Enhancer Function in the 3D Genome

The presence of so-called stripes or flames on Hi-C maps indicates that unidirectional extrusion from an enhancer is quite common.

“The presence of so-called stripes or flames on Hi-C maps indicates that unidirectional extrusion from an enhancer is quite common [116,117,118].”

• cites [116] Vian, 2018, The Energetics and Physiological Impact of Cohesin Extrusion, Cell sourceNot checked — no full text of the source
• cites [117] Hsieh, 2020, Resolving the 3D Landscape of Transcription-Linked Mammalian Chromatin Folding, Mol. Cell sourcePartly supported

  The Results section (“Identification of E-P stripes, dots, and domains”; page unavailable) says stripes or flames have been thought to result from loop extrusion and identifies stripes linking enhancers and promoters. But it also says the stripes studied do not appear to be simply features of loop extrusion. The Discussion proposes extrusion as one possible mechanism, not evidence that unidirectional extrusion from enhancers is common. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [118] Chen, 2023, Structural elements promote architectural stripe formation and facilitate ultra-long-range gene regulation at a human disease locus, Mol. Cell sourceDisputed

  In Results (“A multi-loop model better explains…”; page not provided), the paper says both targeted-loading and random-loading models produce stripes, so stripes do not establish extrusion from an enhancer. The Discussion favors random cohesin loading between loop barriers over the enhancer-loading reel-in model at the SOX9 locus; it does not present enhancer-origin extrusion as common. (LLM, conf=0.87)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

Activation of the Shh gene during neuronal differentiation of mouse embryonic stem cells is accompanied by a decrease in the spatial proximity between Shh and the SBE6 enhancer that controls its work.

“For example, activation of the Shh gene during neuronal differentiation of mouse embryonic stem cells is accompanied by a decrease in the spatial proximity between Shh and the SBE6 enhancer that controls its work [89].”

• cites [89] Benabdallah, 2019, Decreased Enhancer-Promoter Proximity Accompanying Enhancer Activation, Mol. Cell sourceSupported

Current results strongly suggest that the demixing of enhancer- and promoter-associated proteins results in the assembly of a phase condensate that keeps the enhancer and promoter in spatial proximity.

“Current results strongly suggest that the demixing of enhancer- and promoter-associated proteins results in the assembly of a phase condensate that keeps the enhancer and promoter in spatial proximity [97,98,99].”

• cites [97] Li, 2022, Nuclear Protein Condensates and Their Properties in Regulation of Gene Expression, J. Mol. Biol. sourcePartly supported

  The Transcriptional condensates section reports that proteins assemble into phase-separated condensates at enhancers and bring activated enhancers into spatial proximity. It does not say that enhancer- and promoter-associated proteins jointly demix to form a condensate that keeps an enhancer and promoter close. The Chromatin-related condensates section mentions promoter–enhancer loops, but does not establish that condensates maintain them; page numbers are unavailable in our copy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [98] Hnisz, 2017, A Phase Separation Model for Transcriptional Control, Cell sourcePartly supported

  In “Formation of membraneless organelles by phase separation” (page unavailable), the paper hypothesizes that a phase-separated assembly forms at super-enhancers. In “A phase separation model of enhancer assembly and function” (page unavailable), it proposes that the assembly includes factors bound at both enhancers and promoters. It does not establish that such a condensate forms in vivo or keeps enhancers and promoters in spatial proximity, so the claim presents the proposed mechanism more strongly than the source does. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [99] Kantidze, 2020, Weak interactions in higher-order chromatin organization, Nucleic Acids Res. sourceSupported

Some enhancer-promoter loops are destroyed or weakened upon cell treatment with 1,6-hexanediol, an agent that destroys phase condensates.

“Indeed, some enhancer-promoter loops are destroyed or weakened upon cell treatment with 1,6-hexanediol, an agent that destroys phase condensates [106].”

• cites [106] Ulianov, 2021, Suppression of liquid–liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells, Nucleic Acids Res. sourceSupported

Most known transcription factors bind to both enhancers and promoters.

“Most of the known transcription factors bind to both enhancers and promoters [70,71].”

• cites [70] Andersson, 2015, Promoter or enhancer, what’s the difference? Deconstruction of established distinctions and presentation of a unifying model, Bioessays sourceNot checked — no full text of the source
• cites [71] Andersson, 2020, Determinants of enhancer and promoter activities of regulatory elements, Nat. Rev. Genet. sourceCould not verify

  The available copy contains bibliographic information and funding details, but no discussion of transcription-factor binding. Searches for the claim and for shared binding at enhancers and promoters found no passage addressing whether most known transcription factors bind to both. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

The results of several studies suggest the involvement of eRNA in the formation of enhancer-promoter loops.

“The results of several studies suggest the involvement of eRNA in the formation of enhancer-promoter loops, although the specific mechanisms remain unclear [68,69].”

• cites [68] Hsieh, 2014, Enhancer RNAs participate in androgen receptor-driven looping that selectively enhances gene activation, Proc. Natl. Acad. Sci. USA sourceNot checked — no full text of the source
• cites [69] Li, 2013, Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation, Nature sourceSupported

These observations are most easily interpreted in terms of a model that suggests the assembly of a common activator compartment represented by a small nuclear volume occupied by folded chromatin fiber and associated proteins.

