Review

Enhancer talk

Valentina Snetkova

Department of Pathology, New York University School of Medicine, 550 First Avenue, MSB 599, New York, NY 10016, USA

MS 84–171, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

Search for more papers by this author

Jane A Skok

*Author for correspondence:

E-mail Address: Jane.Skok@nyumc.org

Department of Pathology, New York University School of Medicine, 550 First Avenue, MSB 599, New York, NY 10016, USA

Search for more papers by this author

Published Online:27 Mar 2018https://doi.org/10.2217/epi-2017-0157

View article

Abstract

Enhancers are short noncoding segments of DNA (100–1000 bp) that control the temporal and spatial activity of genes in an orientation-independent manner. They can be separated from their target genes by large distances and are thus known as distal regulatory elements. One consequence of the variability in the distance separating enhancers and their target promoters is that it is difficult to determine which elements are involved in the regulation of a particular gene. Moreover, enhancers can be found in clusters in which multiple regulatory elements control expression of the same target gene. However, little is known about how the individual elements contribute to gene expression. Here, we describe how chromatin conformation promotes and constraints enhancer activity. Further, we discuss enhancer clusters and what is known about the contribution of individual elements to the regulation of target genes. Finally, we examine the reliability of different methods used to identify enhancers.

Keywords:

Papers of special note have been highlighted as: • of interest; •• of considerable interest

