Towards a comprehensive catalogue of validated and target-linked human enhancers

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

The classic model is that enhancers recruit cell- and condition-specific TFs and then loop in 3D space to interact with their target promoter.

“The classic model is that enhancers recruit cell- and condition-specific TFs and then loop in 3D space to interact with their target promoter90.”

• cites [90] Dixon, J. R. et al. Chromatin architecture reorganization during stem cell differentiation. Nature 518, 331–336 (2015). This study shows compelling use of Hi-C contact maps to study the dynamics of enhancer–promoter interactions through cellular differentiation. sourcePartly supported

  The Results (Chromatin state and dynamic interactions; Allelic imbalances in gene expression, page not available) describe cell-type-specific enhancer looping and interactions between enhancers and target promoters. The Discussion (page not available) mentions transcription-factor binding sites at cell-type-specific interaction regions, but our copy does not say that enhancers recruit cell- and condition-specific transcription factors as a step preceding those loops. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

Other studies have found enhancer mobility, rather than proximity per se, to be a key determinant of activation.

“Other studies have found enhancer mobility, rather than proximity per se, to be a key determinant of activation154.”

• cites [154] Gu, B. et al. Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements. Science 359, 1050–1055 (2018). sourcePartly supported

  The paper reports greater Fgf5 enhancer mobility in actively transcribing cells and links mobility to transcriptional activity (Abstract; page unavailable). But it does not establish mobility, rather than proximity, as a key determinant of activation. Its proposed mechanism is tentative: mobility may increase enhancer–promoter encounters and reinforce expression after transcription begins. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

The heritable component of common disease risk distributes largely to non-coding regions and appears particularly enriched in enhancers specific to disease-relevant cell types.

“Although most causal genetic variants for Mendelian disorders fall in protein-coding regions, the heritable component of common disease risk distributes largely to non-coding regions, and appears to be particularly enriched in enhancers that are specific to disease-relevant cell types.”

• cites [65] Maurano, M. T. et al. Systematic localization of common disease-associated variation in regulatory DNA. Science 337, 1190–1195 (2012). sourcePartly supported

  The Abstract reports that about 93% of disease- and trait-associated GWAS variants lie in noncoding sequence. The Discussion and ‘De novo identification of pathogenic cell types’ section report enrichment in regulatory DNA active in disease-relevant cell types. The paper measures enrichment in DNase I hypersensitive sites, however, not specifically in enhancers, and does not quantify the heritable component of risk. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

Evolutionary conservation can support the functional candidacy of a region, but not all enhancers are conserved.

“Evolutionary conservation can support the functional candidacy of a region39, but not all enhancers are conserved123-125126.”

• cites [39] Pennacchio, L. A. et al. In vivo enhancer analysis of human conserved non-coding sequences. Nature 444, 499–502 (2006). sourceSupported
• cites 123-125126Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

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

A typical enhancer is probably hundreds of base-pairs in length and acts over a few to tens of kilobases.

“Although the endogenous distributions of enhancer sizes and enhancer–gene distances remain an important topic of exploration, a typical enhancer is probably hundreds of base-pairs in length70,71, and acts over a few to tens of kilobases72.”

• cites [70] Blackwood, E. M. & Kadonaga, J. T. Going the distance: a current view of enhancer action. Science 281, 60–63 (1998). sourceNot checked — no full text of the source
• cites [71] Li, L. & Wunderlich, Z. An enhancer’s length and composition are shaped by its regulatory task. Front. Genet. 8, 63 (2017). sourcePartly supported

  The Introduction says enhancers can range from about 10 to 1,000 base pairs, so a length of hundreds of base pairs is consistent with the source. It does not establish that hundreds of base pairs is typical. The distance claim is attached to a different citation in the citing sentence and is not judged here. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [72] Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell 176, 377–390 (2019). sourcePartly supported

  Citation 72 attaches to the distance claim, not the enhancer-length claim. The Results section, “Distance between Paired Enhancers and Promoters,” reports a median enhancer-to-target-TSS distance of 24.1 kb, supporting a distance of tens of kilobases. But that estimate is limited to high-confidence upstream pairs among the nonintronic enhancers surveyed, so it does not establish the distance for a typical enhancer generally. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

Some enhancers reside in clusters of a handful to even hundreds ('super enhancers'), whereas many are solo.

“Some enhancers reside in clusters of a handful116 to even hundreds (‘super enhancers’76,117), whereas many are solo.”

• cites [116] Levings, P. P. & Bungert, J. The human β-globin locus control region. Eur. J. Biochem. 269, 1589–1599 (2002). sourceNot checked — no full text of the source
• cites [76] Whyte, W. A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013). sourcePartly supported

  The Results section describes super-enhancers as clusters of constituent enhancers and says the vast majority of enhancers in the studied embryonic stem cells are typical enhancers. The passages do not establish that a super-enhancer contains hundreds of enhancers or that the typical enhancers are necessarily solo; no page numbers are available in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [117] Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013). sourceSupported

May combine two claims.

It remains unknown what proportion of candidate enhancers identified solely by biochemical marks are technical false positives or have enhancer-like features but no meaningful impact on expression of cis-encoded genes.

“Although unquestionably useful, it remains unknown what proportion of candidate enhancers identified solely by biochemical marks are technical false positives130-132 or a product of having enhancer-like biochemical features but no meaningful impact on the expression of cis encoded genes133.”

• cites [130] Teytelman, L., Thurtle, D. M., Rine, J. & van Oudenaarden, A. Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins. Proc. Natl Acad. Sci. USA 110, 18602–18607 (2013). sourceNot checked — no full text of the source
• cites [131] Worsley Hunt, R. & Wasserman, W. W. Non-targeted transcription factors motifs are a systemic component of ChIP-seq datasets. Genome Biol. 15, 412 (2014). sourcePartly supported

  The Background (page unavailable) says ChIP-seq peaks can be shaped by technical influences, including non-specific antibody pull-down, and leaves open whether some peaks arise from other causes. That supports the possibility of technical false positives, but the paper examines transcription-factor ChIP-seq peaks, not the proportion of biochemically identified candidate enhancers that are false positives. The separate claim about enhancer-like features having no meaningful effect on cis-gene expression is attached to citation 133, not this source. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [132] Jain, D., Baldi, S., Zabel, A., Straub, T. & Becker, P. B. Active promoters give rise to false positive ‘phantom peaks’ in ChIP-seq experiments. Nucleic Acids Res. 43, 6959–6968 (2015). sourcePartly supported

  The Discussion reports that some ChIP-seq enrichments may be false-positive Phantom Peaks and says it cannot determine which overlapping signals represent genuine binding. The cited paper does not assess candidate enhancers identified solely by biochemical marks or estimate what proportion of them are technical false positives. The separate claim about enhancer-like features without an effect on cis-gene expression is attached to citation 133, not this source. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [133] Diao, Y. et al. A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening. Genome Res. 26, 397–405 (2016). sourceSupported

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

Many enhancers are spatially proximate to their target promoter in 3D, but exceptions have been described.

