassaemia. Nature 306:662–666 sourceNot checked — no full text of the source
• cites [26] Driscoll MC, Dobkin CS, Alter BP (1989) Gamma delta beta-thalassemia due to a de novo mutation deleting the 5’ beta-globin gene activation-region hypersensitive sites. Proc Natl Acad Sci USA 86:7470–7474 sourceSupported
• cites [27] Lettice LA, Heaney SJH, Purdie LA et al (2003) A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum Mol Genet 12:1725–1735 sourcePartly supported

  The “ZRS and polydactyly” section links mutations in a non-coding regulatory element to preaxial polydactyly. It specifies single-base-pair mutations, not enhancer deletions, and does not address beta thalassaemia. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [28] Sharpe J, Lettice L, Hecksher-Sørensen J et al (1999) Identification of Sonic hedgehog as a candidate gene responsible for the polydactylous mouse mutant Sasquatch. Curr Biol 9:97-S1 sourceNot checked — no full text of the source

Pioneer factors often must recruit additional non-pioneer sequence-specific transcription factors for efficient enhancer activation.

“Often, pioneer factors must recruit additional non-pioneer sequence-specific TFs for efficient enhancer activation [30, 31].”

• cites [30] Spitz F, Furlong EEM (2012) Transcription factors: from enhancer binding to developmental control. Nat Rev Genet 13:613–626 sourceNot checked — no full text of the source
• cites [31] Zaret KS, Carroll JS (2011) Pioneer transcription factors: establishing competence for gene expression. Genes Dev 25:2227–2241 sourceSupported

At the DNA level, enhancers represent clusters of binding sites for sequence-specific transcription factors.

“At the DNA level, enhancers represent clusters of binding sites for sequence-specific transcription factors (TFs) (Fig. 1A) (reviewed in [29, 30]).”

• cites [29] Long HK, Prescott SL, Wysocka J (2016) Ever-changing landscapes: transcriptional enhancers in development and evolution. Cell 167:1170–1187 sourceSupported
• cites [30] Spitz F, Furlong EEM (2012) Transcription factors: from enhancer binding to developmental control. Nat Rev Genet 13:613–626 sourceNot checked — no full text of the source

Zelda promotes the formation of local transcription factor hubs involving transcription factors required for developmental patterning, such as Bicoid and Dorsal.

“For instance, Zelda promotes the formation of local TF hubs involving important transcription factors required for developmental patterning, such as Bicoid and Dorsal [51–54].”

• cites [51] Mir M, Reimer A, Haines JE et al (2017) Dense Bicoid hubs accentuate binding along the morphogen gradient. Genes Dev 31:1784–1794 sourceSupported
• cites [52] Mir M, Stadler MR, Ortiz SA et al (2018) Dynamic multifactor hubs interact transiently with sites of active transcription in embryos. Elife 7:e40497 sourcePartly supported

  The Results section ('Bicoid binding events are enriched in Zelda hubs'; page unavailable) reports Bicoid binding enriched within Zelda hubs, supporting that example. The cited paper does not establish Dorsal’s involvement in those hubs, so the Dorsal example is unsupported by this source. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [53] Dufourt J, Trullo A, Hunter J et al (2018) Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs. Nat Commun 9:5194 sourcePartly supported

  The Results and Discussion section (‘Zelda accumulates into subnuclear hubs’; page unavailable) reports local, dynamic Zelda hubs and suggests they could promote cooperation with Bicoid. The Introduction and ‘Zelda fosters temporal transcriptional coordination’ section say Zelda potentiates Bicoid and Dorsal activity, but the paper does not establish that either factor participates in the observed Zelda hubs or that Zelda promotes formation of hubs containing them. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [54] Yamada S, Whitney PH, Huang S-K et al (2019) The drosophila pioneer factor zelda modulates the nuclear microenvironment of a dorsal target enhancer to potentiate transcriptional output. Curr Biol 29:1387-1393.e5 sourceSupported

A 72-bp repeat sequence element in SV40 is an enhancing sequence capable of vastly upregulating gene expression from a plasmid upon transfection into mammalian cells.

“Subsequent landmark experiments by the groups of Walter Schaffner [4] and Pierre Chambon [5] confirmed that a 72-bp repeat sequence element in SV40 [8, 9] was an “enhancing” sequence, capable of vastly upregulating gene expression from a plasmid upon transfection into mammalian cells.”

• cites [4] Banerji J, Rusconi S, Schaffner W (1981) Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell 27:299–308 sourceNot checked — no full text of the source
• cites [5] Moreau P, Hen R, Wasylyk B et al (1981) The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants. Nucleic Acids Res 9:6047–6068. https://doi.org/10.1093/nar/9.22.6047 sourceNot checked — no full text of the source
• cites [8] Benoist C, Chambon P (1981) In vivo sequence requirements of the SV40 early promoter region. Nature 290:304–310 sourceNot checked — no full text of the source
• cites [9] Gruss P, Dhar R, Khoury G (1981) Simian virus 40 tandem repeated sequences as an element of the early promoter. Proc Natl Acad Sci USA 78:943–947 sourceSupported

A subset of transcription factors known as pioneer factors are capable of overcoming closed chromatin constraint through chromatin remodelling.

“A subset of TFs known as pioneer factors are capable of overcoming this constraint through chromatin remodelling (reviewed in [31, 32]).”

• cites [31] Zaret KS, Carroll JS (2011) Pioneer transcription factors: establishing competence for gene expression. Genes Dev 25:2227–2241 sourceSupported
• cites [32] Larson ED, Marsh AJ, Harrison MM (2021) Pioneering the developmental frontier. Mol Cell 81:1640–1650 sourceSupported

At active enhancers, BRD4 co-localises with the Mediator complex, which assembles the pre-initiation complex and RNAP to initiate transcription.

“At active enhancers, the BRD4 protein co-localises with the Mediator complex, which in turn assembles the pre-initiation complex (PIC) and RNAP to initiate transcription [66–68].”

• cites [66] Allen BL, Taatjes DJ (2015) The mediator complex: a central integrator of transcription. Nat Rev Mol Cell Biol 16:155–166 sourcePartly supported

  The “Mediator roles in pol II transcription” and “PIC assembly” sections say that Mediator is generally required for PIC assembly and enables RNA polymerase II recruitment. The “DNA looping” section links Mediator to enhancer activity, but the passages retrieved for BRD4 do not establish that BRD4 co-localises with Mediator at active enhancers. Page numbers are not available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [67] Flanagan PM, Kelleher RJ, Sayre MH et al (1991) A mediator required for activation of RNA polymerase II transcription in vitro. Nature 350:436–438 sourceNot checked — no full text of the source
• cites [68] Kelleher RJ, Flanagan PM, Kornberg RD (1990) A novel mediator between activator proteins and the RNA polymerase II transcription apparatus. Cell 61:1209–1215 sourceNot checked — no full text of the source

SWI/SNF recruits the chromatin activators P300 and CBP, which mediate acetylation of histone H3 lysine 27 through their intrinsic histone acetyltransferase activity.

“SWI/SNF recruits the chromatin activators P300 and CBP, which mediate the acetylation of histone H3 lysine 27 through their intrinsic histone acetyltransferase (HAT) activity [59–62].”

• cites [59] Chan HM, Shikama N, La Thangue NB (2001) Control of gene expression and the cell cycle. Essays Biochem 37:87–96 sourceNot checked — no full text of the source
• cites [60] Jin Q, Yu L-R, Wang L et al (2011) Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation. EMBO J 30:249–262 sourcePartly supported

  The Introduction identifies CBP and p300 as histone acetyltransferase co-activators, and “Substrate and site specificities of CBP/p300” reports that they are required for over 90% of H3K27 acetylation in mouse embryonic fibroblasts. The cited paper does not establish the sentence’s additional claim that SWI/SNF recruits CBP and p300. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [61] Raisner R, Kharbanda S, Jin L et al (2018) Enhancer activity requires CBP/P300 bromodomain-dependent histone H3K27 acetylation. Cell Rep 24:1722–1729 sourceNot checked — no full text of the source
• cites [62] Alver BH, Kim KH, Lu P et al (2017) The SWI/SNF chromatin remodelling complex is required for maintenance of lineage specific enhancers. Nat Commun 8:14648 sourcePartly supported

  The Results section ('SWI/SNF is required for p300 activity at enhancers'; page not provided) says SWI/SNF recruits p300 to enhancer loci and regulates H3K27 acetylation through interaction with this histone acetyltransferase. It does not establish that SWI/SNF also recruits CBP, so the claim's inclusion of CBP is unsupported by the cited Results passages. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Synthetic enhancers recruiting multiple heterotypic transcription factors show stronger transcriptional effects compared with those recruiting high amounts of a single transcription factor.

“Synthetic enhancers recruiting multiple heterotypic TFs showed stronger transcriptional effects compared with those recruiting high amounts of a single TF, providing a functional rationale for combinatorial TF recruitment to natural enhancers [55].”

• cites [55] Smith RP, Taher L, Patwardhan RP et al (2013) Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model. Nat Genet 45:1021–1028 sourceSupported

Many pioneer factors recruit chromatin remodelers.

“The mechanisms of pioneer factor action are still being established [37], but at least some of them (such as FOXA, OCT4 and SOX2/SOX11) have been shown to initiate chromatin opening by direct displacement of histones [38–41], whilst many others recruit chromatin remodelers [42–50].”

• cites [42] Ding J, Xu H, Faiola F et al (2012) Oct4 links multiple epigenetic pathways to the pluripotency network. Cell Res 22:155–167 sourcePartly supported

  The Results and Discussion section (page not available) reports that Oct4 physically interacts with components of several chromatin-remodeling and other epigenetic complexes. It does not establish that many pioneer factors recruit remodelers, or that Oct4’s interactions constitute recruitment to open chromatin. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [43] Pardo M, Lang B, Yu L et al (2010) An expanded Oct4 interaction network: implications for stem cell biology, development, and disease. Cell Stem Cell 6:382–395 sourcePartly supported

  The Discussion (page unavailable) reports that Oct4 copurifies with subunits of the SWI/SNF nucleosome-remodeling complex, supporting an Oct4–remodeler association. It does not show that Oct4 recruits the complex, or establish that many pioneer factors recruit chromatin remodelers. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [44] van den Berg DLC, Snoek T, Mullin NP et al (2010) An Oct4-centered protein interaction network in embryonic stem cells. Cell Stem Cell 6:369–381 sourcePartly supported

  The Results (An Oct4-Centered Interaction Network; page not provided) report that several embryonic-stem-cell transcription factors associate with chromatin-modifying complexes, including SWI/SNF. The Discussion (Multiple Network Connections with Chromatin- and Protein-Modifying Factors; page not provided) proposes that recruitment may occur through initial local targeting, but does not establish that many pioneer factors recruit remodelers. The claim presents that proposed mechanism as a demonstrated, broader finding. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [45] King HW, Klose RJ (2017) The pioneer factor OCT4 requires the chromatin remodeller BRG1 to support gene regulatory element function in mouse embryonic stem cells. Elife 6:e22631 sourcePartly supported

  The Discussion (page unavailable) reports that OCT4 recruits BRG1 to its binding sites and suggests that many pioneer factors may rely on chromatin remodelling. It does not establish that many other pioneer factors recruit remodelers; it presents that broader mechanism as a possibility requiring further study. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [46] Bao X, Rubin AJ, Qu K et al (2015) A novel ATAC-seq approach reveals lineage-specific reinforcement of the open chromatin landscape via cooperation between BAF and p63. Genome Biol 16:284 sourcePartly supported

  The Results section (“BAF and p63 binding reinforce open chromatin”; page not provided) reports that p63 enhances the chromatin targeting of the BAF remodeling complex. This supports an example of a transcription factor recruiting a remodeler, but the paper does not establish that many pioneer factors do so. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [47] Engelen E, Akinci U, Bryne JC et al (2011) Sox2 cooperates with Chd7 to regulate genes that are mutated in human syndromes. Nat Genet 43:607–611 sourceNot checked — no full text of the source
• cites [48] Hu G, Schones DE, Cui K et al (2011) Regulation of nucleosome landscape and transcription factor targeting at tissue-specific enhancers by BRG1. Genome Res 21:1650–1658 sourcePartly supported

  In “BRG1 becomes associated with the GATA1 enhancers during differentiation of HSCs” (page unavailable), the authors say their results are consistent with GATA1 helping recruit BRG1, a chromatin remodeler. The paper does not establish that many pioneer factors recruit remodelers; its recruitment finding concerns GATA1 and is framed as an inference. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [49] Takaku M, Grimm SA, Shimbo T et al (2016) GATA3-dependent cellular reprogramming requires activation-domain dependent recruitment of a chromatin remodeler. Genome Biol 17:36 sourceSupported
• cites [50] Vierbuchen T, Ling E, Cowley CJ et al (2017) AP-1 transcription factors and the BAF complex mediate signal-dependent enhancer selection. Mol Cell 68:1067-1082.e12 sourcePartly supported

  The Results section ('AP-1 TFs recruit the BAF complex to enhancers'; page unavailable) supports recruitment of a chromatin remodeler by AP-1. The Discussion describes AP-1 nucleosome binding as a possible pioneer-factor property, but the paper does not establish that many other pioneer factors recruit remodelers. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Later studies of Drosophila developmental enhancers found evidence for much less rigid motif organisation.

“In contrast, later studies of Drosophila developmental enhancers found evidence for much less rigid motif organisation [90, 91].”

• cites [90] Kulkarni MM, Arnosti DN (2003) Information display by transcriptional enhancers. Development 130:6569–6575 sourceNot checked — no full text of the source
• cites [91] Junion G, Spivakov M, Girardot C et al (2012) A transcription factor collective defines cardiac cell fate and reflects lineage history. Cell 148:473–486 sourceSupported

Homologous enhancers in a pair of distantly related species can retain their function in the non-host organism despite differences in DNA sequence.

“Consistent with this, homologous enhancers in a pair of distantly related species can retain their function in the non-host organism despite differences in DNA sequence [101, 110, 111].”

• cites [101] Wong ES, Zheng D, Tan SZ et al (2020) Deep conservation of the enhancer regulatory code in animals. Science 370:eaax8137 sourceNot checked — no full text of the source
• cites [110] Yang S, Oksenberg N, Takayama S et al (2015) Functionally conserved enhancers with divergent sequences in distant vertebrates. BMC Genomics 16:882 sourceSupported
• cites [111] Hörnblad A, Bastide S, Langenfeld K et al (2021) Dissection of the Fgf8 regulatory landscape by in vivo CRISPR-editing reveals extensive intra- and inter-enhancer redundancy. Nat Commun 12:439 sourceSupported

At non-dimeric sites, physical interactions between co-bound transcription factors were not necessary.

“Interestingly, at non-dimeric sites, physical interactions between co-bound TFs were not necessary [119], consistent with evidence from Drosophila where co-occupied DNA-bound TFs were often spaced quite widely apart (> 50 bp) [118].”

• cites [119] Sönmezer C, Kleinendorst R, Imanci D et al (2021) Molecular co-occupancy identifies transcription factor binding cooperativity in vivo. Mol Cell 81:255-267.e6 sourceSupported

Single molecule footprinting adapted to mammalian cells detected widespread co-occupancy of cooperative transcription factors at enhancers.

“SMF has also recently been adapted to mammalian cells, detecting widespread co-occupancy of cooperative TFs at enhancers [119].”

• cites [119] Sönmezer C, Kleinendorst R, Imanci D et al (2021) Molecular co-occupancy identifies transcription factor binding cooperativity in vivo. Mol Cell 81:255-267.e6 sourcePartly supported

  The Introduction says the authors adapted SMF for mammalian genomes and found DNA co-occupancy widespread at sites of TF cooperativity. The Results describe measurements at mouse cis-regulatory elements, but the passages do not establish that the widespread finding applies specifically to enhancers rather than cis-regulatory elements more broadly. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

The prevalence of flexible enhancer organisation is supported by rapid evolutionary turnover of enhancers, which exceeds that of proximal promoter regions.

“The prevalence of flexible enhancer organisation is also supported by rapid evolutionary turnover of enhancers, which exceeds that of proximal promoter regions [29, 99, 100].”

• cites [29] Long HK, Prescott SL, Wysocka J (2016) Ever-changing landscapes: transcriptional enhancers in development and evolution. Cell 167:1170–1187 sourcePartly supported

  Section I, “General principles of enhancer function” (page unavailable), describes high rates of enhancer turnover and links compensatory motif turnover to flexible enhancer organisation. The passages do not compare enhancer turnover with turnover in proximal promoter regions, so the claimed difference in rates is not supported by this source. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [99] Villar D, Berthelot C, Aldridge S et al (2015) Enhancer evolution across 20 mammalian species. Cell 160:554–566 sourceSupported
• cites [100] Carelli FN, Liechti A, Halbert J et al (2018) Repurposing of promoters and enhancers during mammalian evolution. Nat Commun 9:4066 sourceSupported

Single molecule footprinting can detect the binding of multiple transcription factors on a single DNA molecule.

“First developed in Drosophila, SMF can detect the binding of multiple TFs on a single DNA molecule [116].”

• cites [116] Krebs AR, Imanci D, Hoerner L et al (2017) Genome-wide single-molecule footprinting reveals high RNA polymerase II turnover at paused promoters. Mol Cell 67:411–422.e4 sourcePartly supported

  In Results, “Simultaneous Quantification of Multiple Transcription Initiation Intermediates” (page unavailable), the paper reports measuring multiple promoter-occupancy events on single molecules. It identifies footprints associated with the pre-initiation complex and RNA polymerase II, but does not show detection of multiple individual transcription factors on one molecule. The “Principles of Dual-Enzyme Single-Molecule Footprinting” section says the method was applied in Drosophila, not that SMF was first developed there. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Enhancer conservation is typically modular, commonly preserving the sets of binding sites for the required transcription factors, but often not their exact sequence and orientation.