References

1 Banerji J, Rusconi S, Schaffner W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell 27(2 Pt 1), 299–308 (1981).Crossref, Medline, CAS, Google Scholar
2 Sanyal A, Lajoie BR, Jain G, Dekker J. The long-range interaction landscape of gene promoters. Nature 489(7414), 109–113 (2012).Crossref, Medline, CAS, Google Scholar
3 Lettice LA, Heaney SJ, Purdie LA 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(14), 1725–1735 (2003).Crossref, Medline, CAS, Google Scholar
4 Proudhon C, Snetkova V, Raviram R et al. Active and inactive enhancers cooperate to exert localized and long-range control of gene regulation. Cell Rep. 15(10), 2159–2169 (2016). •• Investigates the contribution of enhancers to 3D organization of a multienhancer hub.Crossref, Medline, CAS, Google Scholar
5 Hewitt SL, Farmer D, Marszalek K et al. Association between the Igk and Igh immunoglobulin loci mediated by the 3′ Igk enhancer induces ‘decontraction’ of the Igh locus in pre-B cells. Nat. Immunol. 9(4), 396–404 (2008).Crossref, Medline, CAS, Google Scholar
6 Pennacchio LA, Bickmore W, Dean A, Nobrega MA, Bejerano G. Enhancers: five essential questions. Nat. Rev. Genet. 14(4), 288–295 (2013).Crossref, Medline, CAS, Google Scholar
7 Buffry AD, Mendes CC, McGregor AP. The functionality and evolution of eukaryotic transcriptional enhancers. Adv. Genet. 96, 143–206 (2016).Crossref, Medline, CAS, Google Scholar
8 Denker A, De Laat W. The second decade of 3C technologies: detailed insights into nuclear organization. Genes Dev. 30(12), 1357–1382 (2016).Crossref, Medline, CAS, Google Scholar
9 De Laat W, Duboule D. Topology of mammalian developmental enhancers and their regulatory landscapes. Nature 502(7472), 499–506 (2013).Crossref, Medline, CAS, Google Scholar
10 Deng WL, Lee J, Wang HX et al. Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor. Cell 149(6), 1233–1244 (2012). •• Forced physical contact between β-globin and its locus control region was shown to be sufficient to recruit RNA polymerase to the β-globin promoters in the absence of GATA1 to activate expression.Crossref, Medline, CAS, Google Scholar
11 Deng WL, Rupon JW, Krivega I et al. Reactivation of developmentally silenced globin genes by forced chromatin looping. Cell 158(4), 849–860 (2014).Crossref, Medline, CAS, Google Scholar
12 Van De Werken HJ, Landan G, Holwerda SJ et al. Robust 4C-seq data analysis to screen for regulatory DNA interactions. Nat. Methods 9(10), 969–972 (2012).Crossref, Medline, CAS, Google Scholar
13 Isoda T, Moore AJ, He Z et al. Non-coding transcription instructs chromatin folding and compartmentalization to dictate enhancer–promoter communication and T cell fate. Cell 171(1), 103–119 (2017).Crossref, Medline, CAS, Google Scholar
14 Fabre PJ, Leleu M, Mormann BH et al. Large scale genomic reorganization of topological domains at the HoxD locus. Genome Biol. 18(1), 149 (2017).Crossref, Medline, Google Scholar
15 Montavon T, Soshnikova N, Mascrez B et al. A regulatory archipelago controls Hox genes transcription in digits. Cell 147(5), 1132–1145 (2011).Crossref, Medline, CAS, Google Scholar
16 Andrey G, Montavon T, Mascrez B et al. A switch between topological domains underlies HoxD genes collinearity in mouse limbs. Science 340(6137), 1234167 (2013).Crossref, Medline, CAS, Google Scholar
17 Jin F, Li Y, Dixon JR et al. A high-resolution map of the three-dimensional chromatin interactome in human cells. Nature 503(7475), 290–294 (2013).Crossref, Medline, CAS, Google Scholar
18 Ghavi-Helm Y, Klein FA, Pakozdi T et al. Enhancer loops appear stable during development and are associated with paused polymerase. Nature 512(7512), 96–100 (2014).Crossref, Medline, CAS, Google Scholar
19 Andrey G, Schopflin R, Jerkovic I et al. Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding. Genome Res. 27(2), 223–233 (2017).Crossref, Medline, CAS, Google Scholar
20 Ren G, Jin W, Cui K et al. CTCF-Mediated enhancer–promoter interaction is a critical regulator of cell-to-cell variation of gene expression. Mol. Cell 67(6), 1049–1058 (2017).Crossref, Medline, CAS, Google Scholar