“Taken together, these observations are most easily interpreted in terms of a model that suggests the assembly of a common activator compartment represented by a small nuclear volume occupied by folded chromatin fiber and associated proteins [105].”

• cites [105] Goel, 2023, Region Capture Micro-C reveals coalescence of enhancers and promoters into nested microcompartments, Nat. Genet. sourcePartly supported

  The Discussion (page not provided) proposes that enhancer–promoter microcompartments are small, punctate A-compartments formed through active chromatin interactions, possibly involving transcription factors and co-factors. It does not describe them as a common activator compartment occupying a small nuclear volume of folded chromatin fiber and associated proteins; that physical description goes beyond the source. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Contacts between architectural proteins can modulate the spatial organization of the genome, including keeping enhancers close to promoters.

“In model experiments, it was convincingly shown that contacts between these proteins can modulate the spatial organization of the genome, including keeping enhancers close to promoters [84,91,92].”

• cites [84] Kyrchanova, 2022, Mechanisms of enhancer-promoter communication and chromosomal architecture in mammals and Drosophila, Front. Genet. sourceSupported
• cites [91] Maksimenko, 2008, Enhancer-Promoter Communication Is Regulated by Insulator Pairing in a Drosophila Model Bigenic Locus, Mol. Cell. Biol. sourceNot checked — no full text of the source
• cites [92] Savitskaya, 2006, Study of Long-Distance Functional Interactions between Su(Hw) Insulators That Can Regulate Enhancer-Promoter Communication in Drosophila melanogaster, Mol. Cell. Biol. sourcePartly supported

  The Discussion describes Su(Hw) insulator pairing as changing whether an enhancer can approach or communicate with a promoter, and proposes that one arrangement may bring them closer. But the paper says the actual positioning of that arrangement is unknown; its experiments establish functional effects, not that protein contacts were shown to keep enhancers physically close to promoters. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

TADs appear to restrict the areas of enhancer action.

“Second, it was demonstrated that eukaryotic chromosomes are partitioned into so-called topologically associated domains (TADs) [35,36,37], which appear to restrict the areas of enhancer action [38,39,40,41].”

• cites [38] Symmons, 2014, Functional and topological characteristics of mammalian regulatory domains, Genome Res. sourceSupported
• cites [39] Kraft, 2015, Disruptions of Topological Chromatin Domains Cause Pathogenic Rewiring of Gene-Enhancer Interactions, Cell sourceSupported
• cites [40] Spielmann, 2016, Breaking TADs: How Alterations of Chromatin Domains Result in Disease, Trends Genet. sourceSupported
• cites [41] Kaiser, 2017, When TADs go bad: Chromatin structure and nuclear organisation in human disease, F1000Research sourceSupported

Active enhancers are often in physical proximity to the promoters they activate, regardless of the genomic distances between these enhancers and promoters.

“First, it was shown that active enhancers are often in physical proximity to the promoters they activate, regardless of the genomic distances between these enhancers and promoters [32,33,34].”

• cites [32] Tolhuis, 2002, Looping and Interaction between Hypersensitive Sites in the Active beta-globin Locus, Mol. Cell sourceNot checked — no full text of the source
• cites [33] Vernimmen, 2007, Long-range chromosomal interactions regulate the timing of the transition between poised and active gene expression, EMBO J. sourcePartly supported

  The Discussion reports 3C evidence consistent with direct physical interaction between remote upstream regulatory elements and the α-globin promoter, with looping coinciding with transcription onset. The Introduction notes that regulatory elements can be tens or hundreds of kilobases from the genes they control. This supports long-range proximity in the studied locus, but not the broader claim that active enhancers are often close to their promoters regardless of genomic distance. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [34] Williamson, 2016, Shh and ZRS enhancer co-localisation is specific to the zone of polarizing activity, Development sourcePartly supported

  The Introduction reports proximity between Shh and its distant ZRS enhancer, and the Results describe greater colocalisation where the enhancer is active. This supports the claim for a long-range enhancer–gene pair, but the paper does not establish that active enhancers are often close to their promoters regardless of genomic distance across enhancers generally. Page numbers are not provided in our copy. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Promoters can have enhancer activity.

“Now, there is convincing evidence that promoters can have enhancer activity [72,73,74,75], while intragenic enhancers can act as alternative promoters [76].”

• cites [72] Dao, 2017, Genome-wide characterization of mammalian promoters with distal enhancer functions, Nat. Genet. sourceNot checked — no full text of the source
• cites [73] Dao, 2018, Transcriptional regulation by promoters with enhancer function, Transcription sourceSupported
• cites [74] Puthier, 2018, Widespread Enhancer Activity from Core Promoters, Trends Biochem. Sci. sourceNot checked — no full text of the source

The level of eRNA transcription correlates with enhancer activity.

“Several studies have shown that the level of eRNA transcription correlates with enhancer activity [60,61].”

• cites [60] Kaikkonen, 2013, Remodeling of the Enhancer Landscape during Macrophage Activation Is Coupled to Enhancer Transcription, Mol. Cell sourceSupported
• cites [61] Mikhaylichenko, 2018, The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription, Genes Dev. sourceSupported

Fusion of the repressive KRAB domain with catalytically inactive Cas9 (dCas9) identified 664 functionally significant out of 78,776 potential enhancer-promoter interactions in the human erythroid cell line K562.