“Instead, enhancer conservation is typically “modular”, commonly preserving the sets of binding sites for the required TFs, but often not their exact sequence and orientation [101–109].”

• cites [101] Wong ES, Zheng D, Tan SZ et al (2020) Deep conservation of the enhancer regulatory code in animals. Science 370:eaax8137 sourceNot checked — no full text of the source
• cites [102] Hare EE, Peterson BK, Iyer VN et al (2008) Sepsid even-skipped enhancers are functionally conserved in Drosophila despite lack of sequence conservation. PLoS Genet 4:e1000106 sourcePartly supported

  The Results sections “Identification of Sepsid Enhancers” and “Binding Site Composition and Organization in Sepsid Enhancers” describe similar collections of predicted binding sites for relevant transcription factors, while finding that few individual sites remain in the same location across families. The Discussion (“Binding Site Turnover and the Regulatory Machinery”) likewise reports extensive rearrangement with conserved enhancer function. These findings support the claim for the eve enhancers studied, but do not establish that this pattern is typical of enhancers generally; the passages also do not specifically assess binding-site orientation. Page numbers are unavailable in our copy. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [103] Schmidt D, Wilson MD, Ballester B et al (2010) Five-vertebrate ChIP-seq reveals the evolutionary dynamics of transcription factor binding. Science 328:1036–1040 sourcePartly supported

  The paper’s discussion (page not provided in our copy) says that tissue-specific regulation can persist through regulatory architectures independent of sequence constraint, and describes nearby compensatory TF-binding events. This supports flexibility in binding-site sequence and location, but the study examines two liver TFs and does not establish that enhancers typically preserve the full set of required TF sites or that site orientation often changes. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [104] Dowell RD (2010) Transcription factor binding variation in the evolution of gene regulation. Trends Genet 26:468–475 sourceNot checked — no full text of the source
• cites [105] Habib N, Wapinski I, Margalit H et al (2012) A functional selection model explains evolutionary robustness despite plasticity in regulatory networks. Mol Syst Biol 8:619 sourceCould not verify

  The Results section on functional conservation of regulatory DNA motifs describes conserved motif–function associations, while the Discussion describes turnover of target genes within functional modules. Neither passage addresses whether individual enhancers preserve sets of transcription-factor binding sites while their exact sequences and orientations change; searches for enhancer conservation, binding-site sequence, and orientation did not establish that claim. (LLM, conf=0.73)

  Experimental — accuracy not yet measured
• cites [106] Arnold CD, Gerlach D, Spies D et al (2014) Quantitative genome-wide enhancer activity maps for five Drosophila species show functional enhancer conservation and turnover during cis-regulatory evolution. Nat Genet 46:685–692 sourcePartly supported

  The Results sections “Specific selection of transcription factor sequence motifs” and “Compensatory changes stabilize regulatory output” link conserved enhancer activity to transcription-factor motifs rather than overall sequence identity, and show that lost motifs can be replaced at different positions. The paper does not establish that binding-site orientation is often unconserved, and its findings concern the assayed Drosophila enhancers rather than enhancers generally. Page numbers are unavailable in our copy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [107] Spivakov M (2014) Spurious transcription factor binding: non-functional or genetically redundant? BioEssays 36:798–806 sourcePartly supported

  In ‘Dose of activation’ as an evolutionary trait (page unavailable), the paper says regulatory regions can retain function despite low base-by-base conservation and turnover of transcription-factor binding sites. In ‘Do “doses” add up at multi-TF enhancers?’ (page unavailable), it describes flexible enhancer organisation, but it does not establish that conserved enhancers commonly preserve the same sets of required transcription factors or address binding-site orientation. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [108] Borok MJ, Tran DA, Ho MCW, Drewell RA (2010) Dissecting the regulatory switches of development: lessons from enhancer evolution in Drosophila. Development 137:5–13 sourceNot checked — no full text of the source
• cites [109] Khoueiry P, Girardot C, Ciglar L et al (2017) Uncoupling evolutionary changes in DNA sequence, transcription factor occupancy and enhancer activity. Elife 6:e28440 sourcePartly supported

  The Results section, “Conserved enhancer occupancy is associated with enhancer function” (page unavailable), says evolutionary constraints likely select for overall enhancer composition rather than the positions of individual binding sites. The Results section, “TF binding site divergence is tolerated in the context of collective TF binding” (page unavailable), also reports conserved Tin occupancy despite motif divergence in enhancers bound by many factors. These findings support flexibility in some enhancers, but do not establish that enhancer conservation is typically modular or that exact binding-site orientation is often not preserved. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Flexibility of enhancer organisation with respect to binding site order and orientation was observed in a massively parallel synthetic enhancer experiment.

“Flexibility of enhancer organisation with respect to binding site order and orientation was also observed in a massively parallel synthetic enhancer experiment [55].”

• cites [55] Smith RP, Taher L, Patwardhan RP et al (2013) Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model. Nat Genet 45:1021–1028 sourcePartly supported

  The Results and Discussion (page numbers unavailable) report that multiple binding-site orders drove strong expression in the massively parallel synthetic regulatory-element experiment, supporting flexibility with respect to order. The passages do not report a test or finding about binding-site orientation, so that part of the claim is unsupported. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Transcription factor binding to DNA appears to be very transient.

“While it is clear that enhancers typically favour the more flexible models of sequence organisation, the degree of necessary cooperation between TFs is less obvious, particularly since TF binding to DNA appears to be very transient [113–117].”

• cites [113] Wilczyński B, Furlong EEM (2010) Dynamic CRM occupancy reflects a temporal map of developmental progression. Mol Syst Biol 6:383 sourcePartly supported

  The Results and discussion report that each of four transcription factors occupies about 50% of its targeted regulatory modules transiently during Drosophila development. This supports changing DNA occupancy over developmental stages, but not the broader implication that transcription-factor binding to DNA is generally short-lived at the molecular level. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [114] Voss TC, Hager GL (2014) Dynamic regulation of transcriptional states by chromatin and transcription factors. Nat Rev Genet 15:69–81 sourceSupported
• cites [115] Chen J, Zhang Z, Li L et al (2014) Single-molecule dynamics of enhanceosome assembly in embryonic stem cells. Cell 156:1274–1285 sourceNot checked — no full text of the source
• cites [116] Krebs AR, Imanci D, Hoerner L et al (2017) Genome-wide single-molecule footprinting reveals high RNA polymerase II turnover at paused promoters. Mol Cell 67:411–422.e4 sourcePartly supported

  In Results, ‘The TATA Box Stabilizes Binding of the PIC’ (page unavailable), the paper says PIC formation at TATA-less promoters may be transient. It does not establish that transcription-factor binding to DNA is generally very transient; it instead reports more frequent PIC binding at TATA-containing promoters and notes that some general transcription factors may form a stable scaffold. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [117] Stergachis AB, Debo BM, Haugen E et al (2020) Single-molecule regulatory architectures captured by chromatin fiber sequencing. Science 368:1449–1454 sourceNot checked — no full text of the source

A deep learning algorithm applied to binding of pluripotency transcription factors at base resolution in mouse embryonic stem cells suggested a soft motif syntax whereby transcription factors help each other to bind directly to the enhancer with a lenient distance-dependency between binding sites.

“Additionally, a deep learning algorithm applied to binding of pluripotency TFs at base resolution in mouse embryonic stem cells (ESCs) suggested a “soft motif syntax” whereby TFs help each other to bind directly to the enhancer with a lenient distance-dependency between binding sites [98].”

• cites [98] Avsec Ž, Weilert M, Shrikumar A et al (2021) Base-resolution models of transcription-factor binding reveal soft motif syntax. Nat Genet 53:354–366 sourceSupported

Transcription factors governing serotonergic neuron differentiation showed synergy despite flexible sequence organisation of their cognate enhancers.

“Similarly, TFs governing serotonergic neuron differentiation showed synergy despite flexible sequence organisation of their cognate enhancers [95].”

• cites [95] Lloret-Fernández C, Maicas M, Mora-Martínez C et al (2018) A transcription factor collective defines the HSN serotonergic neuron regulatory landscape. Elife 7:e32785 sourceSupported

Enhancers can reside in a poised state exhibiting both H3K4me1 and H3K27me3 that is associated with Polycomb repressive complexes.

“In addition to the ‘primed’ and active states, enhancers can also reside in a ‘poised’ state exhibiting both H3K4me1 and H3 lysine 27 trimethylation (H3K27me3) that is associated with Polycomb repressive complexes [131, 135, 136, 138].”

• cites [131] ENCODE Project Consortium, Moore JE, Purcaro MJ et al (2020) Expanded encyclopaedias of DNA elements in the human and mouse genomes. Nature 583:699–710 sourcePartly supported

  The paper’s Extended Data Fig. 3 caption describes a cCRE that may be a poised enhancer in embryonic stem cells, iPSCs and neural progenitor cells. It associates that example with high DNase and H3K4me3 signals, not the claimed combination of H3K4me1 and H3K27me3; the passages found also do not link poised enhancers to Polycomb repressive complexes. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [135] Rada-Iglesias A, Bajpai R, Swigut T et al (2011) A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470:279–283 sourceSupported
• cites [136] Zentner GE, Tesar PJ, Scacheri PC (2011) Epigenetic signatures distinguish multiple classes of enhancers with distinct cellular functions. Genome Res 21:1273–1283 sourceSupported
• cites [138] Koenecke N, Johnston J, Gaertner B et al (2016) Genome-wide identification of Drosophila dorso-ventral enhancers by differential histone acetylation analysis. Genome Biol 17:196 sourceCould not verify

  The Background discusses H3K27ac as a marker of active enhancers, not the claimed combination of H3K4me1 and H3K27me3 at poised enhancers. Searches for those marks, poised enhancers, and Polycomb found only related reference titles, not a passage in this paper stating the claim. The indexed passages have no page numbers. (LLM, conf=0.73)

  Experimental — accuracy not yet measured

Even enhancers with extremely high evolutionary conservation may not require the exact preservation of their sequence for their correct function.

“Furthermore, even enhancers with extremely high evolutionary conservation may not require the exact preservation of their sequence for their correct function, as was recently shown using CRISPR-mediated mutagenesis [112].”

• cites [112] Snetkova V, Ypsilanti AR, Akiyama JA et al (2021) Ultraconserved enhancer function does not require perfect sequence conservation. Nat Genet 53:521–528 sourceSupported

A fine-grained time-course experiment following oestrogen receptor activation in a cancer cell line detected multiple waves of progressive recruitment of at least 46 sequence-specific transcription factors and cofactors, eventually resulting in derepression of the oestrogen-response gene pS2.

“This study detected multiple “waves” of a progressive recruitment of at least 46 sequence-specific TFs and cofactors, including chromatin remodelling complexes and histone acetyl-transferases, eventually resulting in a derepression of an oestrogen-response gene pS2 [87].”

• cites [87] Métivier R, Penot G, Hübner MR et al (2003) Estrogen receptor-α directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115:751–763 sourcePartly supported

  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

At dimeric motif sites, transcription factors tended to bind initially as monomers with subsequent dimerisation presumably stabilising the DNA-protein complex.

“Notably, at dimeric motif sites, TFs tended to bind initially as monomers with subsequent dimerisation presumably stabilising the DNA–protein complex [119].”

• cites [119] Sönmezer C, Kleinendorst R, Imanci D et al (2021) Molecular co-occupancy identifies transcription factor binding cooperativity in vivo. Mol Cell 81:255-267.e6 sourceSupported

Mediator and BRD4 co-localise at very high levels at super-enhancers, long stretches of sequences with gene regulatory activity that generally drive greater levels of gene expression than regular enhancers.

“In particular, Mediator and BRD4 co-localise at very high levels at “super-enhancers” (SEs): long stretches of sequences with gene regulatory activity that generally drive greater levels of gene expression than regular enhancers [69–72].”

• cites [69] Crump NT, Ballabio E, Godfrey L et al (2021) BET inhibition disrupts transcription but retains enhancer–promoter contact. Nat Commun 12:223 sourceSupported
• cites [70] Chapuy B, McKeown MR, Lin CY et al (2013) Discovery and characterization of super-enhancer-associated dependencies in diffuse large B cell lymphoma. Cancer Cell 24:777–790 sourcePartly supported

  The Results section (“Disproportionate binding of BRD4 to overloaded enhancers”; page unavailable) reports exceptionally high BRD4 loading at enhancers larger than typical enhancers. The Discussion (page unavailable) describes them as approximately 12-fold larger. Our copy does not establish co-localization with Mediator or show that these enhancers generally drive higher gene expression than regular enhancers; it reports greater sensitivity to BET inhibition instead. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [71] Di Micco R, Fontanals-Cirera B, Low V et al (2014) Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes. Cell Rep 9:234–247 sourcePartly supported

  The Results sections (page unavailable) report that BRD4 and Mediator subunits localize at super-enhancers of stem-cell genes in human embryonic stem cells, and describe super-enhancers as large enhancer domains. The copy does not establish that super-enhancers generally drive higher gene expression than regular enhancers. It also reports that BRD4 peak signal intensity was similar at super-enhancers and typical enhancers in these cells, although BRD4-binding peaks were more frequent at super-enhancers. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [72] Whyte WA, Orlando DA, Hnisz D et al (2013) Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153:307–319 sourcePartly supported

  The Results sections on large genomic domains and functional attributes describe super-enhancers as unusually long, highly occupied by Mediator, and associated with higher gene expression than typical enhancers. The passages have no page numbers in our copy. They do not establish that BRD4 co-localises with Mediator at super-enhancers. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

The TF collective model was originally formulated in Drosophila, where a set of five transcription factors bound multitudes of enhancers required for heart development that showed no detectable similarity in their sequence organisation beyond the presence of binding motifs for some of these transcription factors.

“The TF collective model was originally formulated in Drosophila, where a set of five TFs bound multitudes of enhancers required for heart development that showed no detectable similarity in their sequence organisation, beyond the presence of binding motifs for some of these TFs [91].”

• cites [91] Junion G, Spivakov M, Girardot C et al (2012) A transcription factor collective defines cardiac cell fate and reflects lineage history. Cell 148:473–486 sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

The stripe 2 enhancer controls even-skipped expression in early embryogenesis via binding of tissue-specific transcription factors.

“The stripe 2 enhancer controls eve expression in early embryogenesis via binding of tissue-specific TFs [121–123].”

• cites [121] Stanojevic D, Small S, Levine M (1991) Regulation of a segmentation stripe by overlapping activators and repressors in the Drosophila embryo. Science 254:1385–1387 sourceNot checked — no full text of the source
• cites [122] Goto T, Macdonald P, Maniatis T (1989) Early and late periodic patterns of even skipped expression are controlled by distinct regulatory elements that respond to different spatial cues. Cell 57:413–422 sourceNot checked — no full text of the source
• cites [123] Small S, Kraut R, Hoey T et al (1991) Transcriptional regulation of a pair-rule stripe in Drosophila. Genes Dev 5:827–839 sourcePartly supported

  The Abstract and Discussion describe the stripe 2 regulatory element as binding factors that activate or repress eve expression in early embryos. They identify these factors as a maternal morphogen and gap proteins with spatially patterned expression, but do not establish that they are tissue-specific transcription factors. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Strengthening transcription factor binding sites at developmental enhancers results in aberrant gene expression patterns.

“Consistent with this, “strengthening” TF binding sites at developmental enhancers results in aberrant gene expression patterns [125].”

• cites [125] Farley EK, Olson KM, Zhang W et al (2015) Suboptimization of developmental enhancers. Science 350:325–328 sourceSupported

Enhancers consistent with the billboard model are likely also widespread in vertebrate genomes.

“Enhancers consistent with the billboard model are likely also widespread in vertebrate genomes [55, 93].”

• cites [55] Smith RP, Taher L, Patwardhan RP et al (2013) Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model. Nat Genet 45:1021–1028 sourcePartly supported

  The Results section (“Reporter expression is influenced by binding site order”; page unavailable) says the tested configurations are largely consistent with the billboard model. The Results section (“Synthetic elements mimic putative liver enhancers”; page unavailable) also finds similar binding-site configurations in 78% of the putative mouse liver enhancers examined. This supports the model’s relevance to native mammalian enhancers, but does not establish that billboard-consistent enhancers are widespread across vertebrate genomes. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [93] Rastegar S, Hess I, Dickmeis T et al (2008) The words of the regulatory code are arranged in a variable manner in highly conserved enhancers. Dev Biol 318:366–377 sourcePartly supported

  In “Highly conserved non-coding sequences and transcriptional regulation” (page unavailable), the paper calls many enhancers loose, variable assemblies of binding sites and identifies that structure as the billboard model. It also finds billboard-like organization in five zebrafish notochord enhancers. Those examples support the possibility, but the paper does not establish how widespread billboard-like enhancers are across vertebrate genomes. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Proposed models for enhancer grammar range from very rigid to flexible, largely dependent on the species, cell types and loci in which they were formulated.

“The various proposed models for enhancer grammar range from very rigid to flexible, largely dependent on the species, cell types and loci in which they were formulated (reviewed in [29]).”

• cites [29] Long HK, Prescott SL, Wysocka J (2016) Ever-changing landscapes: transcriptional enhancers in development and evolution. Cell 167:1170–1187 sourcePartly supported

  In the Introduction, under “Motif organization, enhancer grammar and models of enhancer architecture” (page unavailable), the review contrasts the rigid enhanceosome model with the more flexible billboard model and describes a spectrum between them. It gives examples from particular species and loci, but does not establish that the differences among proposed models are largely dependent on the species, cell types, and loci in which they were formulated. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Co-occupied DNA-bound transcription factors in Drosophila were often spaced quite widely apart, more than 50 bp.