21 Javierre BM, Burren OS, Wilder SP et al. Lineage-specific genome architecture links enhancers and non-coding disease variants to target gene promoters. Cell 167(5), 1369–1384 (2016).Crossref, Medline, CAS, Google Scholar
22 Rubin AJ, Barajas BC, Furlan-Magaril M et al. Lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation. Nat. Genet. 49(10), 1522–1528 (2017).Crossref, Medline, CAS, Google Scholar
23 Bonev B, Mendelson Cohen N, Szabo Q et al. Multiscale 3D genome rewiring during mouse neural development. Cell 171(3), 557–572 (2017).Crossref, Medline, CAS, Google Scholar
24 Phanstiel DH, Van Bortle K, Spacek D et al. Static and dynamic DNA loops form AP-1-bound activation hubs during macrophage development. Mol. Cell 67(6), 1037–1048 (2017).Crossref, Medline, CAS, Google Scholar
25 Allahyar A, Vermeulen C, Bouwman B et al. Locus-specific enhancer hubs and architectural loop collisions uncovered from single allele DNA topologies. bioRxiv (2017).Google Scholar
26 Jiang T, Raviram R, Snetkova V et al. Identification of multi-loci hubs from 4C-seq demonstrates the functional importance of simultaneous interactions. Nucleic Acids Res. 44(18), 8714–8725 (2016). • Employs 4C-seq to detect multiloci contacts that occur in the same cell as opposed to separate pairwise contacts observed in a cell population.Crossref, Medline, CAS, Google Scholar
27 Dixon JR, Selvaraj S, Yue F et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485(7398), 376–380 (2012).Crossref, Medline, CAS, Google Scholar
28 Nora EP, Lajoie BR, Schulz EG et al. Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485(7398), 381–385 (2012).Crossref, Medline, CAS, Google Scholar
29 Nasmyth K. Cohesin: a catenase with separate entry and exit gates? Nat. Cell Biol. 13(10), 1170–1177 (2011).Crossref, Medline, CAS, Google Scholar
30 Nichols MH, Corces VG. A CTCF code for 3D genome architecture. Cell 162(4), 703–705 (2015).Crossref, Medline, CAS, Google Scholar
31 Sanborn AL, Rao SS, Huang SC et al. Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes. Proc. Natl Acad. Sci. USA 112(47), E6456–E6465 (2015).Crossref, Medline, CAS, Google Scholar
32 Fudenberg G, Imakaev M, Lu C, Goloborodko A, Abdennur N, Mirny LA. Formation of chromosomal domains by loop extrusion. Cell Rep. 15(9), 2038–2049 (2016).Crossref, Medline, CAS, Google Scholar
33 Rao SS, Huntley MH, Durand NC et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159(7), 1665–1680 (2014).Crossref, Medline, CAS, Google Scholar
34 Symmons O, Uslu VV, Tsujimura T et al. Functional and topological characteristics of mammalian regulatory domains. Genome Res. 24(3), 390–400 (2014).Crossref, Medline, CAS, Google Scholar
35 Phillips-Cremins JE, Sauria ME, Sanyal A et al. Architectural protein subclasses shape 3D organization of genomes during lineage commitment. Cell 153(6), 1281–1295 (2013).Crossref, Medline, CAS, Google Scholar
36 Beagan JA, Duong MT, Titus KR et al. YY1 and CTCF orchestrate a 3D chromatin looping switch during early neural lineage commitment. Genome Res. 27(7), 1139–1152 (2017).Crossref, Medline, CAS, Google Scholar
37 Narendra V, Rocha PP, An D et al. CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation. Science 347(6225), 1017–1021 (2015).Crossref, Medline, CAS, Google Scholar
38 Narendra V, Bulajic M, Dekker J, Mazzoni EO, Reinberg D. CTCF-mediated topological boundaries during development foster appropriate gene regulation. Genes Dev. 30(24), 2657–2662 (2016).Crossref, Medline, CAS, Google Scholar
39 Lupianez DG, Kraft K, Heinrich V et al. Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions. Cell 161(5), 1012–1025 (2015). •• Demonstrates the importance of a topologically associated domain border for proper physiological development of a limb in both mice and humans.Crossref, Medline, CAS, Google Scholar
40 Hnisz D, Weintraub AS, Day DS et al. Activation of proto-oncogenes by disruption of chromosome neighborhoods. Science 351(6280), 1454–1458 (2016).Crossref, Medline, CAS, Google Scholar
41 Li L, Lyu XW, Hou CH et al. Widespread rearrangement of 3D chromatin organization underlies polycomb-mediated stress-induced silencing. Mol. Cell 58(2), 216–231 (2015).Crossref, Medline, CAS, Google Scholar