“Fusion of the repressive KRAB (Krüppel-associated box) domain with catalytically inactive Cas9 (dCas9) was applied for the identification of 664 functionally significant out of 78,776 potential enhancer-promoter interactions in the human erythroid cell line (K562 cells) [25].”

• cites [25] Gasperini, 2019, A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens, Cell sourcePartly supported

  The Results section on the scaled K562 screen (page unavailable) reports use of CRISPRi and identification of 664 enhancer–gene pairs at a 10% empirical false-discovery threshold. The proof-of-concept Results section identifies the CRISPRi reagent as nuclease-inactive Cas9 tethered to KRAB. Our copy does not substantiate the claimed denominator of 78,776 potential interactions (searched: “78,776” and “78776 candidate enhancer gene pairs tested”). (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Contacts between convergent CTCF binding sites are established by the extrusion of chromatin loops with cohesin.

“Contacts between convergent CTCF binding sites are established by the extrusion of chromatin loops with cohesin [42,46,48,93].”

• cites [42] Fudenberg, 2016, Formation of Chromosomal Domains by Loop Extrusion, Cell Rep. sourcePartly supported

  The Results section on directional CTCF boundaries says that loop extrusion by cohesin can explain contacts at convergent CTCF sites and that the observations support an orientation-dependent mechanism. But the paper presents this as a proposed model, not an established mechanism, so the citing sentence overstates its certainty. Page numbers are unavailable in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [46] Rao, 2014, A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping, Cell sourcePartly supported

  The Results section reports that most loop-anchor CTCF motif pairs are convergent, and the Discussion reports that cohesin subunits occur at over 86% of loop anchors. The paper does not establish loop extrusion as the mechanism; the Discussion instead says the mechanism remains to be clarified and suggests CTCF dimerization. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [48] Wutz, 2017, Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins, EMBO J. sourcePartly supported

  The Results report that 73.8% of the analyzed loops in control cells were anchored at convergent CTCF sites, and the Introduction describes cohesin’s role in loop formation. But the Introduction calls loop extrusion a hypothesis that could explain why convergent sites come together; it does not establish extrusion as the mechanism. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

In eukaryotic cells, transcription occurs in cycles.

“In eukaryotic cells, transcription occurs in cycles [50,51,52].”

• cites [50] Chubb, 2010, Bursts and pulses: Insights from single cell studies into transcriptional mechanisms, Curr. Opin. Genet. Dev. sourceNot checked — no full text of the source
• cites [51] Chubb, 2006, Transcriptional Pulsing of a Developmental Gene, Curr. Biol. sourcePartly supported

  The Conclusions report repeated, discontinuous transcription at a developmentally regulated locus in eukaryotic cells. But the study concerns one locus, not transcription generally across eukaryotic cells, and its pulses were irregular rather than periodic cycles. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [52] Rodriguez, 2020, Transcription in Living Cells: Molecular Mechanisms of Bursting, Annu. Rev. Biochem. sourceNot checked — no full text of the source

The functional significance of the physical approximation of the promoter to the enhancer was demonstrated in experiments on the activation of transcription through the forced formation of enhancer-promoter loops.

“The functional significance of the physical approximation of the promoter to the enhancer was demonstrated in experiments on the activation of transcription through the forced formation of enhancer-promoter loops [87,88].”

• cites [87] Breda, 2016, Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers, Blood sourceSupported
• cites [88] Deng, 2012, Controlling Long-Range Genomic Interactions at a Native Locus by Targeted Tethering of a Looping Factor, Cell sourceSupported

Convergent binding sites for CTCF located at TAD borders block the movement of loop extruders.

“Convergent binding sites for the versatile transcription factor CTCF (CCCTC-binding factor) located at TAD borders [46] block the movement of loop extruders [47,48,49].”

• cites [46] Rao, 2014, A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping, Cell sourceSupported
• cites [47] Nora, 2017, Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization, Cell sourceSupported
• cites [48] Wutz, 2017, Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins, EMBO J. sourceSupported

Spatial proximity between distant genomic elements can only be observed within the folded chromatin fibril.

“However, subsequent studies have shown that spatial proximity between distant genomic elements can only be observed within the folded chromatin fibril [100].”

• cites [100] Gavrilov, 2013, Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub, Nucleic Acids Res. sourcePartly supported

  The Discussion (page not provided) says that characteristic 3C ligation products may be generated only within non-lysed nuclei and proposes a folded chromatin domain or nuclear compartment as a model for an active chromatin hub. That supports proximity being detected in that structural setting, but not the broader claim that spatial proximity between distant genomic elements can only be observed within a folded chromatin fibril. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Eukaryotic chromosomes are partitioned into topologically associated domains (TADs).

“Second, it was demonstrated that eukaryotic chromosomes are partitioned into so-called topologically associated domains (TADs) [35,36,37], which appear to restrict the areas of enhancer action [38,39,40,41].”