“Many enhancers are spatially proximate to their target promoter in 3D109,110, but exceptions have been described111-114.”

• cites [109] Rao, S. S. P. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014). sourceSupported
• cites [110] Weintraub, A. S. et al. YY1 Is a structural regulator of enhancer-promoter loops. Cell 171, 1573–1588.e28 (2017). sourceSupported
• cites [111] Williamson, I. et al. Anterior–posterior differences in HoxD chromatin topology in limb development. Development 139, 3157–3167 (2012). sourceSupported
• cites [112] Ghavi-Helm, Y. et al. Enhancer loops appear stable during development and are associated with paused polymerase. Nature 512, 96–100 (2014). sourceNot checked — no full text of the source
• cites [113] Benabdallah, N. S. et al. Decreased enhancer-promoter proximity accompanying enhancer activation. Mol. Cell 76, 473–484 (2019). sourceSupported
• cites [114] Alexander, J. M. et al. Live-cell imaging reveals enhancer-dependent Sox2 transcription in the absence of enhancer proximity. eLife 8, e41769 (2019). sourceSupported

May combine two claims.

eQTL studies can provide in vivo validation of distally located candidate enhancers while linking them to their target genes.

“The eQTL framework is very powerful, and for variants residing within distally located candidate enhancers, can provide in vivo validation of those enhancers while also linking them to their target genes136.”

• cites [136] GTEx Consortium et al. Genetic effects on gene expression across human tissues. Nature 550, 204–213 (2017). sourcePartly supported

  In “Functional characterization of cis-eQTLs” (page unavailable), the paper reports that eVariants are enriched in predicted enhancers and that more distant secondary eVariants show increased enhancer enrichment and contact with target-gene promoters. This supports using eQTLs to connect candidate enhancer regions with genes, but the passage does not establish in vivo validation of individual candidate enhancers. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

May combine two claims.

The eQTL framework has clear limitations, including reliance on naturally occurring human genetic variation, linkage disequilibrium, and restriction to cell types and tissues practically obtainable from large numbers of individuals.

“On the other hand, the framework has clear limitations, including its reliance on naturally occurring human genetic variation (most enhancers do not harbour common variants that substantially perturb their activity), linkage disequilibrium [G] (multiple variants in a haplotype block may equivalently explain an association), and restriction to cell types and tissues that can be practically obtained”

• cites [137] Strober, B. J. et al. Dynamic genetic regulation of gene expression during cellular differentiation. Science 364, 1287–1290 (2019). sourcePartly supported

  In the paper’s main text (indexed under Abstract; page unavailable), two variants in strong linkage disequilibrium are reported as both associated with SCN5A expression. This illustrates the linkage underlying the cited limitation, but the paper does not say that the variants equivalently explain the association or explicitly identify linkage disequilibrium as a limitation of eQTL analysis. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [138] van der Wijst, M. G. P. et al. Single-cell RNA sequencing identifies celltype-specific cis-eQTLs and co-expression QTLs. Nat. Genet. 50, 493–497 (2018). sourcePartly supported

  The main text (page unavailable) reports near-perfect linkage disequilibrium between an eQTL SNP and a diabetes-associated SNP, supporting the cited point that linkage disequilibrium is relevant to interpreting associations. It does not state that multiple variants in a haplotype block may equivalently explain an association. The Online Methods describe analysis of common variants in donor blood cells, but do not establish the claim about most enhancers or a general restriction to practically obtainable tissues. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

• AssumptionRests on an assumption: Most enhancers do not harbour common variants that substantially perturb their activity.

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

A major component of the heritability of nearly all common diseases partitions to regions with enhancer-like signatures, particularly in disease-relevant cell types.

“It is now widely recognized that a major component of the heritability of nearly all common diseases partitions to these regions, and in particular to regions that have enhancer-like signatures in disease-relevant cell types65.”

• cites [65] Maurano, M. T. et al. Systematic localization of common disease-associated variation in regulatory DNA. Science 337, 1190–1195 (2012). sourcePartly supported

  The section “Disease- and trait-associated variants are concentrated in regulatory DNA” (page unavailable) reports enrichment of GWAS-associated variants in DNase I hypersensitive sites across many diseases and traits. “De novo identification of pathogenic cell types” reports enrichment in particular cell types for Crohn’s disease, multiple sclerosis, and a cardiac trait. The paper does not quantify the share of heritability partitioning to these regions, establish this for nearly all common diseases, or show that the enriched regions specifically have enhancer-like signatures. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

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

Enhancers may have complex relationships with promoters, including feedback loops or competition with neighbouring genes.

“Enhancers may have complex relationships with promoters, including feedback loops or competition with neighbouring genes121,122.”

• cites [121] Cho, S. W. et al. Promoter of lncRNA Gene PVT1 Is a tumor-suppressor DNA boundary element. Cell 173, 1398–1412.e22 (2018). sourcePartly supported

  The Results section reports that PVT1 enhancers preferentially contact the PVT1 promoter, but shift toward the neighbouring MYC promoter when PVT1 is silenced; the Discussion describes this as promoter competition. Our copy does not describe feedback loops between enhancers and promoters, so it supports only the competition part of the claim. No page number is available in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [122] Cinghu, S. et al. Intragenic enhancers attenuate host gene expression. Mol. Cell 68, 104–117.e6 (2017). sourcePartly supported

  The Discussion (page unavailable) proposes an intrinsic negative feedback mechanism between an intragenic enhancer and its host-gene promoter. It also reports that the enhancer can activate distant genes while attenuating its host gene, but it does not describe competition with neighbouring genes. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

There are multiple models for how enhancers interact with their target promoters, including tracking, linking/chaining, short- or long-range looping, transcription factory and hub/condensate models, more than one of which may be correct.

“Finally, there are multiple models for how enhancers interact with their target promoter(s), including tracking, linking/chaining, short- or long-range looping, transcription factory and hub/condensate models (reviewed in 102), more than one of which may be correct.”

• cites [102] Furlong, E. E. M. & Levine, M. Developmental enhancers and chromosome topology. Science 361, 1341–1345 (2018). sourceSupported

May combine two claims.

This is not an inexorable chain of events; for example, stimulus-responsive enhancers may exhibit open chromatin and 3D interactions with their promoters before activation.

“However, it should be emphasized that this is not an inexorable chain of events. For example, stimulus-responsive enhancers may exhibit open chromatin and 3D interactions with their promoters before activation92,93.”