42 Bunting KL, Soong TD, Singh R et al. Multi-tiered reorganization of the genome during B cell affinity maturation anchored by a germinal center-specific locus control region. Immunity 45(3), 497–512 (2016).Crossref, Medline, CAS, Google Scholar
43 Seitan VC, Faure AJ, Zhan Y et al. Cohesin-based chromatin interactions enable regulated gene expression within preexisting architectural compartments. Genome Res. 23(12), 2066–2077 (2013).Crossref, Medline, CAS, Google Scholar
44 Sofueva S, Yaffe E, Chan WC et al. Cohesin-mediated interactions organize chromosomal domain architecture. EMBO J. 32(24), 3119–3129 (2013).Crossref, Medline, CAS, Google Scholar
45 Zuin J, Dixon JR, Van Der Reijden MI et al. Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells. Proc. Natl Acad. Sci. USA 111(3), 996–1001 (2014).Crossref, Medline, CAS, Google Scholar
46 Nora EP, Goloborodko A, Valton A-L et al. Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell 169(5), 930–944 (2017).Crossref, Medline, CAS, Google Scholar
47 Schwarzer W, Abdennur N, Goloborodko A et al. Two independent modes of chromatin organization revealed by cohesin removal. Nature 551(7678), 51–56 (2017).Crossref, Medline, Google Scholar
48 Rao SSP, Huang SC, Glenn St Hilaire B et al. Cohesin loss eliminates all loop domains. Cell 171(2), 305–320; e324 (2017).Crossref, Medline, CAS, Google Scholar
49 Kubo N, Ishii H, Gorkin D et al. Preservation of chromatin organization after acute loss of CTCF in mouse embryonic stem cells. bioRxiv (2017).Google Scholar
50 Long HK, Prescott SL, Wysocka J. Ever-changing landscapes: transcriptional enhancers in development and evolution. Cell 167(5), 1170–1187 (2016).Crossref, Medline, CAS, Google Scholar
51 Whyte WA, Orlando DA, Hnisz D et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153(2), 307–319 (2013).Crossref, Medline, CAS, Google Scholar
52 Hnisz D, Abraham BJ, Lee TI et al. Super-enhancers in the control of cell identity and disease. Cell 155(4), 934–947 (2013).Crossref, Medline, CAS, Google Scholar
53 Hnisz D, Schuijers J, Lin CY et al. Convergence of developmental and oncogenic signaling pathways at transcriptional super-enhancers. Mol. Cell 58(2), 362–370 (2015).Crossref, Medline, CAS, Google Scholar
54 Adam RC, Yang H, Rockowitz S et al. Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice. Nature 521(7552), 366–370 (2015).Crossref, Medline, CAS, Google Scholar
55 Parker SC, Stitzel ML, Taylor DL et al. Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants. Proc. Natl Acad. Sci. USA 110(44), 17921–17926 (2013).Crossref, Medline, CAS, Google Scholar
56 Maeda RK, Karch F. Gene expression in time and space: additive vs hierarchical organization of cis-regulatory regions. Curr. Opin. Genet. Dev. 21(2), 187–193 (2011).Crossref, Medline, CAS, Google Scholar
57 Will AJ, Cova G, Osterwalder M et al. Composition and dosage of a multipartite enhancer cluster control developmental expression of Ihh (Indian hedgehog). Nat. Genet. 49(10), 1539–1545 (2017).Crossref, Medline, CAS, Google Scholar
58 Marinic M, Aktas T, Ruf S, Spitz F. An integrated holo-enhancer unit defines tissue and gene specificity of the Fgf8 regulatory landscape. Dev. Cell 24(5), 530–542 (2013).Crossref, Medline, CAS, Google Scholar
59 Yuh CH, Bolouri H, Davidson EH. Genomic cis-regulatory logic: experimental and computational analysis of a sea urchin gene. Science 279(5358), 1896–1902 (1998).Crossref, Medline, CAS, Google Scholar
60 Romano LA, Wray GA. Conservation of Endo16 expression in sea urchins despite evolutionary divergence in both cis and trans-acting components of transcriptional regulation. Development 130(17), 4187–4199 (2003).Crossref, Medline, CAS, Google Scholar
61 Shin HY, Willi M, Yoo KH et al. Hierarchy within the mammary STAT5-driven Wap super-enhancer. Nat. Genet. 48(8), 904–911 (2016).Crossref, Medline, CAS, Google Scholar
62 Triplett AA, Sakamoto K, Matulka LA, Shen L, Smith GH, Wagner KU. Expression of the whey acidic protein (Wap) is necessary for adequate nourishment of the offspring but not functional differentiation of mammary epithelial cells. Genesis 43(1), 1–11 (2005).Crossref, Medline, CAS, Google Scholar
63 Perry MW, Boettiger AN, Levine M. Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo. Proc. Natl Acad. Sci. USA 108(33), 13570–13575 (2011).Crossref, Medline, CAS, Google Scholar