“Interestingly, at non-dimeric sites, physical interactions between co-bound TFs were not necessary [119], consistent with evidence from Drosophila where co-occupied DNA-bound TFs were often spaced quite widely apart (> 50 bp) [118].”

• cites [118] Rao S, Ahmad K, Ramachandran S (2021) Cooperative binding between distant transcription factors is a hallmark of active enhancers. Mol Cell 81:1651-1665.e4 sourcePartly supported

  The Results section, “Cooperative binding is common at active enhancers,” reports co-binding across spacings of 50–90 bp and significant cooperativity at distances up to 140 bp, supporting the point that Drosophila TFs can co-occupy widely separated sites. But “Many active enhancers contain multiple bound TFs” says ~50 bp was the most observed spacing; the copy does not establish that co-occupied factors were often more than 50 bp apart. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Enhancers devoid of the classic H3K27ac mark have been described.

“For example, enhancers devoid of the classic H3K27ac mark have recently been described [163]; consistent with this, it was shown that this mark is not required for enhancer function, at least in mouse ESCs [164].”

• cites [163] Pradeepa MM, Grimes GR, Kumar Y et al (2016) Histone H3 globular domain acetylation identifies a new class of enhancers. Nat Genet 48:681–686 sourceSupported

Using techniques with antibodies to enhancer- and promoter-associated histone modifications or RNA polymerase has revealed large numbers of 3D enhancer-promoter contacts.

“Using these techniques with antibodies to enhancer- and promoter-associated histone modifications or those to RNA polymerase has also revealed large numbers of 3D enhancer–promoter contacts [244, 295–301].”

• cites [244] Mumbach MR, Satpathy AT, Boyle EA et al (2017) Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements. Nat Genet 49:1602–1612 sourceSupported
• cites [295] Weintraub AS, Li CH, Zamudio AV et al (2017) YY1 Is a structural regulator of enhancer-promoter loops. Cell 171:1573-1588.e28 sourcePartly supported

  The Results section (“Enhancer-Promoter Interactions Depend on YY1 in Living Cells”; page unavailable) reports that H3K27ac HiChIP detected enhancer–promoter interactions; H3K27ac is a modification present at both enhancers and promoters. The paper therefore supports the histone-modification part of the claim, but the passages found do not show contacts detected using antibodies to RNA polymerase. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [296] Pelikan RC, Kelly JA, Fu Y et al (2018) Enhancer histone-QTLs are enriched on autoimmune risk haplotypes and influence gene expression within chromatin networks. Nat Commun 9:2905 sourcePartly supported

  The Methods (HiChIP sequencing and analysis; page unavailable) describe using an antibody to the enhancer-associated H3K27ac mark to identify long-range DNA loops. The Results (HLA-DR3 and -DR15 hQTLs differentially regulate HLA genes; page unavailable) report looping between an enhancer and the HLA-DRB1 promoter. Our copy does not show that promoter-associated histone or RNA-polymerase antibodies revealed contacts, or establish the claimed large number of enhancer–promoter contacts. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [297] Nott A, Holtman IR, Coufal NG et al (2019) Brain cell type-specific enhancer–promoter interactome maps and diseaserisk association. Science 366:1134–1139 sourceSupported
• cites [298] O’Mara TA, Spurdle AB, Glubb DM, Endometrial Cancer Association Consortium (2019) Analysis of promoter-associated chromatin interactions reveals biologically relevant candidate target genes at endometrial cancer risk loci. Cancers 11:1440 sourcePartly supported

  The Methods (§4.3) describe using an H3K27Ac antibody, and the Results (§2.1) report 66,092–449,157 chromatin loops per cell line, with 35%–40% contacting a promoter. This supports the histone-modification example and the large number of contacts, but the paper does not report using antibodies to RNA polymerase. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [299] Shi C, Ray-Jones H, Ding J et al (2021) Chromatin looping links target genes with genetic risk loci for dermatological traits. J Invest Dermatol. https://doi.org/10.1016/j.jid.2021.01.015 sourceSupported
• cites [300] Chen L, Cao W, Aita R et al (2021) Three-dimensional interactions between enhancers and promoters during intestinal differentiation depend upon HNF4. Cell Rep 34:108679 sourceSupported
• cites [301] Kubo N, Ishii H, Xiong X et al (2021) Promoter-proximal CTCF binding promotes distal enhancer-dependent gene activation. Nat Struct Mol Biol 28:152–161 sourcePartly supported

  The Results section (page unavailable) says the authors used H3K4me3 antibodies with PLAC-seq to detect promoter-centered chromatin contacts and found thousands of contacts involving distal enhancers or promoters. This supports the histone-modification portion, but the paper does not report using RNA-polymerase antibodies or enhancer-associated histone antibodies to detect those contacts. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Many developmental enhancers are found in the poised or Polycomb-repressed state in early development.

“Many developmental enhancers are found in the poised or Polycomb-repressed state in early development [139, 142, 143], and the functional role of these states remains an area of active research.”

• cites [139] Ngan CY, Wong CH, Tjong H et al (2020) Chromatin interaction analyses elucidate the roles of PRC2-bound silencers in mouse development. Nat Genet 52:264–272 sourceSupported
• cites [142] Crispatzu G, Rehimi R, Pachano T et al (2021) The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo. Nat Commun 12(1):4344 sourceSupported
• cites [143] Cruz-Molina S, Respuela P, Tebartz C et al (2017) PRC2 facilitates the regulatory topology required for poised enhancer function during pluripotent stem cell differentiation. Cell Stem Cell 20:689-705.e9 sourceNot checked — no full text of the source

The H3K27ac mark is not required for enhancer function, at least in mouse embryonic stem cells.

“For example, enhancers devoid of the classic H3K27ac mark have recently been described [163]; consistent with this, it was shown that this mark is not required for enhancer function, at least in mouse ESCs [164].”

• cites [164] Zhang T, Zhang Z, Dong Q et al (2020) Histone H3K27 acetylation is dispensable for enhancer activity in mouse embryonic stem cells. Genome Biol 21:45 sourceSupported

Some enhancer variants that appear functionally neutral when tested under normal steady-state conditions may show cryptic effects under stress or upon stimulus response.

“Notably, some enhancer variants that appear functionally neutral when tested under normal steady-state conditions may show “cryptic” effects under stress or upon stimulus response [107, 226, 227].”

• cites [107] Spivakov M (2014) Spurious transcription factor binding: non-functional or genetically redundant? BioEssays 36:798–806 sourceSupported
• cites [226] Gibson G, Reed LK (2008) Cryptic genetic variation. Curr Biol 18:R989–R990 sourcePartly supported

  In “How is cryptic genetic variation related to canalization?” (page unavailable), the paper says genetic effects can be suppressed under normal conditions and become visible when the environment changes. It does not specifically discuss enhancer variants, steady-state enhancer tests, or stimulus responses, so those details are not established by this source. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [227] Paaby AB, Rockman MV (2014) Cryptic genetic variation: evolution’s hidden substrate. Nat Rev Genet 15:247–258 sourceSupported

Much natural variation at enhancers has apparently little phenotypic effect due to redundancy both within enhancers and across them.

“Much of this variation has apparently little phenotypic effect due to redundancy both within enhancers (such as through multiple homotypic TF binding sites [191, 193]) and across them (such as through multiple enhancers regulating the same gene, as will be discussed below).”

• cites [191] Spivakov M, Akhtar J, Kheradpour P et al (2012) Analysis of variation at transcription factor binding sites in Drosophila and humans. Genome Biol 13:R49 sourceSupported
• cites [193] Gotea V, Visel A, Westlund JM et al (2010) Homotypic clusters of transcription factor binding sites are a key component of human promoters and enhancers. Genome Res 20:565–577 sourceSupported

3C-derived methods have detected multitudes of enhancer-promoter loops in many human and mouse cell types.

“These enrichment methods have detected multitudes of enhancer–promoter loops in many human and mouse cell types [243, 286–291].”

• cites [243] Javierre BM, Burren OS, Wilder SP et al (2016) Lineage-specific genome architecture links enhancers and non-coding disease variants to target gene promoters. Cell 167:1369-1384.e19 sourceSupported
• cites [286] Montefiori LE, Sobreira DR, Sakabe NJ et al (2018) A promoter interaction map for cardiovascular disease genetics. Elife 7:e35788 sourcePartly supported

  The Introduction says promoter-capture Hi-C has identified thousands of enhancer–promoter contacts in different cell types. The Results report hundreds of thousands of promoter interactions in two human cell types, iPSCs and cardiomyocytes. These passages support the claim for human cells, but do not establish its mouse-cell component. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [287] Schoenfelder S, Mifsud B, Senner CE et al (2018) Divergent wiring of repressive and active chromatin interactions between mouse embryonic and trophoblast lineages. Nat Commun 9:4189 sourcePartly supported

  The Results sections report promoter capture Hi-C detection of hundreds of thousands of promoter contacts in mouse embryonic stem and trophoblast stem cells, including contacts with enhancers. The Introduction mentions studies in human cells, but this paper does not establish the claim's broader scope of multitudes of enhancer–promoter loops across many human and mouse cell types. The supplied copy gives no page numbers. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [288] Novo CL, Javierre B-M, Cairns J et al (2018) Long-range enhancer interactions are prevalent in mouse embryonic stem cells and are reorganized upon pluripotent state transition. Cell Rep 22:2615–2627 sourcePartly supported

  The Results section ('SEs Are Highly Interactive Hubs in ESCs'; page unavailable) reports that the authors used a 3C-based method to detect numerous enhancer–promoter interactions in mouse embryonic stem cells. This supports the mouse-cell example, but this paper does not establish the claim across many human and mouse cell types. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [289] Martin P, McGovern A, Orozco G et al (2015) Capture Hi-C reveals novel candidate genes and complex long-range interactions with related autoimmune risk loci. Nat Commun 6:10069 sourcePartly supported

  The Results (Summary of identified interactions; page unavailable) report thousands of Capture Hi-C interactions in two human cell lines, including some involving promoters. The paper does not establish that these are all enhancer–promoter loops, or report detection across many human and mouse cell types; its experiments used two human cell lines. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [290] Chesi A, Wagley Y, Johnson ME et al (2019) Genome-scale Capture C promoter interactions implicate effector genes at GWAS loci for bone mineral density. Nat Commun 10:1260 sourcePartly supported

  The Results section (page unavailable in our copy) reports 295,422 promoter interactions detected by Capture C in human osteoblasts and also describes a comparison with a human HepG2 cell line. It does not establish detection in many human and mouse cell types; the cited paper’s experiments described here involve human cells, not mouse cells. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [291] Ray-Jones H, Duffus K, McGovern A et al (2020) Mapping DNA interaction landscapes in psoriasis susceptibility loci highlights KLF4 as a target gene in 9q31. BMC Biol 18:47 sourcePartly supported

  The Results section (“Capture Hi-C identified novel gene targets in psoriasis susceptibility loci”; page unavailable) reports an average of 6,593 interactions across three capture experiments in human cell lines, with about 30% involving a transcription start site. The paper also identifies enhancer–promoter interactions using HiChIP in HaCaT cells. It does not establish the claim for many human and mouse cell types; the mouse-cell portion is unsupported by this source. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

In obesity susceptibility, trait-associated SNPs are located within the intron of the FTO gene but lead to the dis-regulation of the distal IRX3 gene.

“A classic example is in obesity susceptibility, where trait-associated SNPs are located within the intron of the FTO gene, but lead to the dis-regulation of the distal IRX3 gene [210].”

• cites [210] Smemo S, Tena JJ, Kim K-H et al (2014) Obesity-associated variants within FTO form long-range functional connections with IRX3. Nature 507:371–375 sourcePartly supported

  The paper’s Abstract places obesity-associated SNPs in FTO introns and reports that they are associated with IRX3 expression in human brain. It also describes a long-range functional connection to IRX3, but treats the possibility that the variants disrupt enhancer activity and alter expression as a possibility; it does not establish the claim’s causal wording that the SNPs lead to IRX3 dysregulation. No page number is supplied in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Genetic variants associated with differential binding of PU.1 underpin GWAS variants associated with blood cell count and autoimmune diseases.

“For example, genetic variants associated with differential binding of PU.1 were found to underpin GWAS variants associated with blood cell count and autoimmune diseases [213–215].”

• cites [213] Waszak SM, Delaneau O, Gschwind AR et al (2015) Population variation and genetic control of modular chromatin architecture in humans. Cell 162:1039–1050 sourceNot checked — no full text of the source
• cites [214] Tehranchi AK, Myrthil M, Martin T et al (2016) Pooled ChIP-Seq links variation in transcription factor binding to complex disease risk. Cell 165:730–741 sourcePartly supported

  In Results, under “bQTLs provide candidate molecular mechanisms for thousands of GWAS-implicated variants” (page unavailable), the paper identifies PU.1 binding QTLs linked to Crohn’s disease, inflammatory bowel disease, atopic dermatitis, and asthma GWAS loci. Our copy does not identify a PU.1 binding variant linked to blood cell count, and presents the disease mechanisms as candidates rather than established causes. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [215] Watt S, Vasquez L, Walter K et al (2021) Genetic perturbation of PU.1 binding and chromatin looping at neutrophil enhancers associates with autoimmune disease. Nat Commun 12:2298 sourceSupported

Certain promoters, termed epromoters, can also function as enhancers.

“Finally, evidence from multiple sources including reporter assays [169, 175, 239, 240], CRISPR [240–242], chromatin mapping [243, 244] and population genetics analysis [207, 245] suggests that certain promoters, termed ‘epromoters’, can also function as enhancers (reviewed in [246]).”

• cites [169] Arnold CD, Gerlach D, Stelzer C et al (2013) Genome-wide quantitative enhancer activity maps identified by STARR-seq. Science 339:1074–1077 sourceNot checked — no full text of the source
• cites [175] Nguyen TA, Jones RD, Snavely AR et al (2016) High-throughput functional comparison of promoter and enhancer activities. Genome Res 26:1023–1033 sourceSupported
• cites [239] Li G, Ruan X, Auerbach RK et al (2012) Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation. Cell 148:84–98 sourceSupported
• cites [240] Dao LTM, Galindo-Albarrán AO, Castro-Mondragon JA et al (2017) Genome-wide characterization of mammalian promoters with distal enhancer functions. Nat Genet 49:1073–1081 sourceNot checked — no full text of the source
• cites [241] Engreitz JM, Haines JE, Perez EM et al (2016) Local regulation of gene expression by lncRNA promoters, transcription and splicing. Nature 539:452–455 sourceSupported
• cites [242] Diao Y, Fang R, Li B et al (2017) A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells. Nat Methods 14:629–635 sourceSupported
• cites [246] Dao LTM, Spicuglia S (2018) Transcriptional regulation by promoters with enhancer function. Transcription 9:307–314 sourceSupported

Enhancers and promoters share many properties.

“Contrary to the initial assumption that enhancers and promoters were biologically distinguishable elements, it is becoming clear that they share many properties (reviewed in [233]).”

• cites [233] Andersson R, Sandelin A (2020) Determinants of enhancer and promoter activities of regulatory elements. Nat Rev Genet 21:71–87 sourceCould not verify

  The available passages identify a review about enhancer and promoter activities, but contain no discussion of whether the two share properties. Searches for shared properties, functional similarities, and a blurred distinction returned only bibliographic text, so the claim cannot be verified from our copy. (LLM, conf=0.70)

  Experimental — accuracy not yet measured

The mammalian β-globin locus developmental β-globin genes are regulated by a powerful tissue-specific enhancer known as a locus control region (LCR).

“These models were initially applied to the mammalian β-globin locus, in which the developmental β-globin genes are regulated by a powerful tissue-specific enhancer known as a locus control region (LCR) [254–257].”

• cites [254] Tuan D, Solomon W, Li Q, London IM (1985) The “beta-like-globin” gene domain in human erythroid cells. Proc Natl Acad Sci USA 82:6384–6388 sourceNot checked — no full text of the source
• cites [255] Reik A, Telling A, Zitnik G et al (1998) The locus control region is necessary for gene expression in the human beta-globin locus but not the maintenance of an open chromatin structure in erythroid cells. Mol Cell Biol 18:5992–6000 sourceSupported
• cites [256] Bender MA, Bulger M, Close J, Groudine M (2000) Beta-globin gene switching and DNase I sensitivity of the endogenous beta-globin locus in mice do not require the locus control region. Mol Cell 5:387–393 sourceSupported
• cites [257] Bungert J, Davé U, Lim KC et al (1995) Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4. Genes Dev 9:3083–3096 sourceSupported

DNA FISH has been used to characterise enhancer-promoter loops for several developmental genes such as Shh and HoxD.

“DNA FISH has been used to characterise enhancer–promoter loops for several developmental genes such as Shh and HoxD [303, 304].”

• cites [303] Amano T, Sagai T, Tanabe H et al (2009) Chromosomal dynamics at the Shh locus: limb bud-specific differential regulation of competence and active transcription. Dev Cell 16:47–57 sourceNot checked — no full text of the source
• cites [304] Williamson I, Eskeland R, Lettice LA et al (2012) Anterior-posterior differences in HoxD chromatin topology in limb development. Development 139:3157–3167 sourceSupported

An element might be classified as a promoter in the cell type-agnostic setting, but as a proximal enhancer-like element in a specific cell type.

“The ENCODE project, for instance, found that an element might be classified as a promoter in the cell type-agnostic setting, but as a proximal enhancer-like element in a specific cell type [131].”