• cites [92] Simeonov, D. R. et al. Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature 549, 111–115 (2017). sourcePartly supported

  The paper reports that stimulation-responsive enhancers can be identified in unstimulated cells and that IL2RA enhancer sites overlap accessible chromatin and elements that loop to the promoter (Abstract, p. unavailable; Extended Data Figure 5, p. unavailable). It does not establish that chromatin accessibility and promoter interactions were both present before activation in the same cells; that timing is the unsupported part. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [93] Ray, J. et al. Chromatin conformation remains stable upon extensive transcriptional changes driven by heat shock. Proc. Natl Acad. Sci. USA 116, 19431–19439 (2019). sourcePartly supported

  The section “Pre-programmed chromatin architecture is conserved across metazoans” (page unavailable) reports that contacts between heat-shock-regulated genes and distal HSF1-bound elements were preformed before heat shock, supporting the pre-activation 3D-interaction claim. Our copy does not establish that those enhancers had open chromatin before activation; the Introduction discusses changes in chromatin accessibility upon heat shock instead. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

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

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

• cites [87] Hnisz, D., Day, D. S. & Young, R. A. Insulated neighborhoods: structural and functional units of mammalian gene control. Cell 167, 1188–1200 (2016). sourceSupported
• cites [88] Maricque, B. B., Chaudhari, H. G. & Cohen, B. A. A massively parallel reporter assay dissects the influence of chromatin structure on cis-regulatory activity. Nat. Biotechnol. 37, 90–95 (2019). sourceSupported
• cites [89] Heinz, S., Romanoski, C. E., Benner, C. & Glass, C. K. The selection and function of cell type-specific enhancers. Nat. Rev. Mol. Cell Biol. 16, 144–154 (2015). sourceSupported

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

At least a few enhancers reside at great distances from their target gene (e.g. ZRS 1 Mb from Shh, MYC enhancer 1.7 Mb downstream), although most are much more proximal.

“At least a few enhancers reside at great distances from their target gene (e.g. the ZRS enhancer located 1 Mb from the Shh gene118, and a MYC enhancer located 1.7 Mb downstream119), although most are much more proximal to their target promoters72.”

• cites [118] Lettice, L. A. et al. 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 (2003). sourceSupported
• cites [119] Bahr, C. et al. Author correction: A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies. Nature 558, E4 (2018). sourceCould not verify

  The cited source is an Author Correction (Nature 558, E4). Its correction notice discusses a duplicated reference, not the location of a MYC enhancer. Searches for “MYC enhancer located 1.7 Mb downstream” and “1.7 Mb downstream of Myc enhancer” found no passage in our copy addressing that distance. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [72] Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell 176, 377–390 (2019). sourceSupported

May combine two claims.

The activity of individual enhancers is generally cell-type-specific or condition-specific, and this specificity is a function of the expression levels of TFs able to bind to it.

“Regardless of any such mechanistic heterogeneity, a common property is that the activity of individual enhancers is generally cell-type-specific or even condition-specific99, and this specificity is a function of the expression levels of the TFs that are able to bind to it89.”

• cites [99] Gosselin, D. et al. An environment-dependent transcriptional network specifies human microglia identity. Science 356, eaal3222 (2017). sourceSupported
• cites [89] Heinz, S., Romanoski, C. E., Benner, C. & Glass, C. K. The selection and function of cell type-specific enhancers. Nat. Rev. Mol. Cell Biol. 16, 144–154 (2015). sourceSupported

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

Genes can be affected by a single enhancer or multiple enhancers acting in concert; conversely, individual enhancers can regulate multiple genes.

“Genes can be affected by a single enhancer or multiple enhancers acting in concert115; conversely, individual enhancers can regulate multiple genes72.”

• cites [115] Osterwalder, M. et al. Enhancer redundancy provides phenotypic robustness in mammalian development. Nature 554, 239–243 (2018). sourceSupported
• cites [72] Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell 176, 377–390 (2019). sourceSupported

May combine two claims.

Correlative approaches can partially overcome this, but such links remain inferential.

“To some degree this can be overcome by correlative approaches (e.g. correlating open chromatin status between promoters and enhancers across large numbers of cell types) but such links remain inferential45,134,135.”

• cites [45] Thurman, R. E. et al. The accessible chromatin landscape of the human genome. Nature 489, 75–82 (2012). sourceSupported
• cites [134] Pliner, H. A. et al. Cicero predicts cis-regulatory DNA Interactions from single-cell chromatin accessibility data. Mol. Cell 71, 858–871.e8 (2018). sourcePartly supported

  The Methods describe Cicero inferring promoter–distal-element links from correlations in chromatin accessibility, and the Limitations section explicitly calls those links putative and says regulatory influence requires downstream experimentation. The cited paper analyzes single cells during myoblast differentiation, however; it does not establish the sentence’s specific example of correlations across large numbers of cell types. No page numbers are available in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [135] Ernst, J. et al. Mapping and analysis of chromatin state dynamics in nine human cell types. Nature 473, 43–49 (2011). sourcePartly supported

  The Results section, “Correlations in activity profiles link enhancers to target genes” (page not provided), says the authors correlated enhancer activity with gene expression across cell types to link enhancers probabilistically to target genes. The Discussion calls these interactions “inferred,” supporting the claim that the links remain inferential. This paper does not describe the specific example of correlating open-chromatin status between promoters and enhancers. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

May combine two claims.

Not all distal conserved elements are detectably enhancers, and far more of the gene-distal non-coding genome is annotated as a regulatory element than is conserved.

“Not all distal conserved elements are detectably enhancers39 and far more of the gene-distal non-coding genome is annotated as a regulatory element than is conserved60,104.”

• cites [39] Pennacchio, L. A. et al. In vivo enhancer analysis of human conserved non-coding sequences. Nature 444, 499–502 (2006). sourceSupported
• cites [60] ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012). This report summarizes the wealth of data generated by the ENCODE Consortium. Hundreds of genome-wide datasets are used across hundreds of cell types and tissues to ascribe a function to the majority of the genome via biochemical annotation. sourcePartly supported

  The citation to this paper is attached to the second clause, not the claim about distal conserved elements and enhancers. The Summary of ENCODE-identified elements (page unavailable) reports that a conservative measure of regulatory-associated bases covers about twice as much of the genome as estimated pan-mammalian constraint, and the Conclusions say many regulatory elements are not constrained. Those passages support the broad comparison, but do not establish it specifically for the gene-distal non-coding genome. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [104] Lindblad-Toh, K. et al. A high-resolution map of human evolutionary constraint using 29 mammals. Nature 478, 476–482 (2011). sourcePartly supported

  The Detection of constrained sequence section reports that constrained elements span 4.2% of the genome. The Chromatin signatures section reports regulatory-associated chromatin states among noncoding constrained elements, but does not directly establish that far more of the gene-distal noncoding genome is annotated as regulatory than conserved. The first clause is attached to a different citation and is not assessed here; page numbers are unavailable in our copy. (LLM, conf=0.74)

  Experimental — accuracy not yet measured

May combine two claims.

Biochemical annotations fail to specify which gene(s) a putative enhancer regulates, let alone the degree of activation conferred.

“Third, biochemical annotations fail to specify which gene(s) a putative enhancer regulates, let alone the degree of activation conferred78,79.”