• cites [35] Nora, 2012, Spatial partitioning of the regulatory landscape of the X-inactivation centre, Nature sourceSupported
• cites [36] Dixon, 2012, Topological domains in mammalian genomes identified by analysis of chromatin interactions, Nature sourcePartly supported

  The Abstract reports topological domains as a pervasive feature of the human and mouse genomes and notes similar partitioning in Drosophila. It suggests this may be a fundamental principle of metazoan genomes, but does not establish the claim for eukaryotic chromosomes generally. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [37] Sexton, 2012, Three-Dimensional Folding and Functional Organization Principles of the Drosophila Genome, Cell sourcePartly supported

  The Introduction describes domains with sharp boundaries in fly embryonic chromosomes, and “Domain-Based Hierarchical Chromosomal Architecture” reports that those chromosomes partition into physical domains. This supports the claim for Drosophila, but the paper’s findings alone do not establish the stated generalization to eukaryotic chromosomes broadly. The later enhancer-action clause has separate citations and is not attributed to this source here. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

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

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

• cites [95] Ulianov, 2017, Activation of the α-globin gene expression correlates with dramatic upregulation of nearby non-globin genes and changes in local and large-scale chromatin spatial structure, Epigenetics Chromatin sourcePartly supported

  The Discussion (page unavailable) reports that CTCF binding and the strength of CTCF-anchored loops decrease during HD3-cell differentiation, supporting a role in local chromatin contacts. It also says TAD and sub-TAD profiles show no dramatic or obvious changes despite the loss of binding, so this paper does not establish that changing CTCF binding profiles play an important role in broader 3D-genome reconfiguration. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [96] Hanssen, 2017, Tissue-specific CTCF–cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo, Nat. Cell Biol. sourcePartly supported

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

  Experimental — accuracy not yet measured

Most enhancers are located at a relatively short distance (up to 50 kb in mammals and up to 10 kb in Drosophila) from target promoters.

“However, most enhancers are located at a relatively short distance (up to 50 kb in mammals and up to 10 kb in Drosophila) from target promoters [21].”

• cites [21] Furlong, 2018, Developmental enhancers and chromosome topology, Science sourceSupported

In mammals and other vertebrates, TADs are formed by the dynamic extrusion of DNA loops, mediated by cohesin motors.

“In mammals and other vertebrates, TADs are formed by the dynamic extrusion of DNA loops [42,43], mediated by cohesin motors [44,45].”

• cites [42] Fudenberg, 2016, Formation of Chromosomal Domains by Loop Extrusion, Cell Rep. sourcePartly supported

  The Introduction (page not available) proposes that loop extrusion forms TADs, and the Results section reports simulations in which TADs consist of dynamic loops. But the paper presents this as a model, identifies cohesin as a possible loop-extruding factor rather than an established motor, and does not establish the mechanism across other vertebrates. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [43] Sanborn, 2015, Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes, Proc. Natl. Acad. Sci. USA sourceNot checked — no full text of the source
• cites [44] Rao, 2017, Cohesin Loss Eliminates All Loop Domains, Cell sourcePartly supported

  The Discussion (“Two mechanisms that guide genome folding”) supports cohesin-dependent loop-domain formation and describes loop extrusion as a proposed mechanism, not an established one. In “The speed of loop extrusion,” the paper says cohesin itself might be a motor but suggests other translocases may be involved; it therefore does not establish that cohesin motors mediate extrusion. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

In some cases, several enhancers form a functionally interdependent network, so that exclusion of any single enhancer from this network results in loss of all enhancer activity.

“In some cases, several enhancers form a functionally interdependent network, so that the exclusion of any single enhancer from this network results in the loss of all enhancer activity [27].”

• cites [27] Farooq, 2021, An interdependent network of functional enhancers regulates transcription and EZH2 loading at the INK4a/ARF locus, Cell Rep. sourceSupported

Enhancers can be located at a considerable distance (up to 1 Mb or more) from target promoters.

“An analysis of the positions of enhancers in the genome showed that they can be located at a considerable distance (up to 1 Mb or more) from the target promoters.”

• cites [21] Furlong, 2018, Developmental enhancers and chromosome topology, Science sourceSupported

Activation of a gene or gene locus by multiple enhancers is quite common.

“Although the number of enhancers in mammalian genomes can be overestimated, activation of a gene or gene locus by multiple enhancers is quite common [26].”

• cites [26] Patrinos, 2004, Multiple interactions between regulatory regions are required to stabilize an active chromatin hub, Genes Dev. sourcePartly supported

  The introductory passage describes several regulatory sites forming an active chromatin hub that interacts with genes in the human β-globin locus. This supports the existence of a locus regulated by multiple elements, but the paper does not establish that activation by multiple enhancers is common across genes or loci. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

In K562 cells, more than 65% of functionally confirmed enhancer-promoter pairs are characterized by the formation of enhancer-promoter loops.

“A recent analysis of enhancer-promoter loops using the micro-C technique demonstrated that in K562 cells, more than 65% of functionally confirmed enhancer-promoter pairs are characterized by the formation of enhancer-promoter loops [90].”

• cites [90] Golov, 2023, A genome-wide nucleosome-resolution map of promoter-centered interactions in human cells corroborates the enhancer-promoter looping model, bioRxiv sourcePartly supported

  The Results report that 61.5% (216/351) of functionally verified enhancer–promoter pairs in K562 had spatial interactions detected, supporting the claim that a majority interact—but not the stated figure of more than 65%. The Abstract and Results identify the method as MChIP-C, not Micro-C. (LLM, conf=0.91)

  Experimental — accuracy not yet measured

Binding of CTCF to the recognition motives on DNA can be regulated through DNA methylation and other ways.

“Binding of CTCF to the recognition motives on DNA can be regulated through DNA methylation and other ways (for example, through competition with other proteins and even with non-coding RNAs such as Jpx (just proximal to XIST) [94]).”