• cites [131] ENCODE Project Consortium, Moore JE, Purcaro MJ et al (2020) Expanded encyclopaedias of DNA elements in the human and mouse genomes. Nature 583:699–710 sourceSupported

A systemic lupus erythematosus-associated variant (TT > A) situated within an enhancer downstream of TNFAIP3 abrogates NF-kB binding, affecting TNFAIP3 expression.

“In another example, a systemic lupus erythematosus-associated variant (termed TT > A) situated within an enhancer downstream of TNFAIP3 has been shown to abrogate NF-kB binding, affecting TNFAIP3 expression [211, 212].”

• cites [211] Adrianto I, Wen F, Templeton A et al (2011) Association of a functional variant downstream of TNFAIP3 with systemic lupus erythematosus. Nat Genet 43:253–258 sourcePartly supported

  The Abstract identifies the SLE-associated TT>A variant downstream of TNFAIP3 and reports reduced binding by a complex containing NF-κB subunits, along with reduced TNFAIP3 expression on the variant-carrying haplotype. It does not show that NF-κB binding was abrogated: the binding-assay passage reports a difference in binding intensity, while the discussion says the precise mechanism linking the variant to expression remains uncertain. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [212] Wang S, Wen F, Wiley GB et al (2013) An enhancer element harboring variants associated with systemic lupus erythematosus engages the TNFAIP3 promoter to influence A20 expression. PLoS Genet 9:e1003750 sourcePartly supported

  The Abstract identifies TT>A as an SLE-associated variant in a downstream enhancer and reports reduced A20 expression, the protein encoded by TNFAIP3. The Results section, “The TT>A risk alleles bind NF-κB subunits with reduced affinity” (page unavailable), finds impaired NF-κB binding—not that binding is abrogated. The claim overstates that effect. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

Cis-regulatory elements exist on a spectrum of enhancer/promoter ability.

“Recently, it has been proposed that cis-regulatory elements exist on a spectrum of enhancer/promoter ability [233, 248].”

• cites [233] Andersson R, Sandelin A (2020) Determinants of enhancer and promoter activities of regulatory elements. Nat Rev Genet 21:71–87 sourceCould not verify

  The supplied passages and indexed matches contain bibliographic information, not the paper’s discussion of regulatory elements. Searches for an enhancer–promoter spectrum, a continuum of activity, and elements functioning as both enhancers and promoters found no substantive passage, so this copy cannot verify the claim. (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites [248] Mikhaylichenko O, Bondarenko V, Harnett D et al (2018) The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription. Genes Dev 32:42–57 sourceSupported

Promoters are sometimes repurposed into enhancers, but at a 13-fold lower rate than enhancer-to-promoter repurposing.

“Promoters are also sometimes repurposed into enhancers, but at a 13-fold lower rate than enhancer-to-promoter [100].”

• cites [100] Carelli FN, Liechti A, Halbert J et al (2018) Repurposing of promoters and enhancers during mammalian evolution. Nat Commun 9:4066 sourcePartly supported

  The Results section, ‘Enhancers are the main source of regulatory repurposing’ (page not specified), reports promoter-to-enhancer repurposing and an approximately 13-fold enrichment in the opposite direction after accounting for enhancer loss. It does not report a 13-fold difference in the raw repurposing rates: that difference was approximately 26-fold in the primate liver analysis. The comparable enrichment in glires was approximately sixfold and not statistically significant. (LLM, conf=0.91)

  Experimental — accuracy not yet measured

eQTL analysis in monocytes stimulated with interferon-γ or lipopolysaccharide revealed thousands of new variants affecting gene expression, which overlapped nearly 250 variants associated with response to infection and susceptibility to renal and immunological disorders.

“For instance, eQTL analysis in monocytes stimulated with interferon-γ or lipopolysaccharide has revealed thousands of new variants affecting gene expression, which overlapped nearly 250 variants associated with response to infection and susceptibility to renal and immunological disorders [228].”

• cites [228] Fairfax BP, Humburg P, Makino S et al (2014) Innate immune activity conditions the effect of regulatory variants upon monocyte gene expression. Science 343:1246949 sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

Some enhancers have undergone genetic mutations allowing them to be repurposed into gene promoters in mammals.

“The “grey area” between enhancers and promoters is supported by evolutionary evidence that some enhancers have undergone genetic mutations allowing them to be repurposed into gene promoters in mammals [100].”

• cites [100] Carelli FN, Liechti A, Halbert J et al (2018) Repurposing of promoters and enhancers during mammalian evolution. Nat Commun 9:4066 sourceSupported

Variants associated with type 1 diabetes were strongly enriched at promoter-connected active enhancers in activated, compared with resting, CD4+ T cells.

“Likewise, variants associated with type 1 diabetes were strongly enriched at promoter-connected active enhancers in activated, compared with resting, CD4+ T cells [229].”

• cites [229] Burren OS, Rubio García A, Javierre B-M et al (2017) Chromosome contacts in activated T cells identify autoimmune disease candidate genes. Genome Biol 18:165 sourcePartly supported

  The Results section (‘PCHi-C-facilitated mapping of candidate disease-causal genes’; page unavailable) reports overlap between fine-mapped autoimmune-disease variants and expressed enhancer RNAs at promoter-interacting regions. The paper also compares activated and non-activated CD4+ T cells, but the passages available do not establish that type 1 diabetes variants were strongly enriched at those enhancers in activated cells relative to resting cells. (LLM, conf=0.74)

  Experimental — accuracy not yet measured

The directionality of a regulatory element's transcriptional output is likely a key feature determining its position on the enhancer/promoter spectrum.

“One key feature determining the position of a regulatory element on this spectrum is likely to be the directionality of its transcriptional output.”

• cites [248] Mikhaylichenko O, Bondarenko V, Harnett D et al (2018) The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription. Genes Dev 32:42–57 sourceSupported

Knockouts of both PRC1 and PRC2 components affect chromosomal contacts between Polycomb-bound loci.

“However, knockouts of both PRC1 and PRC2 components affect chromosomal contacts between Polycomb-bound loci [280, 360].”

• cites [280] Schoenfelder S, Sugar R, Dimond A et al (2015) Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome. Nat Genet 47:1179–1186 sourceSupported
• cites [360] Denholtz M, Bonora G, Chronis C et al (2013) Long-range chromatin contacts in embryonic stem cells reveal a role for pluripotency factors and polycomb proteins in genome organization. Cell Stem Cell 13:602–616 sourcePartly supported

  The Results section, “The preferential co-localization of Polycomb-enriched genomic regions is Eed-dependent” (page unavailable), reports that loss of Eed, a PRC2 component, reduces contacts between Polycomb-occupied regions in embryonic stem cells. The passages do not report a knockout of a PRC1 component, so this source supports only the PRC2 part of the claim. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Deletion or degradation of cohesin or CTCF in differentiated cells has only mild effects on gene expression.

“Surprisingly however, deletion or degradation of cohesin or CTCF in differentiated cells has only mild effects on gene expression [344–349].”

• cites [344] Seitan VC, Faure AJ, Zhan Y et al (2013) Cohesin-based chromatin interactions enable regulated gene expression within preexisting architectural compartments. Genome Res 23:2066–2077 sourcePartly supported

  The Results (page unavailable) report that RAD21 deletion depleted cohesin in noncycling thymocytes and that 1,153 of 17,849 assayed genes were differentially expressed. This supports a limited effect by the proportion of genes affected, but the paper also reports expression changes across the full expression spectrum. It does not test CTCF deletion or degradation, so it cannot support that part of the claim. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [345] Zuin J, Dixon JR, van der Reijden MIJA et al (2014) Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells. Proc Natl Acad Sci USA 111:996–1001 sourceNot checked — no full text of the source
• cites [346] Nora EP, Goloborodko A, Valton A-L et al (2017) Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell 169:930–944.e22 sourcePartly supported

  The Discussion (‘TAD Insulation and Transcriptional Regulation’; page unavailable) describes the immediate transcriptional effects of CTCF depletion as ‘rather limited’ and ‘initially mild.’ But the experiments were in mouse embryonic stem cells, not differentiated cells; the Results also report substantially more differentially expressed genes after four days. This paper does not establish the claimed mild gene-expression effects of cohesin loss in differentiated cells. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [347] Rao SSP, Huang S-C, Glenn St Hilaire B et al (2017) Cohesin loss eliminates all loop domains. Cell 171:305-320.e24 sourcePartly supported

  The Results report that 87% of expressed genes changed in transcription by less than 30% after cohesin degradation, supporting the claim of generally mild effects. The study used HCT-116 colorectal carcinoma cells and did not test CTCF deletion or degradation for its effects on gene expression; it therefore does not establish the full claim about cohesin or CTCF in differentiated cells. Page numbers are not available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [348] Schwarzer W, Abdennur N, Goloborodko A et al (2017) Two independent modes of chromatin organization revealed by cohesin removal. Nature 551:51–56 sourcePartly supported

  In “Transcriptional changes upon TAD loss” (page unavailable), the paper reports that Nipbl deletion in differentiated hepatocytes significantly altered expression of about a thousand genes, while the cells’ regulatory potential was mostly unperturbed. This supports a limited overall effect, but the paper does not establish that the expression changes were uniformly mild or report gene-expression effects from CTCF deletion or degradation. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [349] Thiecke MJ, Wutz G, Muhar M et al (2020) Cohesin-dependent and -independent mechanisms mediate chromosomal contacts between promoters and enhancers. Cell Rep 32:107929 sourcePartly supported

  The Introduction says steady-state gene expression is less affected by architectural-protein depletion, and the Results report that most genes were relatively unperturbed after cohesin depletion. But the Introduction also says cohesin depletion impairs activation of inducible genes, so the unqualified claim about gene expression is broader than the paper supports. The paper’s experiments used HeLa Kyoto cells and protein degradation, not deletion. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

YY1 has a global role in connecting cell type-specific active enhancers and promoters in a range of mammalian cell types including mESCs and neural progenitor cells.

“Recent studies have confirmed its global role in connecting cell type-specific active enhancers and promoters in a range of mammalian cell types including mESCs [295] and neural progenitor cells [365].”

• cites [295] Weintraub AS, Li CH, Zamudio AV et al (2017) YY1 Is a structural regulator of enhancer-promoter loops. Cell 171:1573-1588.e28 sourceSupported
• cites [365] Beagan JA, Duong MT, Titus KR et al (2017) YY1 and CTCF orchestrate a 3D chromatin looping switch during early neural lineage commitment. Genome Res 27:1139–1152 sourcePartly supported

  The Results section reports YY1 enrichment at looping NPC-specific enhancers and genes, and loss of several such loops after Yy1 knockdown. But the paper describes YY1 as a candidate architectural protein and does not establish the claimed global role; it also says it cannot conclusively define YY1’s looping role in ES cells. In the citing sentence, the ES-cell example is attached to a different citation. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

PRC1 mediates chromatin interactions between a subset of active developmental enhancers and promoters in Drosophila.

“Moreover, PRC1 was recently found to mediate chromatin interactions between a subset of active developmental enhancers and promoters in Drosophila [361].”

• cites [361] Loubiere V, Papadopoulos GL, Szabo Q et al (2020) Widespread activation of developmental gene expression characterized by PRC1-dependent chromatin looping. Sci Adv 6:eaax4001 sourceSupported

DNA FISH gives access to the full distribution of inter-locus distances across all analysed cells.

“In contrast, DNA FISH gives access to the full distribution of inter-locus distances across all analysed cells [307].”

• cites [307] Fudenberg G, Imakaev M (2017) FISH-ing for captured contacts: towards reconciling FISH and 3C. Nat Methods 14:673–678 sourceSupported

Divergent gene pairs separated by CTCF sites had lower than expected correlation of gene expression genome-wide, confirming the insulatory role of CTCF on the global scale.

“and it was found that divergent gene pairs separated by CTCF sites had lower than expected correlation of gene expression genome-wide, confirming the insulatory role of CTCF on the global scale [327].”

• cites [327] Xie X, Mikkelsen TS, Gnirke A et al (2007) Systematic discovery of regulatory motifs in conserved regions of the human genome, including thousands of CTCF insulator sites. Proc Natl Acad Sci USA 104:7145–7150 sourceSupported

YY1 and ZNF143 are zinc finger TFs with DNA binding domains that have been implicated as looping factors mediating interactions between active regulatory elements.

“YY1 and ZNF143 are both zinc finger TFs with DNA binding domains that have recently been implicated as looping factors mediating interactions between active regulatory elements [295, 362–365].”

• cites [295] Weintraub AS, Li CH, Zamudio AV et al (2017) YY1 Is a structural regulator of enhancer-promoter loops. Cell 171:1573-1588.e28 sourcePartly supported

  The Introduction and Discussion (page numbers unavailable) identify YY1 as a DNA-binding zinc-finger factor that facilitates enhancer–promoter loops. Our copy does not establish the corresponding claim about ZNF143; a search for ZNF143 returned no relevant passage. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [362] Heidari N, Phanstiel DH, He C et al (2014) Genome-wide map of regulatory interactions in the human genome. Genome Res 24:1905–1917 sourcePartly supported

  The Results section identifies ZNF143 as a zinc finger protein enriched at interacting loci, and the Discussion says the findings implicate it in long-range chromatin interactions. The passages do not establish the same claim for YY1, and the Discussion says ZNF143’s specific role in establishing or maintaining chromatin structure remains to be determined. No page numbers were available in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [363] Bailey SD, Zhang X, Desai K et al (2015) ZNF143 provides sequence specificity to secure chromatin interactions at gene promoters. Nat Commun 2:6186 sourcePartly supported

  The Introduction and Discussion identify ZNF143 as a zinc-finger protein that binds a DNA recognition sequence and helps secure interactions between promoters and distal regulatory elements. The cited paper does not establish the corresponding claim about YY1; a search for YY1 found no relevant passage in our copy. Page numbers were not available. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [364] Ye B-Y, Shen W-L, Wang D et al (2016) ZNF143 is involved in CTCF-mediated chromatin interactions by cooperation with cohesin and other partners. Mol Biol 50:496–503 sourceNot checked — no full text of the source
• cites [365] Beagan JA, Duong MT, Titus KR et al (2017) YY1 and CTCF orchestrate a 3D chromatin looping switch during early neural lineage commitment. Genome Res 27:1139–1152 sourcePartly supported

  The Results section identifies YY1 as a zinc-finger protein enriched at enhancer–gene looping interactions, and the Discussion describes it as a candidate architectural protein. Our copy does not address ZNF143, so it supports the YY1 part of the claim but not the assertion about both factors. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

LDB1 was first identified as a looping factor in the mouse β-globin locus.

“LDB1 was first identified as a looping factor in the mouse β-globin locus [317, 371].”

• cites [317] Song S-H, Hou C, Dean A (2007) A positive role for NLI/Ldb1 in long-range beta-globin locus control region function. Mol Cell 28:810–822 sourceNot checked — no full text of the source
• cites [371] Deng W, Lee J, Wang H et al (2012) Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor. Cell 149:1233–1244 sourcePartly supported

  The Introduction identifies Ldb1 as a factor involved in LCR–β-globin interactions, and the Results show that tethering Ldb1 can produce an LCR–β-globin loop. Our copy does not establish that the mouse β-globin locus was where Ldb1 was first identified as a looping factor; the Introduction cites earlier work on its role there. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Some contacts between PcG-associated regions, including poised promoters and enhancers in ESCs, are retained after these regions are activated during cell differentiation.

“Notably, some contacts between PcG-associated regions, including poised promoters and enhancers in ESCs, are retained after these regions are activated during cell differentiation [141, 143].”

• cites [141] Freire-Pritchett P, Schoenfelder S, Várnai C et al (2017) Global reorganisation of regulatory units upon lineage commitment of human embryonic stem cells. Elife 6:e21926 sourcePartly supported

  The Results section on rewiring and recolouring (Figure 6E–F; page not available) reports that some promoter interactions persist during ESC-to-NEC differentiation, including an IRX3 interaction whose promoter-interacting region changes from repressed to active. The Results section on regulatory elements also reports contacts between poised or Polycomb-associated promoters and similarly marked regions. The copy does not establish that the retained contacts specifically join poised promoters and enhancers that both become activated. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [143] Cruz-Molina S, Respuela P, Tebartz C et al (2017) PRC2 facilitates the regulatory topology required for poised enhancer function during pluripotent stem cell differentiation. Cell Stem Cell 20:689-705.e9 sourceNot checked — no full text of the source

Transgene assays of the human β-globin locus showed that two insulator sites binding CTCF could form a chromatin loop.

“Transgene assays of the human β-globin locus then showed that two insulator sites binding CTCF could form a chromatin loop [332].”

• cites [332] Hou C, Zhao H, Tanimoto K, Dean A (2008) CTCF-dependent enhancer-blocking by alternative chromatin loop formation. Proc Natl Acad Sci U S A 105:20398–20403 sourceSupported

In mammals, PcG-associated promoters and enhancers engage in multitudes of looping contacts, particularly in early development.

“In mammals, PcG-associated promoters and enhancers also engage in multitudes of looping contacts, particularly in early development [139, 141, 276, 357].”