• cites [78] Ghavi-Helm, Y. et al. Highly rearranged chromosomes reveal uncoupling between genome topology and gene expression. Nat. Genet. 51, 1272–1282 (2019). sourceCould not verify

  The Abstract and Discussion report that changes in chromatin topology do not reliably predict gene-expression changes. The Online Methods describe using DNase hypersensitivity and enhancer annotations, but our copy does not assess whether biochemical annotations identify an enhancer’s target genes or quantify the activation it confers. Searches for those specific points found no deciding passage; page numbers were unavailable in our copy. (LLM, conf=0.73)

  Experimental — accuracy not yet measured
• cites [79] Zabidi, M. A. et al. Enhancer-core-promoter specificity separates developmental and housekeeping gene regulation. Nature 518, 556–559 (2015). STARR-seq utilizing different classes of promoters in D. melanogaster shows that enhancers often do not work in a promoter-generic fashion. sourceCould not verify

  The Methods section on enhancer-to-gene assignment (page unavailable) describes proximity-based assignment strategies, and the reported STARR-seq results measure promoter-specific activation. Neither passage assesses whether biochemical annotations can identify an enhancer’s target genes or quantify its activation. Searches for biochemical annotations, target-gene prediction, and quantitative activity found no passage making that comparison. (LLM, conf=0.69)

  Experimental — accuracy not yet measured

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

Enhancers regulating housekeeping genes may act via distinct sets of TFs and cofactors than enhancers regulating developmentally specific genes.

“Enhancers regulating housekeeping genes may act via a distinct sets of TFs and cofactors than enhancers regulating developmentally specific genes79,120.”

• cites [79] Zabidi, M. A. et al. Enhancer-core-promoter specificity separates developmental and housekeeping gene regulation. Nature 518, 556–559 (2015). STARR-seq utilizing different classes of promoters in D. melanogaster shows that enhancers often do not work in a promoter-generic fashion. sourceSupported
• cites [120] Haberle, V. et al. Transcriptional cofactors display specificity for distinct types of core promoters. Nature 570, 122–126 (2019). sourceSupported

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

Through projects such as ENCODE, these annotations underlie the classification of over one million candidate regulatory elements in the human genome as potential enhancers in one or more cell types.

“Through projects such as ENCODE, these annotations underlie the classification of over one million candidate regulatory elements in the human genome as potential enhancers in one or more cell types60,64.”

• cites [60] ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012). This report summarizes the wealth of data generated by the ENCODE Consortium. Hundreds of genome-wide datasets are used across hundreds of cell types and tissues to ascribe a function to the majority of the genome via biochemical annotation. sourcePartly supported

  The Abstract (page unavailable) says ENCODE classified 399,124 regions with enhancer-like features, supporting the claim that its annotations identify candidate enhancers. It does not support the attributed count of over one million potential enhancers; the paper's Genome-wide integration section also describes its enhancer classifications as predictions. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [64] ENCODE Project. SCREEN: search candidate regulatory elements by ENCODE. ENCODE Project http://screen.encodeproject.org/index/about (2019).Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Histone modification and cofactor binding are enriched but not completely predictive of enhancer activity.

“Although enriched, histone modification and cofactor binding is not completely predictive of enhancer activity54,58,72,77,105.”

• cites [54] Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009). sourcePartly supported

  The Discussion reports that p300 cofactor binding predicted tissue-specific enhancer activity in 80% of tested cases, supporting enrichment without perfect prediction. The paper does not establish the same claim for histone modifications; its Discussion mentions epigenetic marks generally but does not test their predictive accuracy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [58] Andersson, R. et al. An atlas of active enhancers across human cell types and tissues. Nature 507, 455–461 (2014). This article reports an integrated analysis of cap analysis of gene expression (CAGE) datasets across hundreds of cell types and tissues performed to generate biochemical annotations of thousands of cell type-specific enhancers by their signatures of bidirectional transcription. sourcePartly supported

  The Results section (“Bidirectional pairs of capped RNAs identify active enhancers”; page unavailable) reports that untranscribed candidate enhancers defined by histone modifications were much less likely to show reporter activity, supporting the point that those marks do not fully predict activity. The Methods section (“Correlation between ENCODE epigenomic data and CAGE-defined enhancers”; page unavailable) says the authors assessed P300 and transcription-factor binding, but the passages do not establish the claim that cofactor binding is enriched yet incompletely predictive. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [72] Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell 176, 377–390 (2019). sourceSupported
• cites [77] Wang, X. et al. High-resolution genome-wide functional dissection of transcriptional regulatory regions and nucleotides in human. Nat. Commun. 9, 5380 (2018). sourcePartly supported

  In Results, “HiDRA elements are enriched in promoters and enhancers” (page unavailable), the paper reports enrichment of active-enhancer-associated histone marks in active fragments. The Introduction and “Regulatory activity outside promoters and enhancers” explain why epigenomic marks do not fully predict reporter activity. The paper discusses enrichment of transcription-factor motifs and binding, but the available passages do not establish the claim specifically for cofactor binding. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [105] Fulco, C. P. et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science 354, 769–773 (2016). sourcePartly supported

  The supplied Abstract passages report that H3K27ac signal distinguished most functional MYC enhancers but did not rank their effects correctly, supporting the point that a histone modification is informative but not fully predictive. They also report transcription-factor binding at the seven enhancers, but do not establish that cofactor binding is enriched or test its predictive value. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

May combine two claims.

A major advantage of MPRAs is that sequences to be tested can simply be synthesized, enabling straightforward saturation mutagenesis and programming synthetic enhancers.

“A major advantage of MPRAs is that sequences to be tested can simply be synthesized, enabling straightforward saturation mutagenesis [G] of enhancers67,184,185 as well as programming synthetic enhancers to inform modelling of their properties186,187.”

• cites [67] Patwardhan, R. P. et al. Massively parallel functional dissection of mammalian enhancers in vivo. Nat. Biotechnol. 30, 265–270 (2012). Along with Melnikov et al. (2012), this study is the first application of an MPRA to enhancer sequences. sourceSupported
• cites [184] Melnikov, A. et al. Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nat. Biotechnol. 30, 271 (2012). Along with Patwardhan et al. (2012), this study shows the first application of an MPRA to variants of enhancer sequences, in addition to coining the term ‘massively parallel reporter assay’. sourceSupported
• cites [185] Kircher, M. et al. Saturation mutagenesis of twenty disease-associated regulatory elements at single base-pair resolution. Nat. Commun. 10, 3583 (2019). sourcePartly supported

  The Results section, “Construction of saturation mutagenesis libraries,” confirms that the authors used MPRAs to test mutations in regulatory elements, including enhancers. But their Methods describe generating sequence variants by error-prone PCR, not by synthesizing the sequences to be tested. The separate claim about programming synthetic enhancers is attached to other citations in the citing sentence. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [186] Grossman, S. R. et al. Systematic dissection of genomic features determining transcription factor binding and enhancer function. Proc. Natl Acad. Sci. USA 114, E1291–E1300 (2017). sourceSupported
• cites [187] Smith, R. P. et al. Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model. Nat. Genet. 45, 1021–1028 (2013). sourceSupported

May combine two claims.