• cites [94] Oh, 2021, Jpx RNA regulates CTCF anchor site selection and formation of chromosome loops, Cell sourceSupported

This specificity seems to be determined by the sets of transcription factors that bind to enhancers and different types of promoters.

“This specificity seems to be determined by the sets of transcription factors that bind to enhancers and different types of promoters [134].”

• cites [134] Zabidi, 2016, Regulatory Enhancer–Core-Promoter Communication via Transcription Factors and Cofactors, Trends Genet. sourceSupported

In the Drosophila melanogaster genome, several enhancers selectively activate only the dpp gene promoter within a 70-kilobase segment containing dpp, Slh, and oaf genes.

“A classic example is the promoters of the dpp, Slh, and oaf genes in Drosophila melanogaster. These genes are located within a 70-kilobase segment of the genome. In the same segment of the genome, there are several enhancers that selectively activate only the dpp gene promoter [135].”

• cites [135] Merli, 1996, Promoter specificity mediates the independent regulation of neighbouring genes, Genes Dev. sourcePartly supported

  The Abstract reports that several dpp enhancers lie near Slh and oaf but do not activate those genes in the normal arrangement. The ‘Mechanisms mediating promoter specificity’ section identifies a dpp promoter they do activate, while also noting that compatible introduced promoters can be activated; thus ‘only’ applies to the native promoters discussed, not to all promoters. The passages do not establish the claimed 70-kilobase span. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

The CTCF sites on the Pcdhα promoters are methylated, preventing CTCF binding and looping.

“However, the CTCF sites on the promoters are methylated, preventing CTCF binding and looping.”

• cites [140] Guo, 2012, CTCF/cohesin-mediated DNA looping is required for protocadherin alpha promoter choice, Proc. Natl. Acad. Sci. USA sourceNot checked — no full text of the source
• cites [141] Canzio, 2019, Antisense lncRNA Transcription Mediates DNA Demethylation to Drive Stochastic Protocadherin α Promoter Choice, Cell sourcePartly supported

  The Introduction (page not supplied) says CTCF sites are hypermethylated in inactive Pcdhα promoters, preventing CTCF/Cohesin binding. It also says promoter–enhancer looping requires that binding. The claim omits the inactive-promoter qualification: the same passage describes active promoters as hypomethylated. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Different enhancers show various levels of specificity, ranging from the ability to activate a wide range of promoters to highly specific activation of a particular promoter.

“Analysis of a number of enhancer-promoter pairs in the mouse genome has demonstrated that different enhancers show various levels of specificity, ranging from the ability to activate a wide range of promoters to highly specific activation of a particular promoter [136].”

• cites [136] Comoglio, 2022, Systematic analysis of intrinsic enhancer-promoter compatibility in the mouse genome, Mol. Cell sourceSupported

There are also enhancers that preferentially activate housekeeping gene promoters.

“There are also enhancers that preferentially activate housekeeping gene promoters. This specificity seems to be determined by the sets of transcription factors that bind to enhancers and different types of promoters [134].”

• cites [134] Zabidi, 2016, Regulatory Enhancer–Core-Promoter Communication via Transcription Factors and Cofactors, Trends Genet. sourceSupported

Even the strongest C-captured contacts are detected in less than 10% of cells.

“This can explain why even the strongest C-captured contacts are detected in less than 10% of cells [107,108,109].”

• cites [107] Finn, 2019, Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization, Cell sourcePartly supported

  In Results, “The steady-state association frequencies of genomic interactors are low” (page unavailable), the paper reports that 102 of 106 pairs colocalized in fewer than 10% of alleles, including many pairs in the top 20% of Hi-C capture frequencies. It does not establish that even the strongest contacts are below 10%: four pairs exceeded that threshold, and the measurement is reported per allele rather than per cell. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [108] Gavrilov, A.A., Golov, A.K., and Razin, S.V. (2013). Actual Ligation Frequencies in the Chromosome Conformation Capture Procedure. PLoS ONE, 8. sourcePartly supported

  The Discussion reports that ligation frequencies for the studied beta-globin promoter–enhancer contacts were in the tenths of a percent, supporting a low detection yield. But the Introduction says 3C cannot estimate the proportion of cells in which two sequences interact, and the Abstract notes that the low yield could be entirely technical. The paper therefore does not establish the claim’s percentage of cells or its extension to the strongest contacts generally. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [109] Gavrilov, 2014, Quantitative analysis of genomic element interactions by molecular colony technique, Nucleic Acids Res. sourcePartly supported

  The Results and Discussion (page unavailable) report that complexes between selected β-globin promoters and enhancers, if present, occurred in fewer than 3% of the chromosomes analyzed. That supports a low frequency for these specific contacts, but not the claim that even the strongest chromosome-conformation-capture contacts occur in fewer than 10% of cells. The Discussion also says that 3C results cannot establish the proportion of cells containing a contact. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Some enhancers show a preference for certain types of promoters.

“Some enhancers show a preference for certain types of promoters. Enhancers were classified according to their ability to preferentially activate promoters containing a DPE (downstream promoter element) or promoters containing a TATA box [133].”

• cites [133] Butler, 2001, Enhancer–promoter specificity mediated by DPE or TATA core promoter motifs, Genes Dev. sourceSupported

In the mammalian protocadherin α (Pcdhα) gene cluster, each promoter contains a CTCF binding site capable of looping with a convergent CTCF binding site on the enhancer.