• cites [139] Ngan CY, Wong CH, Tjong H et al (2020) Chromatin interaction analyses elucidate the roles of PRC2-bound silencers in mouse development. Nat Genet 52:264–272 sourcePartly supported

  The Results section on PRC2-mediated chromatin interactions reports numerous loops involving promoters and distal regions at developmentally regulated genes in mouse embryonic stem cells (page unavailable). The section on PRC2-associated silencers identifies some distal anchors as having poised-enhancer signatures, but says their enhancer activity can emerge during differentiation. It does not establish that active PcG-associated enhancers generally engage in many loops throughout early mammalian development. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [141] Freire-Pritchett P, Schoenfelder S, Várnai C et al (2017) Global reorganisation of regulatory units upon lineage commitment of human embryonic stem cells. Elife 6:e21926 sourceSupported
• cites [276] Hsieh T-HS, Cattoglio C, Slobodyanyuk E et al (2020) Resolving the 3D landscape of transcription-linked mammalian chromatin folding. Mol Cell 78:539-553.e8 sourcePartly supported

  The Results section ('Transcription factors mediate chromatin architecture within TADs'; page unavailable) reports that Polycomb-associated, H3K27me3-rich regions form nested sets of chromatin contacts, supporting the claim’s general point about multiple contacts. It does not establish that the interacting regions are specifically PcG-associated promoters and enhancers, or that these contacts occur particularly in early development. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [357] Rhodes JDP, Feldmann A, Hernández-Rodríguez B et al (2020) Cohesin disrupts polycomb-dependent chromosome interactions in embryonic stem cells. Cell Rep 30:820-835.e10 sourcePartly supported

  The Results (‘Cohesin-Independent Chromosomal Interactions Exist in ESCs’; page unavailable) report numerous Polycomb-associated interactions in embryonic stem cells, but say the interacting domains were mostly associated with promoters. They note that Polycomb domains can also be associated with enhancers without establishing that PcG-associated enhancers engage in multitudes of looping contacts. The cell-type-specific Results further find much weaker or no obvious interactions in the other mammalian cell types examined, so the broad mammalian wording exceeds this evidence. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

CTCF binding has an insulating capacity, effectively blocking signals between enhancers and their target genes in the β-globin and H19/Igf2 loci.

“More unusually, it emerged that CTCF binding has an insulating capacity, effectively blocking signals between enhancers and their target genes in the β-globin and H19/Igf2 loci [323–326].”

• cites [323] Bell AC, West AG, Felsenfeld G (1999) The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell 98:387–396 sourceNot checked — no full text of the source
• cites [324] Saitoh N, Bell AC, Recillas-Targa F et al (2000) Structural and functional conservation at the boundaries of the chicken beta-globin domain. EMBO J 19:2315–2322 sourceSupported
• cites [325] Hark AT, Schoenherr CJ, Katz DJ et al (2000) CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405:486–489 sourceNot checked — no full text of the source
• cites [326] Kanduri C, Pant V, Loukinov D et al (2000) Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive. Curr Biol 10:853–856 sourceNot checked — no full text of the source

CTCF associates with the cohesin complex.

“A key insight into how CTCF mediates chromosomal looping was the discovery of its association with the cohesin complex [132, 334, 335].”

• cites [132] Parelho V, Hadjur S, Spivakov M et al (2008) Cohesins functionally associate with CTCF on mammalian chromosome arms. Cell 132:422–433 sourceNot checked — no full text of the source
• cites [334] Rubio ED, Reiss DJ, Welcsh PL et al (2008) CTCF physically links cohesin to chromatin. Proc Natl Acad Sci USA 105:8309–8314 sourceSupported
• cites [335] Wendt KS, Yoshida K, Itoh T et al (2008) Cohesin mediates transcriptional insulation by CCCTC-binding factor. Nature 451:796–801 sourceNot checked — no full text of the source

Several TFs were required for looping between the LCR and the distal genes, including GATA-1, FOG-1, EKLF and NLI/Ldb1.

“Following the discovery of chromatin looping in the β-globin locus, it was ascertained that several TFs were required for looping between the LCR and the distal genes, including GATA-1, FOG-1, EKLF and NLI/Ldb1 [315–317].”

• cites [315] Vakoc CR, Letting DL, Gheldof N et al (2005) Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1. Mol Cell 17:453–462 sourceSupported
• cites [316] Drissen R, Palstra R-J, Gillemans N et al (2004) The active spatial organization of the beta-globin locus requires the transcription factor EKLF. Genes Dev 18:2485–2490 sourceSupported
• cites [317] Song S-H, Hou C, Dean A (2007) A positive role for NLI/Ldb1 in long-range beta-globin locus control region function. Mol Cell 28:810–822 sourceNot checked — no full text of the source

Forced looping between β-globin and its LCR was shown to rescue β-globin expression in K562 erythroid cells (which instead aberrantly express γ-globin) in a reversible manner.

“Forced looping between β-globin and its LCR was shown to rescue β-globin expression in K562 erythroid cells (which instead aberrantly express γ-globin) in a reversible manner [313].”

• cites [313] Morgan SL, Chang EY, Mariano NC et al (2018) CRISPR-mediated reorganization of chromatin loop structure. J Vis Exp. https://doi.org/10.3791/57457 sourceSupported

Promoter Capture Hi-C-based studies did not observe a significant change in enhancer-promoter contacts upon RNAP inhibition, including in cells rapidly depleted of cohesin or CTCF.

“However, Promoter Capture Hi-C-based studies did not observe a significant change in enhancer–promoter contacts upon RNAP inhibition, including in cells rapidly depleted of cohesin or CTCF [349, 380].”

• cites [349] Thiecke MJ, Wutz G, Muhar M et al (2020) Cohesin-dependent and -independent mechanisms mediate chromosomal contacts between promoters and enhancers. Cell Rep 32:107929 sourcePartly supported

  The Results section on cohesin-dependent and -independent promoter interactions reports that the RNA polymerase II inhibitor triptolide had only a mild effect on promoter interaction profiles. It does not say the effect was statistically insignificant or report that inhibition was tested in cells simultaneously depleted of cohesin or CTCF. A separate Results section reports substantial rewiring of contacts after architectural-protein depletion alone. Page numbers are unavailable in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [380] El Khattabi L, Zhao H, Kalchschmidt J et al (2019) A pliable mediator acts as a functional rather than an architectural bridge between promoters and enhancers. Cell 178:1145-1158.e20 sourcePartly supported

  In Results, ‘The role of Mediator in promoter-enhancer interactions’ (page unavailable), promoter capture Hi-C showed little or no change in enhancer–promoter contacts after PolII and Mediator depletion, and no further change when they were depleted alongside cohesin. The paper reports rapid cohesin depletion, but not rapid CTCF depletion; loss of CTCF-dependent loops after cohesin depletion is not the same experiment. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Transcription at stimulus-response genes was shown to disrupt chromatin loops, potentially through destabilising cohesin association with the chromatin fibre by RNAP machinery.

“For example, transcription at stimulus–response genes was shown to disrupt chromatin loops [387, 388], potentially through destabilising cohesin association with the chromatin fibre by RNAP machinery [388].”

• cites [387] Jubb AW, Boyle S, Hume DA, Bickmore WA (2017) Glucocorticoid receptor binding induces rapid and prolonged large-scale chromatin decompaction at multiple target loci. Cell Rep 21:3022–3031 sourcePartly supported

  The Results report chromatin decompaction at glucocorticoid-responsive loci, but the Discussion says dissolution of preformed loops cannot be excluded—not that transcription was shown to disrupt loops. The Results and Discussion also say decompaction can occur without transcription. Our copy does not identify destabilisation of cohesin by RNAP machinery as a mechanism (searched: cohesin). (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [388] Heinz S, Texari L, Hayes MGB et al (2018) Transcription elongation can affect genome 3D structure. Cell 174:1522-1536.e22 sourceSupported

Neither Hi-C nor Micro-C detected a widespread enrichment for enhancer-promoter interactions in early Drosophila embryos irrespective of the activity of these elements.

“For example, neither Hi-C nor Micro-C detected a widespread enrichment for enhancer–promoter interactions in early Drosophila embryos irrespective of the activity of these elements [393].”

• cites [393] Ing-Simmons E, Vaid R, Bing XY et al (2021) Independence of chromatin conformation and gene regulation during Drosophila dorsoventral patterning. Nat Genet 53:487–499 sourceSupported

Transcription can either disrupt or stabilise specific enhancer-promoter loops.

“Whilst it is established that transcription helps shape up higher-level genome conformation (reviewed in [386]), there is also evidence that it can either disrupt or stabilise specific enhancer–promoter loops.”

• cites [386] van Steensel B, Furlong EEM (2019) The role of transcription in shaping the spatial organization of the genome. Nat Rev Mol Cell Biol 20:327–337 sourceSupported

Removing BRD4 (and, consequently, Mediator) from chromatin using chemical inhibitors did not perturb the majority of enhancer-promoter contacts, despite major changes in transcription.

“Nonetheless, removing BRD4 (and, consequently, Mediator) from chromatin using chemical inhibitors did not perturb the majority of enhancer–promoter contacts, despite major changes in transcription [69].”

• cites [69] Crump NT, Ballabio E, Godfrey L et al (2021) BET inhibition disrupts transcription but retains enhancer–promoter contact. Nat Commun 12:223 sourcePartly supported

  The Introduction and Results report major transcriptional changes while enhancer–promoter contacts remained largely intact after BET inhibitor treatment. However, the Discussion says the drugs reduced BRD4 and Mediator binding but left detectable binding, so describing both proteins as removed from chromatin overstates the treatment’s effect. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Evidence for chromatin LLPS and its functional significance in gene control is still incomplete.

“Whilst LLPS in general has been demonstrated both in vitro and in vivo [421, 422], evidence for chromatin LLPS and its functional significance in gene control is still incomplete.”

• cites [421] Nott TJ, Petsalaki E, Farber P et al (2015) Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol Cell 57:936–947 sourceSupported
• cites [422] Elbaum-Garfinkle S, Kim Y, Szczepaniak K et al (2015) The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc Natl Acad Sci USA 112:7189–7194 sourceSupported

Rapid removal of chromatin-bound Mediator and RNAP does not significantly affect chromosomal architecture, including enhancer-promoter interactions, in spite of triggering widespread transcriptional changes and cell cycle arrest.

“However, it was shown recently that rapid removal of chromatin-bound Mediator and RNAP does not significantly affect chromosomal architecture, including enhancer–promoter interactions, in spite of triggering widespread transcriptional changes and cell cycle arrest [380].”

• cites [380] El Khattabi L, Zhao H, Kalchschmidt J et al (2019) A pliable mediator acts as a functional rather than an architectural bridge between promoters and enhancers. Cell 178:1145-1158.e20 sourceSupported

An explanation for TAD formation and maintenance has been provided by the model of chromosomal loop extrusion.

“an explanation has eventually been provided by the model of chromosomal loop extrusion [342, 435].”

• cites [342] Sanborn AL, Rao SSP, Huang S-C et al (2015) Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes. Proc Natl Acad Sci USA 112:E6456–E6465 sourceNot checked — no full text of the source
• cites [435] Fudenberg G, Imakaev M, Lu C et al (2016) Formation of chromosomal domains by loop extrusion. Cell Rep 15:2038–2049 sourceSupported

A proximal enhancer of the Shh gene moves further away from the Shh promoter upon activation, according to both 3C-derived methods and imaging.

“In contrast, a proximal enhancer of the Shh gene moves further away from the Shh promoter upon activation, according to both 3C-derived methods and imaging [407].”

• cites [407] Benabdallah NS, Williamson I, Illingworth RS et al (2019) Decreased enhancer-promoter proximity accompanying enhancer activation. Mol Cell 76:473-484.e7 sourceSupported

High-concentration hubs were capable of transactivation and recruitment of RNAP even without LLPS.

“Furthermore, since high-concentration hubs were capable of transactivation and recruitment of RNAP even without LLPS, the question remains if LLPS is necessary for any genomic locus [412].”

• cites [412] Chong S, Dugast-Darzacq C, Liu Z et al (2018) Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science 361:eaar2555 sourcePartly supported

  The ‘LCD hubs interact with RNA Pol II’ section reports polymerase enrichment in LCD hubs, and the Discussion reports transactivation at native chromosomal loci without detectable phase separation. The paper says phase separation was not detected at endogenous expression levels; it does not establish that LLPS was absent, as the claim’s unqualified wording implies. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Enhancer-promoter loops dissociated tenfold faster in the absence of transcription than when the reporter gene was being transcribed, suggesting that transcription can stabilise contact between these elements.

“Moreover, enhancer–promoter loops dissociated tenfold faster in the absence of transcription than when the reporter gene was being transcribed, suggesting that transcription can stabilise contact between these elements [311].”

• cites [311] Chen H, Levo M, Barinov L et al (2018) Dynamic interplay between enhancer–promoter topology and gene activity. Nat Genet 50:1296–1303 sourceSupported

In mammalian differentiation models, extensive remodelling of enhancer-promoter contacts is typically observed consistently with enhancer activation.

“In mammalian differentiation models, in contrast, extensive remodelling of enhancer–promoter contacts is typically observed consistently with enhancer activation [141, 394–397].”

• cites [141] Freire-Pritchett P, Schoenfelder S, Várnai C et al (2017) Global reorganisation of regulatory units upon lineage commitment of human embryonic stem cells. Elife 6:e21926 sourceSupported
• cites [394] Rubin AJ, Barajas BC, Furlan-Magaril M et al (2017) Lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation. Nat Genet 49:1522–1528 sourcePartly supported

  The indexed passages (section labeled Abstract; no page given) report that enhancer–promoter contacts gained strength alongside enhancer H3K27ac activation during epidermal differentiation. But the paper studies this differentiation model, not what is typically observed across mammalian models, and it also reports that 65% of differentiation-induced genes had exclusively stable, pre-established contacts. The broader claim of typical extensive remodelling therefore overstates this source. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [395] Siersbæk R, Madsen JGS, Javierre BM et al (2017) Dynamic rewiring of promoter-anchored chromatin loops during adipocyte differentiation. Mol Cell 66:420-435.e5 sourcePartly supported

  The Results and Discussion report extensive promoter–enhancer loop rewiring coupled to enhancer activation during 3T3-L1 adipocyte differentiation. But the paper studies one differentiation model and says previous studies had not reached a clear consensus on loop plasticity; it does not establish that this is typically observed across mammalian differentiation models. Page numbers are unavailable in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [396] Oudelaar AM, Beagrie RA, Gosden M et al (2020) Dynamics of the 4D genome during in vivo lineage specification and differentiation. Nat Commun 11:2722 sourceSupported
• cites [397] Dixon JR, Jung I, Selvaraj S et al (2015) Chromatin architecture reorganization during stem cell differentiation. Nature 518:331–336 sourcePartly supported

  The Results section, ‘Chromatin state and dynamic interactions’ (page unavailable), reports changing long-range interactions during lineage specification associated with H3K4me1 and enhancer density. It does not establish that the extensively remodelled contacts are specifically enhancer–promoter contacts or that their remodelling consistently accompanies enhancer activation. The separate ‘Allelic imbalances in gene expression’ results link enhancer activity to promoter contacts, but examine allelic differences rather than contact remodelling during differentiation. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Live cell imaging over a ~25 min interval could not detect close proximity between the SLC and the Sox2 promoter, even during the bursts of Sox2 transcription.

“However, live cell imaging over a ~ 25 min interval could not detect close proximity between the SLC and the Sox2 promoter, even during the bursts of Sox2 transcription [402].”

• cites [402] Alexander JM, Guan J, Li B et al (2019) Live-cell imaging reveals enhancer-dependent Sox2 transcription in the absence of enhancer proximity. eLife 8:e41769 sourcePartly supported

  The Results section (“Sox2 transcription is not associated with SCR proximity”; page unavailable) reports bursts without close Sox2–SCR proximity and no significant change in distance across a 25-minute window centered on burst initiation. But it also reports prolonged proximity in some transcriptionally active cells, so the categorical claim that imaging could not detect close proximity even during bursts overstates the finding. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Synthetic activation of the Shh promoter, bypassing the need for enhancer activation and causing increased transcription, did not lead to increased distance.

“Notably, synthetic activation of the Shh promoter, bypassing the need for enhancer activation and causing increased transcription, did not lead to increased distance [407].”

• cites [407] Benabdallah NS, Williamson I, Illingworth RS et al (2019) Decreased enhancer-promoter proximity accompanying enhancer activation. Mol Cell 76:473-484.e7 sourceSupported

Knockdown of eRNAs resulted in decreased enhancer-promoter proximity in human cells.

“Knockdown of eRNAs resulted in decreased enhancer–promoter proximity in human cells [81, 390].”

• cites [81] Hsieh C-L, Fei T, Chen Y et al (2014) Enhancer RNAs participate in androgen receptor-driven looping that selectively enhances gene activation. Proc Natl Acad Sci USA 111:7319–7324 sourceNot checked — no full text of the source
• cites [390] Li W, Notani D, Ma Q et al (2013) Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation. Nature 498:516–520 sourceSupported

In Drosophila, pronounced changes in enhancer activity during embryonic development seem to occur without prominent changes in their 3D conformation.

“In Drosophila, pronounced changes in enhancer activity during embryonic development seem to occur without prominent changes in their 3D conformation [392, 393].”

• cites [392] Ghavi-Helm Y, Klein FA, Pakozdi T et al (2014) Enhancer loops appear stable during development and are associated with paused polymerase. Nature 512:96–100 sourceNot checked — no full text of the source
• cites [393] Ing-Simmons E, Vaid R, Bing XY et al (2021) Independence of chromatin conformation and gene regulation during Drosophila dorsoventral patterning. Nat Genet 53:487–499 sourceSupported

Commonly used in vivo approaches such as FRAP and sensitivity to 1,6-hexanediol are not always indicative of LLPS.

“commonly used in vivo approaches like fast fluorescence recovery after photobleaching (FRAP) and sensitivity to treatment with 1,6-hexanediol are not always indicative of LLPS [423].”

• cites [423] Alberti S, Gladfelter A, Mittag T (2019) Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell 176:419–434 sourceSupported

Under the loop extrusion model, cohesin in interphase nuclei continuously extrudes chromosomal loops until it encounters a boundary, which prevents further extrusion.