The authors developed a related approach introducing ~28 gRNAs per cell, enabling 5,779 candidate enhancers to be evaluated in a single experiment.

“As scRNA-seq is costly and individual enhancers most likely only regulate one or a few genes in cis, we developed a related approach wherein ~28 gRNAs were introduced per cell, enabling 5,779 candidate enhancers to be evaluated in a single experiment72.”

• cites [72] Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell 176, 377–390 (2019). sourceSupported

• AssumptionRests on an assumption: Individual enhancers most likely only regulate one or a few genes in cis.

May combine two claims.

Further challenges for non-coding variant interpretation include linkage disequilibrium, the possibility that more than one variant contributes to a given association, the need to match cell type with disease, and unclear mechanisms by which non-coding variants exert effects on disease risk.

“Further challenges include linkage disequilibrium, the possibility that more than one variant contributes to a given association, the need to match the cell type in which functional characterization and/or biochemical annotation is being carried out with the disease in question, and the fact that the mechanisms by which non-coding variants exert their effects on disease risk remain unclear223.”

• cites [223] Boyle, E. A., Li, Y. I. & Pritchard, J. K. An expanded view of complex traits: from polygenic to omnigenic. Cell 169, 1177–1186 (2017). 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

May combine two claims.

At least as usually implemented, MPRAs remain limited by several factors, including length constraints and cost of DNA synthesis or complexity of shotgun libraries, the confounding effect of the reporter's minimal promoter, and the use of episomes whose chromatin may differ from that of the genome.

“However, at least as they are usually implemented, MPRAs remain limited by several factors, including the length constraints and cost of DNA synthesis or the immense complexity of shotgun libraries, the confounding effect of the reporter’s minimal promoter, and the use of episomes whose chromatin may have different properties than that of the genome189.”

• cites [189] Gilbert, N. & Allan, J. Supercoiling in DNA and chromatin. Curr. Opin. Genet. Dev. 25, 15–21 (2014). sourceCould not verify

  The “Supercoiling domains” and “Effects of supercoiling on DNA” sections discuss DNA topology and chromatin, not limitations of massively parallel reporter assays. Searches for MPRAs, episomes, minimal promoters, DNA synthesis costs, and shotgun libraries found no passage addressing the attributed claim. (LLM, conf=0.75)

  Experimental — accuracy not yet measured

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

Activating domains dCas9-VPR or dCas9-p300 have been used to scan for poised enhancers in CRISPRa.

“Activating domains (dCas9–VPR or dCas9–p300) have also been used to scan for poised enhancers in an approach termed CRISPR activation or ‘CRISPRa’92,208(Figure 2d).”

• cites [92] Simeonov, D. R. et al. Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature 549, 111–115 (2017). sourcePartly supported

  The Abstract reports using CRISPRa to scan genomic regions and identify stimulation-responsive enhancers in unstimulated cells. But it specifies dCas9-VP64, not either dCas9-VPR or dCas9-p300; the Methods section on cell generation likewise identifies dCas9-VP64. No page numbers are available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [208] Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561 (2017). sourcePartly supported

  The Results section (“Epigenetic Activation Reveals Gene Regulatory Elements”; page unavailable) describes a dCas9-p300 gain-of-function screen for regulatory elements near HER2, and the Discussion says dCas9-p300 can activate expression from distal elements. Our copy does not describe those elements as poised enhancers or report using dCas9-VPR. The cited paper therefore supports the dCas9-p300 screening component, but not the full wording of the claim. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

The vast majority of biochemically nominated candidate enhancers, when perturbed by CRISPRi, do not result in detectable changes in expression of genes located in cis.

“How are we to explain the fact that the vast majority of biochemically nominated candidate enhancers, when perturbed by CRISPRi, do not result in detectable changes in expression of genes located in cis72?”

• cites [72] Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell 176, 377–390 (2019). sourceSupported

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

Single-cell ATAC-seq has enabled in vivo profiling of accessible chromatin at the scale of a whole organism.

“For example, single-cell ATAC-seq has enabled the in vivo profiling of accessible chromatin at the scale of a whole organism82,83.160,161.”

• cites [82] Cusanovich, D. A. et al. Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing. Science 348, 910–914 (2015). sourcePartly supported

  The Abstract describes ATAC-seq-based chromatin accessibility profiling in more than 15,000 single cells. The experiments described in our copy use mixtures of cultured human and mouse cell lines, not in vivo profiling across a whole organism; the paper discusses broader tissue characterization only as a future possibility. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [83] Buenrostro, J. D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486–490 (2015). sourcePartly supported

  The Main section (page unavailable) reports that scATAC-seq maps accessible chromatin in individual cells. Its experiments used captured cells from cell lines; the paper does not report in vivo profiling at the scale of a whole organism. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [160] Cusanovich, D. A. et al. A single-cell atlas of in vivo mammalian chromatin accessibility. Cell 174, 1309–1324.e18 (2018). sourceSupported
• cites [161] Cusanovich, D. A. et al. The cis-regulatory dynamics of embryonic development at single-cell resolution. Nature 555, 538–542 (2018). sourceSupported

May combine two claims.

CRISPR technology has recently made it much more straightforward to delete genomic sequences in the mouse, enabling new insights into enhancer redundancy and consequences of disrupting TADs.

“Second, CRISPR technology has recently made it much more straightforward to delete genomic sequences in the mouse, enabling new insights into aspects such as enhancer redundancy115 and the consequences of disrupting TADs220.”

• cites [115] Osterwalder, M. et al. Enhancer redundancy provides phenotypic robustness in mammalian development. Nature 554, 239–243 (2018). sourceSupported
• cites [220] Lupiáñez, D. G. et al. Disruptions of topological chromatin domains cause pathogenic rewiring of gene–enhancer interactions. Cell 161, 1012–1025 (2015). This article demonstrates that rearrangement across TAD boundaries can cause a pathogenic phenotype in humans; the study also uses CRISPR–Cas9 to effectively reconstruct human patient genome rearrangements in mouse models. sourceSupported

May combine two claims.

Most MPRAs only test for enhancer activity using a single promoter, or at best a handful, which could contribute to a high false negative rate.

“The fact that most MPRAs only test for enhancer activity using a single promoter, or at best a handful79, could contribute to a high false negative rate.”