“In the mammalian protocadherin α (Pcdhα) gene cluster, each promoter contains a CTCF binding site capable of looping with a convergent CTCF binding site on the enhancer.”

• cites [140] Guo, 2012, CTCF/cohesin-mediated DNA looping is required for protocadherin alpha promoter choice, Proc. Natl. Acad. Sci. USA sourceNot checked — no full text of the source
• cites [141] Canzio, 2019, Antisense lncRNA Transcription Mediates DNA Demethylation to Drive Stochastic Protocadherin α Promoter Choice, Cell sourceSupported

Enhancers were classified according to their ability to preferentially activate promoters containing a DPE or promoters containing a TATA box.

“Enhancers were classified according to their ability to preferentially activate promoters containing a DPE (downstream promoter element) or promoters containing a TATA box [133].”

• cites [133] Butler, 2001, Enhancer–promoter specificity mediated by DPE or TATA core promoter motifs, Genes Dev. sourceSupported

During the search of a target promoter, enhancers meet other genes that are not activated by this enhancer.

“During the “search” of a target promoter (regardless of the “searching mechanism”), enhancers meet other genes that, however, are not activated by this enhancer [21,128,129].”

• cites [21] Furlong, 2018, Developmental enhancers and chromosome topology, Science sourceSupported
• cites [128] Schoenfelder, 2019, Long-range enhancer–promoter contacts in gene expression control, Nat. Rev. Genet. sourceNot checked — no full text of the source
• cites [129] Sanyal, 2012, The long-range interaction landscape of gene promoters, Nature sourcePartly supported

  The paper’s discussion of Figure 2d reports enhancer-like elements looping to TSSs, including some classified as non-expressed; its discussion of Figure 3b finds that most distal-element interactions are not with the nearest TSS (page unavailable in our copy). These findings support encounters with genes other than an enhancer’s apparent target, but the paper does not establish a temporal promoter ‘search’ or show that a particular enhancer fails to activate each gene it contacts. It also cautions that some TSSs classified as non-expressed may be expressed at low levels. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Enhancers that control the transcription of many inducible genes are often located in close spatial proximity to these genes in the nuclear space, even in the absence of an inducer.

“Enhancers that control the transcription of many inducible genes are often located in close spatial proximity to these genes in the nuclear space, even in the absence of an inducer [81,125].”

• cites [81] Jin, 2013, A high-resolution map of the three-dimensional chromatin interactome in human cells, Nature sourceSupported
• cites [125] Comoglio, 2018, Thrombopoietin signaling to chromatin elicits rapid and pervasive epigenome remodeling within poised chromatin architectures, Genome Res. sourceSupported

The ability to establish contacts between enhancers and promoters is partially controlled at the level of chromosome organization into TADs, which in many cases limit the scope of enhancers.

“The ability to establish contacts between enhancers and promoters is partially controlled at the level of chromosome organization into topologically associated domains, which in many cases limit the scope of enhancers [38,41,130,131].”

• cites [38] Symmons, 2014, Functional and topological characteristics of mammalian regulatory domains, Genome Res. sourceSupported
• cites [41] Kaiser, 2017, When TADs go bad: Chromatin structure and nuclear organisation in human disease, F1000Research sourceSupported
• cites [130] Razin, 2017, Gene functioning and storage within a folded genome, Cell. Mol. Biol. Lett. sourceSupported

Stimulation of promoter activity by forced looping with an enhancer is a strong argument in favor of the loop model.

“Stimulation of promoter activity by forced looping with an enhancer is a strong argument in favor of this model [87,88,110,111].”

• cites [87] Breda, 2016, Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers, Blood sourceSupported
• cites [88] Deng, 2012, Controlling Long-Range Genomic Interactions at a Native Locus by Targeted Tethering of a Looping Factor, Cell sourceSupported
• cites [110] Morgan, 2017, Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping, Nat. Commun. sourceSupported

Selective demethylation of the CTCF binding site on a particular Pcdhα promoter allows that promoter to establish contact with the enhancer, resulting in activation of the expression of that particular Pcdhα gene.

“Selective demethylation of the CTCF binding site on a particular promoter allows that promoter to establish contact with the enhancer, resulting in activation of the expression of that particular Pcdhα gene [140,141].”

• cites [140] Guo, 2012, CTCF/cohesin-mediated DNA looping is required for protocadherin alpha promoter choice, Proc. Natl. Acad. Sci. USA sourceNot checked — no full text of the source
• cites [141] Canzio, 2019, Antisense lncRNA Transcription Mediates DNA Demethylation to Drive Stochastic Protocadherin α Promoter Choice, Cell sourceSupported

Cohesin depletion significantly suppresses the activation of inducible genes, especially when the enhancer is located at a significant distance from the promoter.

“At the same time, cohesin depletion significantly suppresses the activation of inducible genes, especially when the enhancer is located at a significant distance from the promoter [120,121,122].”