“Under this model, cohesin in interphase nuclei continuously extrudes chromosomal loops until it encounters a boundary, which prevents further extrusion.”

• cites [342] Sanborn AL, Rao SSP, Huang S-C et al (2015) Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes. Proc Natl Acad Sci USA 112:E6456–E6465 sourceNot checked — no full text of the source
• cites [435] Fudenberg G, Imakaev M, Lu C et al (2016) Formation of chromosomal domains by loop extrusion. Cell Rep 15:2038–2049 sourceSupported

LDB1 forms loops independently of both cohesin and CTCF.

“In contrast to YY1 and ZNF143, LDB1 has been shown to form loops independently of both cohesin and CTCF [371, 375].”

• cites [371] Deng W, Lee J, Wang H et al (2012) Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor. Cell 149:1233–1244 sourcePartly supported

  The Results section reports that tethering Ldb1’s self-association domain produces an LCR–β-globin promoter loop, and the Discussion identifies Ldb1 self-association as a force behind long-range interactions. Neither passage establishes that these loops form independently of cohesin or CTCF; searches for both terms found no such test or conclusion in our copy. Page numbers were not available. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [375] Krivega I, Dale RK, Dean A (2014) Role of LDB1 in the transition from chromatin looping to transcription activation. Genes Dev 28:1278–1290 sourcePartly supported

  The Abstract and Results (Figure 3; page not specified) show that LDB1 and its dimerization domain support β-globin LCR–promoter looping. They do not establish that this looping is independent of cohesin or CTCF; the paper discusses those proteins as factors in other looping mechanisms. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Metazoan genomes are organised into megabase-scale segregated domains that are often invariant between cell types, termed TADs.

“metazoan genomes are organised into megabase-scale segregated domains that are often invariant between cell types, termed topologically associating domains (TADs) [432–434].”

• cites [432] Dixon JR, Selvaraj S, Yue F et al (2012) Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485:376–380 sourceSupported
• cites [433] Nora EP, Lajoie BR, Schulz EG et al (2012) Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485:381–385 sourceSupported
• cites [434] Ji X, Dadon DB, Powell BE et al (2016) 3D chromosome regulatory landscape of human pluripotent cells. Cell Stem Cell 18:262–275 sourceSupported

Permissive contacts were observed in mammalian cells upon TNF-induced enhancer activation.

“In addition, permissive contacts were also observed in mammalian cells upon TNF-induced enhancer activation [398].”

• cites [398] Jin F, Li Y, Dixon JR et al (2013) A high-resolution map of the three-dimensional chromatin interactome in human cells. Nature 503:290–294 sourceSupported

Mechanisms by which LADs promote transcriptional repression may include sequestration of genes and their regulatory elements in heterochromatin foci.

“Mechanisms by which LADs promote transcriptional repression are not fully understood, but may include sequestration of genes and their regulatory elements in heterochromatin foci [462].”

• cites [462] van Steensel B, Belmont AS (2017) Lamina-associated domains: links with chromosome architecture, heterochromatin, and gene repression. Cell 169:780–791 sourceSupported

The insulating effect of TAD boundaries may be inversely dependent on enhancer strength.

“Recent mechanistic experiments suggest that the insulating effect of TAD boundaries may be inversely dependent on enhancer strength [405].”

• cites [405] Zuin J, Roth G, Zhan Y et al (2021) Nonlinear control of transcription through enhancer–promoter interactions. BioRxiv. https://doi.org/10.1101/2021.04.22.440891 sourcePartly supported

  The Main text reports that a weaker enhancer was completely insulated by a CTCF site, whereas the stronger full-length enhancer was not. It also says insulation is context-dependent, but does not establish an inverse relationship specifically for TAD boundaries; it reports complete TAD-boundary insulation even for the strong enhancer. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

The majority of specific enhancer-promoter loops are highly enriched within, compared with across, TAD boundaries.

“The majority of specific enhancer–promoter loops follow this pattern and are highly enriched within, compared with across, TAD boundaries [277, 401, 405].”

• cites [277] Rao SSP, Huntley MH, Durand NC et al (2014) A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159:1665–1680 sourcePartly supported

  The Results section on promoter-anchored loops reports many enhancer–promoter loops, and the Results section on domain boundaries reports that loops often demarcate domains (page numbers unavailable in our copy). Neither passage establishes that a majority of enhancer–promoter loops lie within TAD boundaries or that they are enriched within versus across those boundaries. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [401] Bonev B, Mendelson Cohen N, Szabo Q et al (2017) Multiscale 3D genome rewiring during mouse neural development. Cell 171:557-572.e24 sourceSupported
• cites [405] Zuin J, Roth G, Zhan Y et al (2021) Nonlinear control of transcription through enhancer–promoter interactions. BioRxiv. https://doi.org/10.1101/2021.04.22.440891 sourceSupported

Cis-regulatory contacts emerge prior to the formation of TADs in murine cells exiting mitosis.

“Cis-regulatory contacts were also shown to emerge prior to the formation of TADs in murine cells exiting mitosis [455].”

• cites [455] Zhang H, Emerson DJ, Gilgenast TG et al (2019) Chromatin structure dynamics during the mitosis-to-G1 phase transition. Nature 576:158–162 sourceSupported

Many computational algorithms for Hi-C analysis explicitly correct for distance decay, identifying significant interactions based on how unusual their contact frequency is at a given distance.

“many computational algorithms for the analysis of data from Hi-C and related techniques explicitly correct for distance decay, identifying “significant interactions” based on how unusual their contact frequency is at a given distance [277, 468–471].”

• cites [277] Rao SSP, Huntley MH, Durand NC et al (2014) A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159:1665–1680 sourcePartly supported

  The Experimental Procedures section (“Annotation of Peaks”; page unavailable) describes an algorithm that identifies statistically significant contact frequencies by comparing each Hi-C pixel with its surrounding neighborhoods. That supports the unusual-contact-frequency part of the claim. The paper does not establish that many algorithms explicitly correct for distance decay or test contacts against an expectation at a given genomic distance; its stated comparison is with local background. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [468] Kaul A, Bhattacharyya S, Ay F (2020) Identifying statistically significant chromatin contacts from Hi-C data with FitHiC2. Nat Protoc 15:991–1012 sourceSupported
• cites [469] Bhattacharyya S, Chandra V, Vijayanand P, Ay F (2019) Identification of significant chromatin contacts from HiChIP data by FitHiChIP. Nat Commun 10:4221 sourceSupported
• cites [470] Holgersen EM, Gillespie A, Leavy OC et al (2021) Identifying high-confidence capture Hi-C interactions using CHiCANE. Nat Protoc 16:2257–2285 sourceNot checked — no full text of the source
• cites [471] Cairns J, Freire-Pritchett P, Wingett SW et al (2016) CHiCAGO: robust detection of DNA looping interactions in capture Hi-C data. Genome Biol 17:127 sourceSupported

A massively parallel reporter assay in Drosophila cells revealed distinct groups of enhancers that preferred one of two types of core promoter, derived from either a housekeeping or a developmental gene and enriched for different TF binding motifs.

“a massively parallel reporter assay in Drosophila cells revealed distinct groups of enhancers that preferred one of two types of core promoter, derived from either a housekeeping or a developmental gene and enriched for different TF binding motifs [247].”

• cites [247] Zabidi MA, Arnold CD, Schernhuber K et al (2015) Enhancer–core-promoter specificity separates developmental and housekeeping gene regulation. Nature 518:556–559 sourceSupported

Enhancer trap experiments in Drosophila showed that some endogenous enhancers had a preference for either a TATA- or a DPE-containing promoter.

“enhancer trap experiments in Drosophila that used randomly integrated reporter genes driven by a TATA- and a DPE-containing promoter, respectively, showed that some endogenous enhancers had a preference for either of the two promoters [475].”

• cites [475] Butler JE, Kadonaga JT (2001) Enhancer–promoter specificity mediated by DPE or TATA core promoter motifs. Genes Dev 15:2515–2519 sourceSupported

Active promoters and enhancers can likely give rise to TAD boundaries even in the absence of convergent CTCF sites.

“active promoters and enhancers can likely give rise to TAD boundaries even in the absence of convergent CTCF sites [401].”

• cites [401] Bonev B, Mendelson Cohen N, Szabo Q et al (2017) Multiscale 3D genome rewiring during mouse neural development. Cell 171:557-572.e24 sourcePartly supported

  In Results, “Transcription Is Correlated with but Not Sufficient to Induce Insulation at TAD Boundaries De Novo” (page not provided), the paper identifies CTCF-negative TAD boundaries near active promoters. It does not establish that active enhancers give rise to boundaries, and its gene-activation experiment found that transcriptional activation alone was insufficient to create one. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Around one-sixth of the genome undergoes compartment changes during the 24 h circadian cycle in mouse adult liver.

“around 1/6th of the genome undergoes compartment changes during the 24 h circadian cycle in mouse adult liver [458].”

• cites [458] Furlan-Magaril M, Ando-Kuri M, Arzate-Mejía RG et al (2021) The global and promoter-centric 3D genome organization temporally resolved during a circadian cycle. Genome Biol 22:162 sourceSupported

Capture Hi-C analyses in multiple cell types consistently detect enhancer-promoter contacts that cross TAD boundaries.

“Capture Hi-C analyses in multiple cell types consistently detect enhancer–promoter contacts that cross TAD boundaries [141, 243].”

• cites [141] Freire-Pritchett P, Schoenfelder S, Várnai C et al (2017) Global reorganisation of regulatory units upon lineage commitment of human embryonic stem cells. Elife 6:e21926 sourceSupported
• cites [243] Javierre BM, Burren OS, Wilder SP et al (2016) Lineage-specific genome architecture links enhancers and non-coding disease variants to target gene promoters. Cell 167:1369-1384.e19 sourcePartly supported

  The Results (Promoter Capture Hi-C; page not provided) report that about a third of PCHi-C interactions crossed TAD boundaries in all eight cell types examined. But that analysis covers promoter interactions generally; it does not establish that the boundary-crossing contacts specifically joined enhancers to promoters. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

TADs correspond to only one level of a hierarchy of loop domains, with super-/sub-TADs and insulated neighbourhoods potentially corresponding to other levels.

“TADs correspond to only one level of a hierarchy of such “loop domains”, with super-/sub-TADs and insulated neighbourhoods potentially corresponding to other levels [434, 441, 442].”

• cites [434] Ji X, Dadon DB, Powell BE et al (2016) 3D chromosome regulatory landscape of human pluripotent cells. Cell Stem Cell 18:262–275 sourcePartly supported

  The Discussion describes TADs as nested sets of CTCF–CTCF loops, and Results (“Schematics of TAD structures”) identifies insulated neighborhoods within TADs. These passages support multiple levels of loop organization, but do not establish the proposed place of super-TADs in that hierarchy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [441] Dowen JM, Fan ZP, Hnisz D et al (2014) Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes. Cell 159:374–387 sourcePartly supported

  The Introduction describes a hierarchy that includes TADs and gene loops, and the Discussion places CTCF-associated loop substructures within TADs. This supports insulated neighborhoods and sub-TAD structures as possible levels below TADs, but the paper does not address super-TADs as another level. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [442] Dixon JR, Gorkin DU, Ren B (2016) Chromatin domains: the unit of chromosome organization. Mol Cell 62:668–680 sourcePartly supported

  The Nature of TADs section (page unavailable) describes hierarchical TAD organization, with sub-TADs and insulation neighborhoods at smaller scales. It does not mention super-TADs, and it leaves open whether TADs reflect looping events or “loop domains,” so it does not fully support the proposed hierarchy of loop domains. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Disruption of TADs can cause promiscuous enhancer-promoter contacts and ectopic gene expression leading to developmental aberrations and disease.

“disruption of TADs can cause promiscuous enhancer–promoter contacts and ectopic gene expression leading to developmental aberrations and disease (reviewed in [445])”

• cites [445] Lupiáñez DG, Spielmann M, Mundlos S (2016) Breaking TADs: how alterations of chromatin domains result in disease. Trends Genet 32:225–237 sourcePartly supported

  In “TADs and Disease,” the review reports that disrupted TAD boundaries permit ectopic enhancer–promoter interactions and gene misexpression that cause malformations. But “Additional information” cautions that enhancers are not simply promiscuous or nonselective: only a subset of accessible genes responds. The claim is supported except for that characterization of the contacts. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Targeted deletions of TAD boundary-forming CTCF sites in the Shh locus did not entirely disrupt developmental regulation of Shh expression.

“targeted deletions of TAD boundary-forming CTCF sites in the Shh locus did not entirely disrupt the developmental regulation of Shh expression [450, 451].”

• cites [450] Williamson I, Kane L, Devenney PS et al (2019) Developmentally regulated expression is robust to TAD perturbations. Development 146:dev179523 sourceSupported
• cites [451] Paliou C, Guckelberger P, Schöpflin R et al (2019) Preformed chromatin topology assists transcriptional robustness of during limb development. Proc Natl Acad Sci USA 116:12390–12399 sourceSupported

In an ectopic context, the SCR enhancer-Sox2 promoter pair did not show prominent specific interactions, despite clearly detectable transcriptional effects of SCR.

“in the ectopic context this enhancer–promoter pair did not show prominent specific interactions, despite the clearly detectable transcriptional effects of SCR [405].”

• cites [405] Zuin J, Roth G, Zhan Y et al (2021) Nonlinear control of transcription through enhancer–promoter interactions. BioRxiv. https://doi.org/10.1101/2021.04.22.440891 sourceSupported

Experiments using finer-scale mapping with Micro-C have revealed even smaller, nested stripes that may occur independently of loop extrusion.

“experiments using finer-scale mapping with Micro-C have revealed even smaller, nested stripes that may occur independently of loop extrusion [276].”

• cites [276] Hsieh T-HS, Cattoglio C, Slobodyanyuk E et al (2020) Resolving the 3D landscape of transcription-linked mammalian chromatin folding. Mol Cell 78:539-553.e8 sourceSupported

Analysis in the Shh locus suggests that within-TAD chromosomal contacts do not show a pronounced distance dependence.

“analysis in the Shh locus suggests that within-TAD chromosomal contacts there do not show a pronounced distance dependence [467].”

• cites [467] Symmons O, Pan L, Remeseiro S et al (2016) The Shh topological domain facilitates the action of remote enhancers by reducing the effects of genomic distances. Dev Cell 39:529–543 sourceSupported

Even in interphase nuclei, genomic distance continues to be a reasonable predictor of functional enhancer-promoter contacts, particularly in the short to medium range (<100 kb).

“even in interphase nuclei, genomic distance continues to be a reasonable predictor of functional enhancer–promoter contacts, particularly in the short to medium range (< 100 kb), as evidenced by high-throughput enhancer perturbation data [183, 184].”

• cites [183] Fulco CP, Nasser J, Jones TR et al (2019) Activity-by-contact model of enhancer–promoter regulation from thousands of CRISPR perturbations. Nat Genet 51:1664–1669 sourcePartly supported

  The paper’s results (indexed under Abstract; page unavailable) report that 84% of regulatory distal elements were within 100 kb of their target promoter, supporting the distance-range part of the claim. But the same passage says distance-threshold predictions performed poorly: only 13% of distal elements within 100 kb of an expressed promoter had a regulatory effect. It does not establish the claim specifically for interphase nuclei. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [184] Nasser J, Bergman DT, Fulco CP et al (2021) Genome-wide enhancer maps link risk variants to disease genes. Nature. https://doi.org/10.1038/s41586-021-03446-x sourceSupported

In mitosis, genomic distance becomes the predominant factor governing the frequency of chromosomal contacts across the genome.

“In mitosis, where TADs and compartments dissolve, genomic distance becomes the predominant factor governing the frequency of chromosomal contacts across the genome [459].”

• cites [459] Naumova N, Imakaev M, Fudenberg G et al (2013) Organization of the mitotic chromosome. Science 342:948–953 sourceSupported

Considerable TAD re-arrangements observed in Drosophila balancer chromosomes compared with wild-type result in changes in expression of only about 10% of genes.

“the considerable TAD re-arrangements observed in Drosophila balancer chromosomes compared with their wild-type counterparts result in changes in the expression of only ~ 10% of genes [449].”

• cites [449] Ghavi-Helm Y, Jankowski A, Meiers S et al (2019) Highly rearranged chromosomes reveal uncoupling between genome topology and gene expression. Nat Genet 51:1272–1282 sourcePartly supported

  The Results section reports that 512 of 5,357 testable genes (9.6%) differed in expression between balancer and wild-type haplotypes. But that figure covers the chromosomes’ extensive genomic rearrangements overall, not changes caused specifically by TAD rearrangements. The TAD analysis reports differential expression for 23 of 161 expressed genes in resized TADs and 29 of 237 testable genes near perturbed boundaries. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Contacts from active chromatin and heterochromatin regions appear largely segregated into separate compartments (A and B).

“contacts from active chromatin and heterochromatin regions appear largely segregated into separate compartments (referred to as A and B, respectively) [272].”

• cites [272] Lieberman-Aiden E, van Berkum NL, Williams L et al (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326:289–293 sourceSupported

Even within the same TAD, enhancers are not always fully promiscuous and can sometimes skip nearby genes in favour of more distal targets.

“Even within the same TAD, enhancers are not always fully promiscuous and can sometimes “skip” nearby genes, in favour of more distal targets [472, 473].”