• cites [79] Zabidi, M. A. et al. Enhancer-core-promoter specificity separates developmental and housekeeping gene regulation. Nature 518, 556–559 (2015). STARR-seq utilizing different classes of promoters in D. melanogaster shows that enhancers often do not work in a promoter-generic fashion. sourcePartly supported

  The study tested enhancer activity with two core promoters, and its results show that many enhancers were much more active with one than the other (Abstract; page not provided). It therefore supports the relevance of testing more than one promoter, but does not establish the cited claim that most MPRAs use only one or a handful of promoters. The possible false-negative consequence is framed as the citing authors’ inference. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

May combine two claims.

MPRAs have been applied to assess biochemically annotated candidate enhancers, candidate enhancers harbouring variants that potentially mediate eQTLs, and even scans of the entire human genome.

“MPRAs have been applied to assess biochemically annotated candidate enhancers77,176-178, candidate enhancers harbouring variants that potentially mediate eQTLs179-182, and even scans of the entire human genome108,183.”

• cites [77] Wang, X. et al. High-resolution genome-wide functional dissection of transcriptional regulatory regions and nucleotides in human. Nat. Commun. 9, 5380 (2018). sourcePartly supported

  This source is cited for the genome-wide-scan portion of the sentence. The Introduction reports a genome-wide HiDRA assay, but says its approximately 7 million tested fragments were preferentially selected from accessible chromatin in GM12878 cells. The Results (‘HiDRA experimental method overview’; page unavailable) likewise say the library targeted accessible DNA rather than randomly sampling the entire genome, so the claim overstates its scope. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [176] Vockley, C. M. et al. Direct GR binding sites potentiate clusters of TF binding across the human genome. Cell 166, 1269–1281.e19 (2016). sourceSupported
• cites [177] Vanhille, L. et al. High-throughput and quantitative assessment of enhancer activity in mammals by CapStarr-seq. Nat. Commun. 6, 6905 (2015). sourceSupported
• cites [178] Klein, J. C., Keith, A., Agarwal, V., Durham, T. & Shendure, J. Functional characterization of enhancer evolution in the primate lineage. Genome Biol. 19, 99 (2018). sourceSupported
• cites [179] Ulirsch, J. C. et al. Systematic functional dissection of common genetic variation affecting red blood cell traits. Cell 165, 1530–1545 (2016). sourcePartly supported

  The Introduction and Experimental Procedures describe an MPRA screening variants linked to red-blood-cell GWAS hits, and the Discussion reports that some tested variants lie within endogenous enhancers. The paper does not say these candidates were selected because they might mediate eQTLs; it mentions eQTL studies in the Introduction as a separate way to identify target genes. This verdict concerns the enhancer-variant clause to which this source is attached, not the separately cited whole-genome scans. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [180] Tewhey, R. et al. Direct identification of hundreds of expression-modulating variants using a multiplexed reporter assay. Cell 172, 1132–1134 (2018). sourceNot checked — no full text of the source
• cites [181] Vockley, C. M. et al. Massively parallel quantification of the regulatory effects of noncoding genetic variation in a human cohort. Genome Res. 25, 1206–1214 (2015). sourceSupported
• cites [182] Klein, J. C. et al. Functional testing of thousands of osteoarthritis-associated variants for regulatory activity. Nat. Commun. 10, 2434 (2019). sourcePartly supported

  The Introduction says MPRAs have been used to characterize variants underlying eQTLs, and the Results identify two tested osteoarthritis-associated SNPs as eQTLs. This paper’s own MPRA, however, selected variants associated with osteoarthritis GWAS signals rather than candidate enhancers selected for potentially mediating eQTLs. The cited paper therefore supports the general eQTL application, but not that more specific description of its screen. (LLM, conf=0.81)

  Experimental — accuracy not yet measured
• cites [108] Muerdter, F. et al. Resolving systematic errors in widely used enhancer activity assays in human cells. Nat. Methods 15, 141–149 (2018). sourceSupported
• cites [183] Liu, Y. et al. Functional assessment of human enhancer activities using whole-genome STARR-sequencing. Genome Biol. 18, 219 (2017). sourceSupported

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

The initial CRISPR screens of regulatory elements delivered an individual gRNA per cell.

“The initial CRISPR screens of regulatory elements delivered an individual gRNA per cell195.”

• cites [195] Canver, M. C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197 (2015). This study is the first CRISPR-based screen to identify functional non-coding sequence within an enhancer. sourceSupported

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

A clear strength of MPRAs is their ability to simultaneously test large numbers of sequences for regulatory activity via a relatively straightforward, widely accessible toolkit.

“A clear strength of MPRAs is their ability to simultaneously test large numbers of sequences for regulatory activity via a relatively straightforward, widely accessible toolkit (i.e. oligonucleotide synthesis, molecular biology, cell culture, and sequencing)175.”

• cites [175] Gasperini, M., Starita, L. & Shendure, J. The power of multiplexed functional analysis of genetic variants. Nat. Protoc. 11, 1782–1787 (2016). sourcePartly supported

  The Abstract says massively parallel reporter assays map regulatory sequence–function relationships and can assess hundreds to thousands of variants in one experiment. The Construction of a variant library and Sequencing to count variant frequency sections describe oligonucleotide libraries and sequencing, but the paper does not establish that the toolkit is relatively straightforward or widely accessible; it describes library construction as challenging. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

CRISPRi has been used in multiple single-gene screens of regulatory elements.

“CRISPRi has subsequently been used in multiple single-gene screens of regulatory elements105,208,209.”

• cites [105] Fulco, C. P. et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science 354, 769–773 (2016). sourceSupported
• cites [208] Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561 (2017). sourceSupported
• cites [209] Fulco, C. P. et al. Activity-by-contact model of enhancer–promoter regulation from thousands of CRISPR perturbations. Nat. Genet. 51, 1664–1669 (2019). sourceSupported

May combine two claims.

Automatic sequence-based enhancer annotation is helpful but performs modestly for predicting enhancers and the contexts in which they are active.

“Consequent to these limitations, automatic sequence-based enhancer annotation is helpful and worthwhile129, but performs modestly for predicting enhancers and the contexts in which they are active.”

• cites [129] Van Loo, P. & Marynen, P. Computational methods for the detection of cis-regulatory modules. Brief. Bioinform. 10, 509–524 (2009). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S2 (checkable).

In light of this heterogeneity, a 'one-size-fits-all' set of annotations to catalogue enhancers seems problematic.

“In light of this heterogeneity, a ‘one-size-fits-all’ set of annotations to catalogue enhancers seems problematic.”

Probably not a substantial claim: may not pass S2 (checkable).

The field lacks a clear framework for identifying enhancers, with different subfields using different definitions and criteria.

“As a consequence, the field lacks a clear framework for identifying enhancers, with different subfields (e.g. biochemistry, genomics, etc.) using different definitions and criteria, even though we are all ostensibly studying the same underlying biological phenomenon.”

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

Enhancers are widely presumed to be the most numerically prevalent class of non-coding DNA regulatory element.

“Enhancers are only one class of non-coding DNA regulatory element, although they are widely presumed to be the most numerically prevalent (Box 2).”