• cites [120] Calderon, 2022, Cohesin-dependence of neuronal gene expression relates to chromatin loop length, Elife sourcePartly supported

  The Results (pages unavailable) report that a subset of late-response genes failed to reach normal expression after stimulation in cohesin-deficient neurons, and that these genes formed significantly longer chromatin loops. But most immediate-early genes and some late-response genes remained fully inducible. The observed tendency toward longer enhancer–promoter loops specifically did not reach statistical significance, so the claim overstates both the general effect on inducible genes and the evidence for enhancer–promoter distance. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [121] Aljahani, 2022, Analysis of sub-kilobase chromatin topology reveals nano-scale regulatory interactions with variable dependence on cohesin and CTCF, Nat. Commun. sourcePartly supported

  The Results report reduced expression of several genes in mouse embryonic stem cells after cohesin depletion, and the Discussion reports little change in enhancer–promoter interactions below 5 kb but reductions in interactions and expression at loci spanning about 20 and 50 kb. The paper did not test activation of inducible genes in those experiments; it describes the importance of cohesin for gene expression upon stimuli as consistent with other reports. Thus, the specific claim about significantly suppressed activation of inducible genes goes beyond its findings. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [122] Cuartero, 2018, Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation, Nat. Immunol. sourcePartly supported

  The Discussion (page unavailable) says cohesin is critically required for inducible gene expression in primary myeloid cells, supporting the claim that depletion impairs activation. But the Results section on partial rescue (page unavailable) links cohesin dependence to genes near enhancers; our copy does not establish that suppression is especially strong when an enhancer is far from its promoter. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

The probability of establishing spatial proximity between promoter and enhancer sharply decreases with increasing genomic distance between them.

“The first scenario is supported by the results of model experiments, which demonstrate that the probability of establishing spatial proximity between promoter and enhancer sharply decreases with increasing genomic distance between them [113].”

• cites [113] Zuin, 2022, Nonlinear control of transcription through enhancer–promoter interactions, Nature sourceSupported

A recently developed oligopainting strategy combined with super-resolution microscopy allows the path of the chromatin fibril to be traced at relatively long genomic loci.

“A recently developed oligopainting strategy [149] combined with super-resolution microscopy allows the path of the chromatin fibril to be traced at relatively long genomic loci [150,151].”

• cites [149] Beliveau, 2015, Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes, Nat. Commun. sourceSupported
• cites [150] Bintu, 2018, Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells, Science sourceSupported
• cites [151] Su, 2020, Genome-Scale Imaging of the 3D Organization and Transcriptional Activity of Chromatin, Cell sourcePartly supported

  The Results section, “High-Resolution Chromatin Tracing of Whole Chromosomes” (page unavailable), reports tracing chromosome 21 across approximately 650 loci using oligonucleotide probes and sequential imaging, supporting the claim that long genomic regions can be traced. The paper describes a fluorescence microscope and sequential FISH, but our copy does not establish that this tracing used super-resolution microscopy. The separate oligopainting citation [149] is not assessed here. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

SDS extraction, used in most C-methods, solubilizes a significant portion of histones, which should lead to chromatin decompactization even in the presence of formaldehyde crosslinks.

“SDS extraction, used in most C-methods, solubilizes a significant portion of histones (Golov et al., unpublished results), which should lead to chromatin decompactization even in the presence of formaldehyde crosslinks.”

Mechanisms that establish and maintain enhancer-promoter proximity may be fundamentally different.

“Therefore, mechanisms that establish and maintain enhancer-promoter proximity may be fundamentally different (see Section 5 for further discussion).”

The looping model for enhancer-promoter communication received strong confirmation after the development of methods for studying spatial organization of the genome using proximity ligation.

“As applied to enhancers in vertebrate animals, this model received strong confirmation after the development of methods for studying the spatial organization of the genome using the proximity ligation technique [29].”

• cites [29] Dekker, 2002, Capturing Chromosome Conformation, Science sourceNot checked — no full text of the source

Intragenic enhancers can act as alternative promoters.

“Now, there is convincing evidence that promoters can have enhancer activity [72,73,74,75], while intragenic enhancers can act as alternative promoters [76].”

• cites [76] Kowalczyk, 2012, Intragenic Enhancers Act as Alternative Promoters, Mol. Cell sourceNot checked — no full text of the source

Promoters with enhancer activity possess some epigenetic signatures typical for enhancers, such as p300 binding and a high H3K27ac/H3K4me3 ratio.

“Interestingly, promoters with enhancer activity possess some epigenetic signatures typical for enhancers, such as p300 binding and a high H3K27ac/H3K4me3 ratio [72].”

• cites [72] Dao, 2017, Genome-wide characterization of mammalian promoters with distal enhancer functions, Nat. Genet. sourceNot checked — no full text of the source

Once spatial contacts between enhancers and promoters are established, they can be maintained by mechanisms that do not rely on DNA loop extrusion.

“Once spatial contacts between enhancers and promoters are established, they can be maintained by mechanisms that do not rely on DNA loop extrusion.”

The estimates of the number of enhancers are made by identifying potential enhancers based on analysis of epigenetic profiles without confirmation of enhancer activity in functional tests.

“It should be noted, however, that the above estimates of the number of enhancers are made by identifying potential enhancers based on the analysis of epigenetic profiles without confirmation of enhancer activity in functional tests.”

A model of an activator chromatin compartment provides the possibility of transferring activating factors from an enhancer to a promoter without establishing direct contact between these elements.

“A model of an activator chromatin compartment is substantiated, which provides the possibility of transferring activating factors from an enhancer to a promoter without establishing direct contact between these elements.”

The extrusion of chromatin loops will contribute to the convergence of promoters and enhancers.

“Accordingly, the extrusion of chromatin loops will contribute to the convergence of promoters and enhancers.”

More typical is the juxtaposition of the enhancer and the target promoter during transcriptional activation.