• cites [472] Sanyal A, Lajoie BR, Jain G, Dekker J (2012) The long-range interaction landscape of gene promoters. Nature 489:109–113 sourcePartly supported

  The passage on genomic order (section labeled Abstract; page unavailable) reports that only about 7% of looping interactions involve the nearest TSS, and the concluding passage says interactions can skip genes. This supports the point that nearby genes can be bypassed, but the paper does not establish that the observed cases occur within the same TAD. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [473] Furlong EEM, Levine M (2018) Developmental enhancers and chromosome topology. Science 361:1341–1345 sourceSupported

Relocation of LADs away from the nuclear periphery is associated with activation of the respective regulatory elements and their increased proximity.

“This relocation is associated with the activation of the respective regulatory elements and their increased proximity [463].”

• cites [463] Robson MI, de Las Heras JI, Czapiewski R et al (2017) Constrained release of lamina-associated enhancers and genes from the nuclear envelope during T-cell activation facilitates their association in chromosome compartments. Genome Res 27:1126–1138 sourceSupported

Some A compartment regions consisting of H3K9me2-marked chromatin localise at the nuclear periphery.

“some A compartment regions consisting of H3K9me2-marked chromatin were also found to localise at the nuclear periphery [464].”

• cites [464] Smith CL, Poleshko A, Epstein JA (2021) The nuclear periphery is a scaffold for tissue-specific enhancers. Nucleic Acids Res 49:6181–6195 sourceSupported

Loss of TAD insulation between limb-specific enhancers associated with EPHA4 expression and adjacent genes including WNT6 and PAX3 results in their misexpression and abnormal limb development in both mice and humans.

“loss of TAD insulation between limb-specific enhancers associated with EPHA4 expression and a set of adjacent genes, including WNT6 and PAX3, results in their misexpression and abnormal limb development in both mice and humans [446].”

• cites [446] Lupiáñez DG, Kraft K, Heinrich V et al (2015) Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions. Cell 161:1012–1025 sourcePartly supported

  The Results sections on structural changes and enhancer interactions report Pax3 and Wnt6 misexpression in mutant mouse limbs and new contacts with enhancers in the Epha4 TAD. The Discussion section on conservation reports corresponding abnormal chromatin interactions in patient fibroblasts, but does not demonstrate WNT6 or PAX3 misexpression in developing human limbs; it presents the link to human limb phenotypes as a plausible explanation, not an established result. Page numbers were not provided in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

This preference was consistent with the tissue-invariant or tissue-specific activity of the enhancers themselves.

“Notably, this preference was consistent with the tissue-invariant or tissue-specific activity of the enhancers themselves [247].”

• cites [247] Zabidi MA, Arnold CD, Schernhuber K et al (2015) Enhancer–core-promoter specificity separates developmental and housekeeping gene regulation. Nature 518:556–559 sourceSupported

PCHi-C showed that active promoters contacted a median of two active enhancers in human primary blood cells.

“PCHi-C showed that active promoters contacted a median of two active enhancers in human primary blood cells [243].”

• cites [243] Javierre BM, Burren OS, Wilder SP et al (2016) Lineage-specific genome architecture links enhancers and non-coding disease variants to target gene promoters. Cell 167:1369-1384.e19 sourcePartly supported

  The Results section, “Promoter Capture Hi-C” (page unavailable), reports PCHi-C experiments in 17 human primary blood cell types, but gives a median of four interactions per promoter fragment per cell type. The paper also discusses contacts between active promoters and active enhancers, but the passages found do not establish a median of two active enhancers per active promoter. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Genome Architecture Mapping (GAM) enables probing multi-way chromosomal contacts.

“Genome Architecture Mapping (GAM) is one technique that enables probing multi-way chromosomal contacts [497].”

• cites [497] Beagrie RA, Scialdone A, Schueler M et al (2017) Complex multi-enhancer contacts captured by genome architecture mapping. Nature 543:519–524 sourceSupported

Cohesin-dependent enhancers tend to be longer-range than their cohesin-independent counterparts.

“Cohesin-dependent enhancers also tend to be longer-range than their cohesin-independent counterparts [301, 349, 514].”

• cites [301] Kubo N, Ishii H, Xiong X et al (2021) Promoter-proximal CTCF binding promotes distal enhancer-dependent gene activation. Nat Struct Mol Biol 28:152–161 sourcePartly supported

  The Results section (page not provided) reports that CTCF-dependent genes generally have long-range enhancer–promoter contacts (≥100 kb), whereas CTCF-independent genes appear to rely on short-range contacts (≤50 kb), especially in neural precursor cells. This supports the distance comparison, but the paper tests dependence on CTCF loss, not cohesin loss, so it does not establish the claim specifically for cohesin-dependent versus cohesin-independent enhancers. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [349] Thiecke MJ, Wutz G, Muhar M et al (2020) Cohesin-dependent and -independent mechanisms mediate chromosomal contacts between promoters and enhancers. Cell Rep 32:107929 sourceSupported
• cites [514] Kane L, Williamson I, Flyamer IM et al (2021) Cohesin is required for long-range enhancer action. Biorxiv. https://doi.org/10.1101/2021.06.24.449812 sourceSupported

Advances in super-resolution imaging enable visualisation of multiple interacting loci to the resolution of a few kilobases in single cells.

“advances in super-resolution imaging (reviewed in [500]) now enable the visualisation of multiple interacting loci to the resolution of a few kilobases in single cells [448, 501–503].”

• cites [448] Bintu B, Mateo LJ, Su J-H et al (2018) Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science 362:eaau1783 sourcePartly supported

  The section “Multiplexed super-resolution FISH imaging for chromatin tracing” reports 3D super-resolution images of multiple chromatin segments in individual cells. Each segment spans 30 kb, however, so this paper does not support the claimed resolution of a few kilobases. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [501] Cardozo Gizzi AM, Cattoni DI, Fiche J-B et al (2019) Microscopy-based chromosome conformation capture enables simultaneous visualization of genome organization and transcription in intact organisms. Mol Cell 74:212-222.e5 sourceSupported
• cites [502] Nir G, Farabella I, Pérez Estrada C et al (2018) Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling. PLoS Genet 14:e1007872 sourceSupported
• cites [503] Mateo LJ, Murphy SE, Hafner A et al (2019) Visualizing DNA folding and RNA in embryos at single-cell resolution. Nature 568:49–54 sourceSupported

In an enhancer chain, not all enhancers need to contact the gene promoter directly; the signal could be passed along the chain.

“In this mechanism, not all of the enhancers need to contact the gene promoter directly; rather, the signal could be passed along the chain.”

• cites [486] Patrinos GP, de Krom M, de Boer E et al (2004) Multiple interactions between regulatory regions are required to stabilize an active chromatin hub. Genes Dev 18:1495–1509 sourcePartly supported

  In 'Deletion of the β-globin promoter combined with LCR HS2 and HS3 deletion' (page unavailable), the paper suggests that HS3 interacts with sequences other than the β-globin promoter and that this interaction matters for transcription. This supports the possibility that a regulatory element need not contact the promoter directly, but the paper does not describe a signal being passed along an enhancer chain. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [487] Gavrilov AA, Razin SV (2008) Spatial configuration of the chicken alpha-globin gene domain: immature and active chromatin hubs. Nucleic Acids Res 36:4629–4640 sourcePartly supported

  The Discussion (page not provided) reports that the active αA promoter showed little association with upstream regulatory elements, supporting the possibility of promoter activity without direct contact with each element. It does not propose an enhancer chain or say that a signal passes along one. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [488] Markenscoff-Papadimitriou E, Allen WE, Colquitt BM et al (2014) Enhancer interaction networks as a means for singular olfactory receptor expression. Cell 159:543–557 sourcePartly supported

  The Results section, “Extensive cis and trans interactions between potential OR enhancers in OSNs” (page unavailable), reports frequent enhancer–enhancer contacts, and the Discussion proposes that multiple enhancers contribute to OR transcription. The paper does not establish or explicitly propose that a signal passes along an enhancer chain so that some enhancers need not contact the promoter directly. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [489] Ing-Simmons E, Seitan VC, Faure AJ et al (2015) Spatial enhancer clustering and regulation of enhancer-proximal genes by cohesin. Genome Res 25:504–513 sourceSupported

Hubs detected by these techniques sometimes contain multiple promoters, corroborating evidence for promoter-promoter contacts from conventional 3C-derived methods.

“in addition to multiple enhancers, hubs detected by these techniques sometimes contain multiple promoters, corroborating the evidence for promoter-promoter contacts from the conventional 3C-derived methods [453, 498].”

• cites [453] Espinola SM, Götz M, Bellec M et al (2021) Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development. Nat Genet 53:477–486 sourceSupported
• cites [498] Oudelaar AM, Davies JOJ, Hanssen LLP et al (2018) Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains. Nat Genet 50:1744–1751 sourceSupported

Recent evidence suggests that some promoter-promoter contacts likely have a direct cis-regulatory role similarly to enhancer-promoter contacts.

“recent evidence that promoter-promoter contacts within nested stripe domains preferentially involve transcriptionally active, rather than inactive, promoters [276], as well as the existence of epromoters (discussed above), suggest that some promoter-promoter contacts likely have a direct cis-regulatory role similarly to enhancer–promoter contacts.”

• cites [276] Hsieh T-HS, Cattoglio C, Slobodyanyuk E et al (2020) Resolving the 3D landscape of transcription-linked mammalian chromatin folding. Mol Cell 78:539-553.e8 sourceSupported
• cites [493] Zhu I, Song W, Ovcharenko I, Landsman D (2021) A model of active transcription hubs that unifies the roles of active promoters and enhancers. Nucleic Acids Res 49:4493–4505 sourcePartly supported

  The paper’s ‘Validation of the model with Hi-C data’ section reports promoter–promoter interactions in active gene clusters and proposes that nearby active promoters might act as enhancers for one another. That supports the suggested cis-regulatory role, but our copy does not establish the cited sentence’s more specific claim about contacts within nested stripe domains preferentially involving active rather than inactive promoters. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

In the mammalian Hox gene cluster, activation of genes with crucial roles in development is tightly controlled at each developmental stage by complex enhancer networks.

“in the well-characterised mammalian Hox gene cluster, activation of genes with crucial roles in development is tightly controlled at each developmental stage by complex enhancer networks [481, 482].”

• cites [481] Montavon T, Soshnikova N, Mascrez B et al (2011) A regulatory archipelago controls Hox genes transcription in digits. Cell 147:1132–1145 sourcePartly supported

  The Introduction describes the developmental importance and sequential expression of mammalian Hox genes. The Discussion reports that several regulatory elements collectively control Hoxd transcription in digits. The paper does not establish that complex enhancer networks control activation across all mammalian Hox clusters at each developmental stage. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [482] Lonfat N, Montavon T, Darbellay F et al (2014) Convergent evolution of complex regulatory landscapes and pleiotropy at Hox loci. Science 346:1004–1006 sourceNot checked — no full text of the source

Deletion or inhibition of any one of the promoter-interacting enhancers caused disruption of H3K27ac and eRNA levels on the other enhancers in the network.

“deletion or inhibition of any one of the promoter-interacting enhancers caused disruption of H3K27ac and eRNA levels on the other enhancers in the network [491].”

• cites [491] Farooq U, Saravanan B, Islam Z et al (2021) An interdependent network of functional enhancers regulates transcription and EZH2 loading at the INK4a/ARF locus. Cell Rep 34:108898 sourceSupported

There is evidence for enhancer-enhancer interactions that create an enhancer chain or network.

“there is also evidence for enhancer–enhancer interactions that create an enhancer “chain” or network [486–489].”

• cites [486] Patrinos GP, de Krom M, de Boer E et al (2004) Multiple interactions between regulatory regions are required to stabilize an active chromatin hub. Genes Dev 18:1495–1509 sourcePartly supported

  The Discussion describes a chromatin hub formed by interactions among regulatory hypersensitive sites, supporting the network aspect of the claim. But the paper says it remains unclear whether individual sites in the locus control region interact with one another or instead interact simultaneously with a gene; it does not establish an enhancer–enhancer chain. Our copy provides section labels but no page numbers. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [487] Gavrilov AA, Razin SV (2008) Spatial configuration of the chicken alpha-globin gene domain: immature and active chromatin hubs. Nucleic Acids Res 36:4629–4640 sourcePartly supported

  The Results (page not available in our copy) report a clear interaction between the LCR-like MRE and a downstream enhancer. The Discussion describes a chromatin hub involving multiple regulatory elements, but does not establish an enhancer-only chain or network; that part of the claim goes beyond the paper's evidence. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [488] Markenscoff-Papadimitriou E, Allen WE, Colquitt BM et al (2014) Enhancer interaction networks as a means for singular olfactory receptor expression. Cell 159:543–557 sourceSupported
• cites [489] Ing-Simmons E, Seitan VC, Faure AJ et al (2015) Spatial enhancer clustering and regulation of enhancer-proximal genes by cohesin. Genome Res 25:504–513 sourceSupported

Hi-M was employed in the developing Drosophila embryo to show that cis-regulatory elements cooperate to form hubs early in development prior to transcription, facilitated by the pioneer factor Zelda.

“Hi-M was recently employed in the developing Drosophila embryo to show that cis-regulatory elements cooperate to form hubs early in development prior to transcription, facilitated by the pioneer factor Zelda [453].”

• cites [453] Espinola SM, Götz M, Bellec M et al (2021) Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development. Nat Genet 53:477–486 sourceSupported

In the β-globin locus, interactions between the LCR and the γ-globin or the β-globin genes can switch very rapidly on the same DNA strand.

“in the β-globin locus, interactions between the LCR and the γ-globin or the β-globin genes can switch very rapidly on the same DNA strand [505].”

• cites [505] Bartman CR, Hsu SC, Hsiung CC-S et al (2016) Enhancer regulation of transcriptional bursting parameters revealed by forced chromatin looping. Mol Cell 62:237–247 sourceSupported

Evidence from live imaging shows that an enhancer located in-between two reporter genes can initiate simultaneous transcriptional bursting from both gene promoters.

“evidence from live imaging shows that an enhancer located in-between two reporter genes can initiate simultaneous transcriptional bursting from both gene promoters [310].”

• cites [310] Fukaya T, Lim B, Levine M (2016) Enhancer control of transcriptional bursting. Cell 166:358–368 sourceSupported

A similar network of contacts is detectable between active promoters of multiple histone genes in many mammalian cell types.

“A similar network of contacts is also detectable between the active promoters of multiple histone genes in many mammalian cell types [243, 279, 471, 492].”

• cites [243] Javierre BM, Burren OS, Wilder SP et al (2016) Lineage-specific genome architecture links enhancers and non-coding disease variants to target gene promoters. Cell 167:1369-1384.e19 sourcePartly supported

  The Results section, “Promoter Capture Hi-C” (page unavailable), reports promoter-to-promoter contacts across 17 human primary blood cell types. It does not identify a network among active histone-gene promoters or establish its presence in many mammalian cell types. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [279] Schoenfelder S, Furlan-Magaril M, Mifsud B et al (2015) The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements. Genome Res 25:582–597 sourcePartly supported

  In Results > Promoter capture Hi-C (page unavailable), the paper reports contact enrichment between the Hist1h4h and Hist1h2ai loci in mouse embryonic stem cells and fetal liver cells. Results > Promoter–promoter 3D interaction networks reports preferential contacts among promoters more broadly. These passages do not establish a network specifically among active histone-gene promoters in many mammalian cell types. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [471] Cairns J, Freire-Pritchett P, Wingett SW et al (2016) CHiCAGO: robust detection of DNA looping interactions in capture Hi-C data. Genome Biol 17:127 sourcePartly supported

  In Results, under “Promoter interactions detected by CHiCAGO: validation and key properties” (page unavailable), the paper reports frequent contacts between histone-gene promoters on chromosome 6 in GM12878 cells. It does not establish that those promoters are active or that the same network is detectable in many mammalian cell types; its findings about active histone marks concern promoter-interacting fragments generally, not this histone-gene network. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [492] Wang Q, Sawyer IA, Sung M-H et al (2016) Cajal bodies are linked to genome conformation. Nat Commun 7:10966 sourcePartly supported

  The Results section, “Many inter-chromosomal interactions of HIST1 are CB proximal” (page not available), reports contacts involving histone gene clusters on chromosomes 1 and 6 in HeLa cells. It does not establish a network specifically between active histone-gene promoters or show that such contacts occur in many mammalian cell types. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Analyses in human ESCs and early ES-derived neural progenitors found that enhancers within a CRU tended to have the same activity state in a given cell type.

“Analyses in human ESCs and early ES-derived neural progenitors found that, as expected, enhancers within a CRU tended to have the same activity state in a given cell type [141].”

• cites [141] Freire-Pritchett P, Schoenfelder S, Várnai C et al (2017) Global reorganisation of regulatory units upon lineage commitment of human embryonic stem cells. Elife 6:e21926 sourceSupported

In the Drosophila Hox locus, one enhancer regulates either pb or zen2 depending on developmental stage via distinct chromatin loops.

“An example of the former is found in the Drosophila Hox locus, where one enhancer regulates either pb or zen2 depending on the developmental stage via distinct chromatin loops [484].”

• cites [484] Miller SW, Posakony JW (2020) Disparate expression specificities coded by a shared Hox-C enhancer. Elife 9:e39876 sourcePartly supported

  The Results and Figure 7 model in the Discussion show that EO053 contributes to zen2 expression earlier and pb expression later. The Discussion (‘Serving separate promoters’; page unavailable) proposes separate looping interactions with the two promoters, but presents those loops as a hypothesis, not an established mechanism as the citing sentence implies. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

CRISPR-based approaches have confirmed that promoters often receive signals from multiple functional enhancers.

“CRISPR-based approaches have confirmed that promoters often receive signals from multiple functional enhancers [479, 480].”