• AssumptionThe authors state this as an assumption: Enhancers are the most numerically prevalent class of non-coding DNA regulatory element.

May combine two claims.

TF sequence motifs alone are poorly predictive, as only a small fraction of potential TF binding sites in the genome are typically bound in a cell type where the TF is expressed.

“TF sequence motifs alone are poorly predictive, as only a small fraction of potential TF binding sites in the genome are typically bound in a cell type where the TF is expressed68.”

• cites [68] Spitz, F. & Furlong, E. E. M. Transcription factors: from enhancer binding to developmental control. Nat. Rev. Genet. 13, 613–626 (2012). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

There may be in vivo enhancers that are non-canonically marked or have contextual dependencies not maintained in a reporter assay.

“Finally, there may be in vivo enhancers that are non-canonically marked, or that have contextual dependencies that are not maintained in a reporter assay.”

Probably not a substantial claim: may not pass S4 (based).

There may be sequences that activate transcription in a reporter assay but do not meaningfully do so in vivo.

“There may be sequences that activate transcription in the context of a reporter assay but do not meaningfully do so in vivo.”

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

Our present understanding is that enhancers are bound by cell-type-specific TFs, associated with open chromatin, and flanked by histones carrying H3K27ac and/or H3K4me1 modifications.

“Our present understanding is that enhancers are bound by cell-type-specific TFs, are associated with regions of open chromatin, and are flanked by histones carrying H3K27ac and/or H3K4me1 modifications.”

• cites [68] Spitz, F. & Furlong, E. E. M. Transcription factors: from enhancer binding to developmental control. Nat. Rev. Genet. 13, 613–626 (2012). sourceNot checked — no full text of the source
• cites [69] Shlyueva, D., Stampfel, G. & Stark, A. Transcriptional enhancers: from properties to genome-wide predictions. Nat. Rev. Genet. 15, 272–286 (2014). sourceNot checked — no full text of the source

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

The recruited TFs directly or indirectly facilitate chromatin remodelling and recruitment of the basal transcriptional machinery at the promoter, thereby enhancing transcription.

“The recruited TFs directly or indirectly (e.g. via a co-activator) facilitate chromatin remodelling and recruitment of the basal transcriptional machinery at the promoter (Figure 1a), thereby enhancing transcription91.”

• cites [91] Thanos, D. & Maniatis, T. Virus induction of human IFNβ gene expression requires the assembly of an enhanceosome. Cell 83, 1091–1100 (1995). sourceNot checked — no full text of the source

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

Enhancers interact with their cognate promoters in 3D space and can be latent, primed, or active.

“They interact with their cognate promoters in 3D space, and can be latent, primed, or active68,69.”

• cites [68] Spitz, F. & Furlong, E. E. M. Transcription factors: from enhancer binding to developmental control. Nat. Rev. Genet. 13, 613–626 (2012). sourceNot checked — no full text of the source
• cites [69] Shlyueva, D., Stampfel, G. & Stark, A. Transcriptional enhancers: from properties to genome-wide predictions. Nat. Rev. Genet. 15, 272–286 (2014). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

New data types based on MPRAs or genome editing are complicating the picture, particularly as biochemical annotations and functional data are not always in agreement.

“New data types, e.g. based on massively parallel reporter assays (MPRAs) or genome editing, are further complicating the picture, particularly as biochemical annotations and functional data are not always in agreement.”

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

The human gene catalogue is essentially complete, but there is no equivalently vetted inventory of bona fide human enhancers.

“The human gene catalogue is essentially complete, but we lack an equivalently vetted inventory of bona fide human enhancers.”

• AssumptionRests on an assumption: The human gene catalogue is essentially complete.

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

Methods for profiling chromatin state in single cells are advancing quickly and have the potential to overcome the challenge of cellular heterogeneity.

“However, methods for profiling chromatin state in single cells are advancing quickly and have the potential to overcome this challenge.”

May combine two claims.

The presence of a motif for an expressed TF does not mean that it is bound, and even if it is, not all binding is functional.

“Furthermore, the presence of a motif for an expressed TF does not mean that it is bound, and even if it is, not all binding is functional68.”

• cites [68] Spitz, F. & Furlong, E. E. M. Transcription factors: from enhancer binding to developmental control. Nat. Rev. Genet. 13, 613–626 (2012). sourceNot checked — no full text of the source

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

The mechanistic details of how enhancers activate expression of their target genes have yet to be fully worked out.

“Although we have many clues, the mechanistic details of how enhancers activate expression of their target genes have yet to be fully worked out (Figure 1a).”

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

Over one million candidate cis-regulatory elements with enhancer-like signatures have been catalogued, collectively spanning ~16% of the human genome.

“This has resulted in the cataloguing of over one million candidate cis-regulatory elements with enhancer-like signatures; these collectively span ~16% of the human genome64.”

• cites [64] ENCODE Project. SCREEN: search candidate regulatory elements by ENCODE. ENCODE Project http://screen.encodeproject.org/index/about (2019).Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Hundreds-of-thousands of candidate enhancers have been nominated by biochemical annotations, but only a handful are validated and confidently linked to their target genes.

“Hundreds-of-thousands of candidate enhancers have been nominated by biochemical annotations; however, only a handful of these are validated and confidently linked to their target gene(s).”

Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

There is a tremendous amount still unknown about the repertoire of human enhancers, including where they reside, how they work, and what genes they mediate effects through.

“However, despite their clear importance to both basic and disease biology, there is a tremendous amount that we still do not understand about the repertoire of human enhancers, including where they reside, how they work, and what genes they mediate their effects through.”

Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

Our understanding of core enhancer characteristics, based largely on a few paradigmatic examples, is being challenged by studies suggesting a more heterogeneous landscape.

“Rather, our understanding of the core characteristics of enhancers, based largely on a few paradigmatic examples, is being challenged by studies that suggest a more heterogeneous landscape.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

eQTL studies may represent our only hope for comprehensively observing the consequences of human enhancer disruption, given the diversity of epigenomic contexts during development.

“On one hand, given the diversity of epigenomic contexts traversed during development, eQTL studies may represent our only hope for comprehensively observing the consequences of human enhancer disruption (as all engineered mutations will be in models such as cell lines, organoids, or mice).”

May combine two claims.

Synthetic CRISPRi or CRISPRa perturbations may lead to false positives or false negatives.

“This may lead to false positives (e.g. through spreading of KRAB repressive effects, or unnatural activation by VPR) or false negatives (e.g. an active enhancer that is not susceptible to CRISPRi-mediated inactivation).”

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

Single-cell methods may enable identification of candidate enhancers active in extremely specific developmental contexts or heterogeneously active within a single cell type.

“Single-cell methods may also enable the identification of candidate enhancers that appear to be active in extremely specific developmental contexts, or heterogeneously active within a single cell type.”

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

In a coding screen, NHEJ-induced indels at a single DSB are likely to cause a frameshift and complete loss of gene function.