“However, more typical is the juxtaposition of the enhancer and the target promoter during transcriptional activation.”

Remote enhancers in the mouse β-globin gene domain form a single complex to which the genes that need to be activated are recruited.

“It has been shown that remote enhancers in the mouse β-globin gene domain form a single complex to which the genes that need to be activated are recruited [32].”

• cites [32] Tolhuis, 2002, Looping and Interaction between Hypersensitive Sites in the Active beta-globin Locus, Mol. Cell sourceNot checked — no full text of the source

The spatial organization of the genome plays an important role in controlling promoter activation by enhancers.

“Recent advances in the study of the 3D genome have shown that the spatial organization of the genome plays an important role in controlling promoter activation by enhancers.”

The results of visualization of enhancers and target promoters in fixed and living cells do not support the assumption that the enhancer is in direct contact with the target promoter.

“The results of visualization of enhancers and target promoters in fixed and living cells do not support the assumption that the enhancer is in direct contact with the target promoter.”

After the establishment of spatial communication between the enhancer and the promoter, cohesin is most likely not involved in keeping the enhancer and promoter in spatial proximity.

“After the establishment of spatial communication between the enhancer and the promoter (the formation of a common activator compartment), cohesin is most likely not involved in keeping the enhancer and promoter in spatial proximity.”

Enhancers possess selectivity towards certain promoters even within a TAD.

“However, enhancers possess selectivity towards certain promoters even within a topologically associated domain.”

An enhancer that activates the odorant receptor genes establishes communication only with the gene whose promoter is derepressed.

“Thus, an enhancer that activates the odorant receptor genes establishes communication only with the gene whose promoter is derepressed [132].”

• cites [132] 2017, Regulatory Features for Odorant Receptor Genes in the Mouse Genome, Front. Genet.Not checked yet

  Not checked in the free run.

If the distance between the enhancer and the promoter is too large, several extrusion complexes can be loaded, which can organize the segment of the chromatin fibril separating them into a rosette of several loops, again bringing the enhancer closer to the promoter.

“If the distance between the enhancer and the promoter is too large, then several extrusion complexes can be loaded, which can organize the segment of the chromatin fibril separating the enhancer and promoter into a rosette of several loops, again bringing the enhancer closer to the promoter (Figure 2C).”

Promoters repressed through epigenetic mechanisms cannot communicate with enhancers.

“Promoters repressed through epigenetic mechanisms cannot communicate with enhancers.”

The loop model for establishing E-P communication is currently the most reasonable.

“Despite some uncertainty about how close an enhancer should be to an activated promoter, the loop model for establishing E-P communication is currently the most reasonable.”

When the enhancer and the target promoter are located at a considerable distance along the genome, the establishment of a spatial contact between them because of a random search seems unlikely.

“In the case when the enhancer and the target promoter are located at a considerable distance (along the genome), the establishment of a spatial contact between them because of a random search seems unlikely.”

In Drosophila melanogaster embryos, many developmental enhancers establish spatial contacts with their target promoters prior to the beginning of transcription from these promoters.

“Consistently, in Drosophila melanogaster embryos, many developmental enhancers establish spatial contacts with their target promoters prior to the beginning of transcription from these promoters [126].”

• cites [126] Klein, 2014, Enhancer loops appear stable during development and are associated with paused polymerase, Nature sourceNot checked — no full text of the source

DNA looping by the cohesin motor can play an important role in bringing the enhancer closer to the promoter since the search in three dimensions turns into scanning along the chromatin fiber.

“In this situation, the DNA looping by the cohesin motor can play an important role in bringing the enhancer closer to the promoter since the search in three dimensions turns into scanning along the chromatin fiber.”

If the enhancer and promoter have convergently directed CTCF binding sites, bidirectional extrusion of the loop starting anywhere between them will eventually bring the enhancer closer to the promoter.

“Another scenario is that the enhancer and promoter have convergently directed CTCF binding sites. In this case, bidirectional extrusion of the loop starting anywhere between the enhancer and the promoter will eventually bring the enhancer closer to the promoter (Figure 2B).”

In Drosophila, the operation of the DNA loop extrusion mechanism has not been demonstrated yet.

“In Drosophila, the operation of the DNA loop extrusion mechanism has not been demonstrated yet.”

The juxtaposition of enhancer and promoter is not always sufficient to activate the promoter.

“It should be noted that the juxtaposition of enhancer and promoter is not always sufficient to activate the promoter.”

Loading cohesin onto the enhancer and subsequent unidirectional extrusion of the chromatin loop will move the enhancer along the DNA molecule until it collides with the promoter.

“Loading cohesin onto the enhancer and subsequent unidirectional extrusion of the chromatin loop will move the enhancer along the DNA molecule until it collides with the promoter (Figure 2A).”

The mechanism of transcription factor binding cannot explain the selective activation of certain promoters in clusters of paralogous genes since the promoters of all genes in a cluster contain binding sites for the same transcription factors.

“However, this mechanism cannot explain the selective activation of certain promoters in clusters of paralogous genes since the promoters of all genes in a cluster contain binding sites for the same transcription factors.”

Classical loop domains (TADs) with a spot at the top of the triangle on Hi-C interaction maps are not observed in Drosophila.

“Classical loop domains (TADs) with a spot at the top of the triangle on Hi-C interaction maps are not observed in Drosophila.”