• cites [479] Fulco CP, Munschauer M, Anyoha R et al (2016) Systematic mapping of functional enhancer–promoter connections with CRISPR interference. Science 354:769–773 sourcePartly supported

  The paper’s concluding summary (page not specified in our copy) says CRISPRi revealed coordinated activity of multiple enhancers controlling a single gene at the MYC and GATA1 loci. That supports the existence of such connections, but the study of these loci does not establish that promoters often receive signals from multiple functional enhancers generally. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [480] Osterwalder M, Barozzi I, Tissières V et al (2018) Enhancer redundancy provides phenotypic robustness in mammalian development. Nature 554:239–243 sourceSupported

Tri-C and MC-4C provided evidence for higher-order hubs containing three and more regulatory elements in the α- and β-globin loci.

“Both technologies, applied in the α- and β- globin loci respectively, provided evidence for higher-order hubs, containing three and more regulatory elements [499, 485].”

• cites [499] Oudelaar AM, Harrold CL, Hanssen LLP et al (2019) A revised model for promoter competition based on multi-way chromatin interactions at the α-globin locus. Nat Commun 10:5412 sourceSupported
• cites [485] Allahyar A, Vermeulen C, Bouwman BAM et al (2018) Enhancer hubs and loop collisions identified from single-allele topologies. Nat Genet 50:1151–1160 sourceSupported

Contacts between promoters of Hox genes in ESCs (in which they are inactive) depend on Polycomb Repressive complex 1.

“contacts between the promoters of Hox genes in ESCs (in which they are inactive) that were shown to depend on Polycomb Repressive complex 1 [280].”

• cites [280] Schoenfelder S, Sugar R, Dimond A et al (2015) Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome. Nat Genet 47:1179–1186 sourceSupported

Physical contacts between enhancers and promoters are often highly dynamic.

“Evidence from live-imaging studies at candidate loci suggests that physical contacts between enhancers and promoters are often highly dynamic [310, 311, 448].”

• cites [310] Fukaya T, Lim B, Levine M (2016) Enhancer control of transcriptional bursting. Cell 166:358–368 sourceSupported
• cites [311] Chen H, Levo M, Barinov L et al (2018) Dynamic interplay between enhancer–promoter topology and gene activity. Nat Genet 50:1296–1303 sourcePartly supported

  The live-imaging results describe changes in enhancer–promoter distance and transitions between topological states at a Drosophila reporter locus (Necessity of sustained physical proximity for transcription; Transcription enhances stability of the paired conformation; pages unavailable). They do not establish that direct physical contacts are often highly dynamic across loci. The Discussion instead emphasizes persistent proximity during sustained transcription and treats direct contact as a possibility, not an observed event. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [448] Bintu B, Mateo LJ, Su J-H et al (2018) Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science 362:eaau1783 sourceCould not verify

  The chromatin-interactions section reports cell-to-cell variation in contacts between chromatin loci, but not changes over time at enhancer–promoter pairs. The Methods Summary describes imaging hybridized cells rather than live imaging. Our copy therefore does not establish that enhancer–promoter contacts are often highly dynamic. (LLM, conf=0.73)

  Experimental — accuracy not yet measured

Biophysical modelling suggests that the same enhancer may have vastly unequal effects on different promoters depending on their intrinsic sensitivity to enhancer-promoter contacts, irrespective of the properties of a given enhancer.

“biophysical modelling suggests that the same enhancer may have vastly unequal effects on different promoters depending on their intrinsic sensitivity to enhancer–promoter contacts (irrespectively of the properties of a given enhancer) [406].”

• cites [406] Xiao J, Hafner A, Boettiger AN (2020) How subtle changes in 3D structure can create large changes in transcription. BioRxiv. https://doi.org/10.1101/2020.10.22.351395 sourceSupported

The LCR in the β-globin locus can simultaneously engage two adult globin genes, Hbb-b1 and Hbb-b2, while skipping over embryonic globin genes, in mouse fetal liver cells.

“the LCR in the β-globin locus can simultaneously engage two adult globin genes, Hbb-b1 and Hbb-b2, while skipping over embryonic globin genes, in mouse fetal liver cells [485].”

• cites [485] Allahyar A, Vermeulen C, Bouwman BAM et al (2018) Enhancer hubs and loop collisions identified from single-allele topologies. Nat Genet 50:1151–1160 sourceSupported

The chromatin state of the promoter at dual-state CRUs typically corresponded to the majority state of its enhancers, consistent with competition of opposing regulatory effects at the promoters.

“the chromatin state of the promoter at dual-state CRUs typically corresponded to the majority state of its enhancers, consistent with a competition of opposing regulatory effects at the promoters.”

• cites [141] Freire-Pritchett P, Schoenfelder S, Várnai C et al (2017) Global reorganisation of regulatory units upon lineage commitment of human embryonic stem cells. Elife 6:e21926 sourcePartly supported

  The Results section on CRU reorganisation reports that a promoter’s chromatin state generally matched the most prevalent state among its interacting regions. The Discussion section on regulation by multiple enhancers interprets this as possible ‘majority vote’ signal integration, but does not establish competition between opposing regulatory effects; it also offers independent enhancer action and promoter-state turnover as possible explanations. Page numbers are unavailable in our copy. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Cohesin/CTCF-associated enhancers are good candidates for pioneer enhancers, most importantly because of the selective requirement of cohesin for inducible gene control.

“Theoretically, cohesin/CTCF-associated enhancers are good candidates for pioneer enhancers for a number of reasons, most important of which is the selective requirement of cohesin for inducible gene control [513].”

• cites [513] Cuartero S, Weiss FD, Dharmalingam G et al (2018) Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation. Nat Immunol 19:932–941 sourceSupported

20% of CRUs detected in human ESCs and early ES-derived neural progenitors were dual-state, containing a mixture of active and poised or Polycomb-repressed enhancers.

“with 20% of CRUs detected in these cells being “dual-state”, i.e. containing a mixture of active and poised or Polycomb-repressed enhancers [141].”

• cites [141] Freire-Pritchett P, Schoenfelder S, Várnai C et al (2017) Global reorganisation of regulatory units upon lineage commitment of human embryonic stem cells. Elife 6:e21926 sourceSupported

When an enhancer comes into proximity with a compatible promoter, it transmits a dose of activation that results in a certain level of transcriptional output.

“when an enhancer comes into proximity with a compatible promoter, it transmits to it a “dose of activation” that results in a certain level of transcriptional output [107].”

• cites [107] Spivakov M (2014) Spurious transcription factor binding: non-functional or genetically redundant? BioEssays 36:798–806 sourcePartly supported

  In “The output of TF binding as a ‘dose of activation’” (page unavailable), the paper proposes that TF binding sites can contribute activating doses to promoters through DNA looping and that promoters respond to the total dose received. The citing sentence captures that model but states it categorically for an enhancer, whereas the paper presents it as a possibility and describes transcriptional output as a response to inputs from multiple binding sites. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Hi-M combines visualisation of chromatin interactions and transcription in single cells.

“Hi-M is one such technique that combines visualisation of chromatin interactions and transcription in single cells [501, 504].”

• cites [501] Cardozo Gizzi AM, Cattoni DI, Fiche J-B et al (2019) Microscopy-based chromosome conformation capture enables simultaneous visualization of genome organization and transcription in intact organisms. Mol Cell 74:212-222.e5 sourceSupported
• cites [504] Cardozo Gizzi AM, Espinola SM, Gurgo J et al (2020) Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M. Nat Protoc 15:840–876 sourceNot checked — no full text of the source

The number of enhancers correlates with the expression level of a gene across cell types.

“the number of enhancers correlates with the expression level of a gene across cell types [243, 278, 279, 349].”

• cites [243] Javierre BM, Burren OS, Wilder SP et al (2016) Lineage-specific genome architecture links enhancers and non-coding disease variants to target gene promoters. Cell 167:1369-1384.e19 sourcePartly supported

  The Results section (“Enhancer Activity Associates with Lineage-Specific Gene Expression”; page not provided) reports that promoter interactions with active enhancers have an additive association with gene expression across cell types. The STAR Methods section specifies that the analysis counted interacting active enhancers, not enhancers generally, and was restricted to genes with active promoters in all samples. The claim omits those qualifications. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [278] Mifsud B, Tavares-Cadete F, Young AN et al (2015) Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C. Nat Genet 47:598–606 sourceNot checked — no full text of the source
• cites [279] Schoenfelder S, Furlan-Magaril M, Mifsud B et al (2015) The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements. Genome Res 25:582–597 sourceSupported
• cites [349] Thiecke MJ, Wutz G, Muhar M et al (2020) Cohesin-dependent and -independent mechanisms mediate chromosomal contacts between promoters and enhancers. Cell Rep 32:107929 sourcePartly supported

  The Results section (page unavailable) reports that changes in the number of promoter-connected active enhancers were associated with the direction of transcriptional change after cohesin depletion. It does not establish that the number of enhancers correlates with a gene’s expression level across cell types; the reported association concerns changes within the studied cells. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Enhancers increase the frequency of transcriptional bursts without significantly affecting their amplitude and duration.

“Evidence from live imaging and single-cell transcriptomics indicates that enhancers increase the frequency of transcriptional bursts without significantly affecting their amplitude and duration [310, 505, 516].”

• cites [310] Fukaya T, Lim B, Levine M (2016) Enhancer control of transcriptional bursting. Cell 166:358–368 sourcePartly supported

  The Abstract and Results (Differential bursting frequencies) report that stronger enhancers generally produce more bursts with similar amplitudes and durations. However, the Results identify a significant reduction in burst amplitude for the Kr CD2 enhancer, so the claim that amplitude is not significantly affected needs that exception. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [505] Bartman CR, Hsu SC, Hsiung CC-S et al (2016) Enhancer regulation of transcriptional bursting parameters revealed by forced chromatin looping. Mol Cell 62:237–247 sourcePartly supported

  The Results section reports that forced LCR–promoter contacts increased β-globin burst fraction without changing burst size, supporting the frequency and amplitude portions for that intervention. But enhancer deletion had a lesser yet significant effect on burst size, and the Discussion says burst duration could not be determined definitively. The paper therefore does not support the unqualified claim that enhancers leave amplitude and duration unaffected. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [516] Larsson AJM, Johnsson P, Hagemann-Jensen M et al (2019) Genomic encoding of transcriptional burst kinetics. Nature 565:251–254 sourcePartly supported

  In the passage on Sox2 enhancer deletion (indexed as Abstract; page unavailable), deletion significantly reduced burst frequency without a significant change in burst size. The paper infers burst size from parameters it says cannot be individually identified across much of the parameter space, so its result does not establish that burst amplitude and duration were each unaffected. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

The ABC model showed good performance in predicting transcriptional effects of CRISPRi-mediated enhancer perturbations in dozens of loci in human cell lines, particularly for more proximal enhancers.

“The ABC model showed a good performance in predicting the transcriptional effects of CRISPRi-mediated enhancer perturbations (one-by-one) in dozens of loci in human cell lines, particularly for the more proximal enhancers [183, 184].”

• cites [183] Fulco CP, Nasser J, Jones TR et al (2019) Activity-by-contact model of enhancer–promoter regulation from thousands of CRISPR perturbations. Nat Genet 51:1664–1669 sourcePartly supported

  The paper’s main-text passage labeled Abstract (page unavailable) reports that ABC scores correlated with measured effects of CRISPR perturbations on gene expression and outperformed alternative predictors. The Methods report CRISPRi-FlowFISH screens for 30 genes in K562 cells. Although the paper says most regulatory enhancers were within 100 kb of their target promoters, the passages do not establish that ABC performed better for proximal than for more distant enhancers. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [184] Nasser J, Bergman DT, Fulco CP et al (2021) Genome-wide enhancer maps link risk variants to disease genes. Nature. https://doi.org/10.1038/s41586-021-03446-x sourcePartly supported

  In “ABC enhancer-gene maps in 131 biosamples” (page unavailable), the paper reports that ABC performed well at classifying regulatory connections against 5,755 CRISPR-tested element–gene pairs across 11 cell types and states. It does not establish that performance was particularly good for proximal enhancers: Extended Data Fig. 4 reports different positive rates by distance and cautions against comparing precision–recall curves across distance groups. The reported evaluation classifies regulatory effects rather than predicting their magnitude. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Promoters may have a memory (hysteresis) of enhancer action.

“This way, promoters may have a “memory” (hysteresis) of enhancer action [405, 406], reviving some early debates on this topic [527].”

• cites [405] Zuin J, Roth G, Zhan Y et al (2021) Nonlinear control of transcription through enhancer–promoter interactions. BioRxiv. https://doi.org/10.1101/2021.04.22.440891 sourceSupported
• cites [406] Xiao J, Hafner A, Boettiger AN (2020) How subtle changes in 3D structure can create large changes in transcription. BioRxiv. https://doi.org/10.1101/2020.10.22.351395 sourceSupported

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].”

• cites [517] Hay D, Hughes JR, Babbs C et al (2016) Genetic dissection of the α-globin super-enhancer in vivo. Nat Genet 48:895–903 sourceSupported
• cites [518] Dukler N, Gulko B, Huang Y-F, Siepel A (2016) Is a super-enhancer greater than the sum of its parts? Nat Genet 49:2–3 sourcePartly supported

  In “To the Editor” (page unavailable), the authors find that enhancer-knockout data from the α-globin and Wap loci are consistent with a model without explicit interactions between enhancers. But their preferred linear-logistic model is not a model in which individual enhancers contribute additively to transcriptional output; it fits both data sets better than the directly additive model. The claim captures the lack of clear evidence for interactions but blurs that distinction. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Sub-additivity may underlie some cases of functional enhancer redundancy, such as for Drosophila shadow enhancers that can back up important developmental enhancers and enable them to accumulate deleterious mutations.

“Sub-additivity may also underlie some cases of functional enhancer redundancy, such as for Drosophila “shadow enhancers” that can “back up” important developmental enhancers and enable them to accumulate deleterious mutations [520–522].”

• cites [520] Hong J-W, Hendrix DA, Levine MS (2008) Shadow enhancers as a source of evolutionary novelty. Science 321:1314 sourcePartly supported

  In “Shadow Enhancers as a Source of Evolutionary Novelty” (page unavailable), the paper describes Drosophila shadow enhancers with activity overlapping primary enhancers and suggests they can explain why deleting a well-defined enhancer sometimes has no apparent phenotype. It also proposes that shadow enhancers can evolve new regulatory activities without disrupting core developmental functions. Our copy does not address sub-additivity or say that shadow enhancers enable primary enhancers to accumulate deleterious mutations. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [521] Barolo S (2012) Shadow enhancers: frequently asked questions about distributed cis-regulatory information and enhancer redundancy. BioEssays 34:135–141 sourcePartly supported

  The Main Text describes Drosophila shadow enhancers that can compensate for the loss of another enhancer under normal laboratory conditions, supporting the backup example. The Evolvability section discusses the possibility that redundancy permits regulatory change, but does not say that sub-additivity enables important enhancers to accumulate deleterious mutations; it also cautions that enhancers needed for robustness may not be free to change. Page numbers are unavailable in our copy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [522] Cannavò E, Khoueiry P, Garfield DA et al (2016) Shadow enhancers are pervasive features of developmental regulatory networks. Curr Biol 26:38–51 sourcePartly supported

  The Results section (“Shadow Enhancers Can Buffer the Effects of Natural Sequence Variation”; page unavailable) says redundant enhancers can compensate for mutations that disable a partner, supporting the backup role. Our copy does not link that role to sub-additivity or establish that backup enables important developmental enhancers to accumulate deleterious mutations; the Discussion (“How Are Redundant Enhancers Maintained during Evolution?”; page unavailable) instead reports no support for a related hypothesis that shadow enhancers accumulate mutations under relaxed constraint. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

In the ABC model, each enhancer is considered separately and the dose of activation it contributes to a promoter depends on its individual activity in cis and the frequency of the respective enhancer-promoter contact.

“In ABC, each enhancer is considered separately and the “dose” of activation it contributes to a promoter depends on its individual activity in cis and the frequency of the respective enhancer–promoter contact [183, 184].”

• cites [183] Fulco CP, Nasser J, Jones TR et al (2019) Activity-by-contact model of enhancer–promoter regulation from thousands of CRISPR perturbations. Nat Genet 51:1664–1669 sourceSupported
• cites [184] Nasser J, Bergman DT, Fulco CP et al (2021) Genome-wide enhancer maps link risk variants to disease genes. Nature. https://doi.org/10.1038/s41586-021-03446-x sourceSupported

The frequency of enhancer-promoter contacts may have non-linear effects on transcriptional output.

“It also follows from these models that the frequency of enhancer–promoter contacts may have non-linear effects on transcriptional output, consistent with experimental observations [405, 503].”

• cites [405] Zuin J, Roth G, Zhan Y et al (2021) Nonlinear control of transcription through enhancer–promoter interactions. BioRxiv. https://doi.org/10.1101/2021.04.22.440891 sourceSupported
• cites [503] Mateo LJ, Murphy SE, Hafner A et al (2019) Visualizing DNA folding and RNA in embryos at single-cell resolution. Nature 568:49–54 sourcePartly supported

  In “Cis contacts predict transcription” (page unavailable), the paper reports that enhancer–promoter proximity weakly predicts whether nascent transcription is present. It does not establish how contact frequency affects the amount of transcription or show a non-linear relationship, so that part of the attributed claim is unsupported. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Transcription itself is discontinuous, occurring in bursts during which RNAP complexes transcribe one by one from the TSS producing one or more transcripts, followed by a period of transcriptional silence.

“transcription itself is discontinuous, occurring in “bursts” during which RNAP complexes transcribe one by one from the TSS producing one or more transcripts, followed by a period of transcriptional silence (reviewed in [515]).”