“In a coding screen, the indels resulting from NHEJ at a single DSB are likely to result in a frameshift and the gene’s complete loss of function.”

May combine two claims.

In vivo reporter assays test elements outside of their native genomic context and do not link elements to their endogenous target genes.

“The disadvantages of in vivo reporter assays are similar to MPRAs, including that elements are tested outside of their native genomic context, and the elements are not linked to their endogenous target gene(s).”

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

Heterogeneous classes of data will be available for many non-coding variants but will not always agree.

“Heterogeneous classes of data will be available for many variants (e.g. biochemical annotations, molecular QTLs, computational predictions of variant effects, MPRAs, and CRISPR perturbation) but will not always agree.”

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

Wholesale deletions of candidate enhancers are unambiguously disruptive of a bounded region.

“By contrast, wholesale deletions of candidate enhancers are unambiguously disruptive of a bounded region.”

Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

It may simply not be realistic to test every candidate enhancer with every functional approach in every cell type of interest.

“A further challenge is that although technologies are rapidly improving, it may simply not be realistic to test every candidate enhancer with every functional approach in every cell type of interest.”

May combine two claims.

Relying on epigenetic perturbations rather than genetic ones bypasses many limitations of non-coding CRISPR screens, allowing all alleles in a given cell to be more consistently perturbed.

“Relying on epigenetic perturbations, rather than genetic ones, bypasses many of these limitations, e.g. allowing all alleles in a given cell to be more consistently perturbed.”

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

An MPRA tests the functional activity of thousands of candidate regulatory sequences in a single experiment.

“An MPRA tests the functional activity of thousands of candidate regulatory sequences in a single experiment.”

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

Practitioners of MPRAs, including the authors, typically fail to confirm that each 'positive' element fully meets the original Banerji definition (i.e. active in both orientations and from many positions).

“Practitioners of MPRAs, including ourselves, typically fail to confirm that each ‘positive’ element fully meets the original Banerji definition (i.e. active in both orientations and from many positions).”

May combine two claims.

The ability of single guide scans to fully tile a region is limited by the distribution of PAM sites and the non-random distribution of NHEJ-mediated mutations.

“In this vein, the ability of single guide scans to fully ‘tile’ a region is limited both by the distribution of protospacer adjacent motif [G] (PAM) sites and the non-random distribution of NHEJ-mediated mutations.”

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

Single-cell methods have the potential to replace conventional bulk 1D and 3D biochemical assays.

“Overall, single-cell methods have the potential to replace conventional bulk 1D and 3D biochemical assays.”

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

The first whole-transcriptome screen delivered one CRISPRi perturbation per cell, targeting 71 candidate enhancers across 7 genomic loci.

“The first such screen delivered one CRISPRi perturbation per cell, targeting 71 candidate enhancers across 7 genomic loci216.”

• cites [216] Xie, S., Duan, J., Li, B., Zhou, P. & Hon, G. C. Multiplexed engineering and analysis of combinatorial enhancer activity in single cells. Mol. Cell 66, 285–299.e5 (2017). This study reports the first use of single-cell RNA-seq to phenotype pools of candidate enhancer perturbations in a ‘whole-transcriptome’ screen. sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Putative enhancers identified by CRISPR screens may fail to show activity in MPRAs.

“On the other side of the balance sheet, putative enhancers identified by CRISPR screens may fail to show activity in MPRAs.”

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

Small indels are probably capable of disrupting TF binding sites within an enhancer only if they directly overlap the binding site.

“Although small indels are probably capable of disrupting TF binding sites within an enhancer, they might only do so if directly overlapping the binding site itself.”

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

All of the aforementioned methods (biochemical annotations, MPRAs, CRISPR screens, etc.) are performed in vitro on cell lines and are therefore only capable of accessing a limited number of biological contexts.

“All of the aforementioned methods (biochemical annotations, MPRAs, CRISPR screens, etc.) are performed in vitro on cell lines, and are therefore only capable of accessing a limited number of biological contexts.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

Many of the challenges highlighted here apply not only to candidate enhancers, but also to non-coding variants located within them.

“We note that many of the challenges highlighted here apply not only to candidate enhancers, but also to non-coding variants located within them.”

Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

Advances in scalable methods for biochemical annotation and functional characterization of regulatory elements are paving the way to a comprehensive catalogue of human enhancers.

“Advances in scalable methods for the biochemical annotation and functional characterization of regulatory elements are paving the way to a comprehensive catalogue of human enhancers.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

The proposed definition may not be easily adaptable to candidate enhancers that overlap promoters or protein-coding regions.

“Additionally, we also note that our definition may not be easily adaptable to candidate enhancers that overlap promoters or protein-coding regions.”

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

Like the biochemical annotations on which they are based, any such candidate links will still lack functional validation.

“However, like the biochemical annotations on which they are based, any such candidate links will still lack functional validation.”

May combine two claims.

Long-deletion CRISPR screens deliver pairs of gRNAs per cell targeting closely located sites, which can result in clean deletion of the intervening sequence.

“Such ‘long deletion’ scans deliver pairs of gRNAs per cell that target closely located sites, which can result in clean deletion of the intervening sequence.”

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

Multiplex whole-transcriptome screens may be limited to epigenetic perturbation, particularly if large numbers of DSBs are toxic to cells.

“Furthermore, such multiplex screens may be limited to epigenetic perturbation, particularly if large numbers of DSBs are toxic to cells.”

May combine two claims.

The further apart the pair of gRNA cuts, the less often full deletion occurs; for a 365 bp deletion, full deletion occurs ~20% of the time.

“However, a challenge is that the further apart the pair of cuts induced by the gRNAs, the less often full deletion occurs, e.g. ~20% for a 365 bp deletion204.”

• cites [204] Gasperini, M. et al. CRISPR/Cas9-mediated scanning for regulatory elements required for HPRT1 expression via thousands of large, programmed genomic deletions. Am. J. Hum. Genet. 101, 192–205 (2017). sourceNot checked — no full text of the source

May combine two claims.

Conventional MPRAs fail to capture how each sequence affects and is affected by its genomic neighbourhood, nor which promoter(s) an 'active' enhancer endogenously affects.

“Conventional MPRAs also fail to capture how each sequence affects and is affected by its genomic neighbourhood, nor which promoter(s) an ‘active’ enhancer endogenously affects.”

May combine two claims.

MPRAs test each sequence entirely out of context, which is both a strength (isolating the sequence) and a weakness (properties observed out-of-context may be irrelevant in native context).

“The fact that MPRAs test each sequence of interest entirely out of context is on one hand a strength, as it isolates that sequence in order to study its properties independently of that context. But it is also a weakness in that properties observed out-of-context may be irrelevant when that native context is restored.”

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

eQTL studies are powerful for assessing in vivo effects but are limited in critical ways.

“As discussed above, eQTL studies are powerful for assessing in vivo effects, but are limited in critical ways.”