64 Bothma JP, Garcia HG, Ng S, Perry MW, Gregor T, Levine M. Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo. Elife 4, e07956 (2015). • Explores how pairs of enhancers controlling Gap gene expression in Drosophila embryo exhibit different modes of behavior ranging from additive to synergistic.Crossref, Google Scholar
65 Hong JW, Hendrix DA, Levine MS. Shadow enhancers as a source of evolutionary novelty. Science 321(5894), 1314–1314 (2008).Crossref, Medline, CAS, Google Scholar
66 Zeitlinger J, Zinzen RP, Stark A et al. Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo. Gene Dev. 21(4), 385–390 (2007).Crossref, Medline, CAS, Google Scholar
67 Frankel N, Davis GK, Vargas D, Wang S, Payre F, Stern DL. Phenotypic robustness conferred by apparently redundant transcriptional enhancers. Nature 466(7305), 490–493 (2010).Crossref, Medline, CAS, Google Scholar
68 Perry MW, Boettiger AN, Bothma JP, Levine M. Shadow enhancers foster robustness of Drosophila gastrulation. Curr. Biol. 20(17), 1562–1567 (2010).Crossref, Medline, CAS, Google Scholar
69 Hay D, Hughes JR, Babbs C et al. Genetic dissection of the alpha-globin super-enhancer in vivo. Nat. Genet. 48(8), 895–903 (2016).Crossref, Medline, CAS, Google Scholar
70 Roldan E, Fuxa M, Chong W et al. Locus ‘decontraction’ and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene. Nat. Immunol. 6(1), 31–41 (2005).Crossref, Medline, CAS, Google Scholar
71 Skok JA, Gisler R, Novatchkova M, Farmer D, De Laat W, Busslinger M. Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes. Nat. Immunol. 8(4), 378–387 (2007).Crossref, Medline, CAS, Google Scholar
72 Jhunjhunwala S, Van Zelm MC, Peak MM et al. The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions. Cell 133(2), 265–279 (2008).Crossref, Medline, CAS, Google Scholar
73 Proudhon C, Hao B, Raviram R, Chaumeil J, Skok JA. Long-range regulation of V(D)J recombination. Adv. Immunol. 128, 123–182 (2015).Crossref, Medline, CAS, Google Scholar
74 Queen C, Baltimore D. Immunoglobulin gene-transcription is activated by downstream sequence elements. Cell 33(3), 741–748 (1983).Crossref, Medline, CAS, Google Scholar
75 Cockerill PN, Garrard WT. Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell 44(2), 273–282 (1986).Crossref, Medline, CAS, Google Scholar
76 Meyer KB, Neuberger MS. The immunoglobulin kappa-locus contains a second, stronger b-cell-specific enhancer which is located downstream of the constant region. Embo J. 8(7), 1959–1964 (1989).Crossref, Medline, CAS, Google Scholar
77 Liu ZM, George-Raizen JB, Li S, Meyers KC, Chang MY, Garrard WT. Chromatin structural analyses of the mouse Igkappa gene locus reveal new hypersensitive sites specifying a transcriptional silencer and enhancer. J. Biol. Chem. 277(36), 32640–32649 (2002).Crossref, Medline, CAS, Google Scholar
78 Inlay M, Alt FW, Baltimore D, Xu Y. Essential roles of the kappa light chain intronic enhancer and 3′ enhancer in kappa rearrangement and demethylation. Nat. Immunol. 3(5), 463–468 (2002).Crossref, Medline, CAS, Google Scholar
79 Inlay MA, Gao HH, Odegard VH, Lin T, Schatz DG, Xu Y. Roles of the Ig kappa light chain intronic and 3′ enhancers in Igk somatic hypermutation. J. Immunol. 177(2), 1146–1151 (2006).Crossref, Medline, CAS, Google Scholar
80 Xiang Y, Garrard WT. The downstream transcriptional enhancer, Ed, positively regulates mouse Ig kappa gene expression and somatic hypermutation. J. Immunol. 180(10), 6725–6732 (2008).Crossref, Medline, CAS, Google Scholar
81 Zhou X, Xiang Y, Garrard WT. The Igkappa gene enhancers, E3′ and Ed, are essential for triggering transcription. J. Immunol. 185(12), 7544–7552 (2010).Crossref, Medline, CAS, Google Scholar
82 Qian J, Wang Q, Dose M et al. B cell super-enhancers and regulatory clusters recruit AID tumorigenic activity. Cell 159(7), 1524–1537 (2014).Crossref, Medline, CAS, Google Scholar
83 Huang Y, Koues OI, Zhao JY et al. cis-regulatory circuits regulating NEK6 kinase overexpression in transformed b cells are super-enhancer independent. Cell Rep. 18(12), 2918–2931 (2017). • Reveals that only a subset of enhancers predicted to affect NEK6 expression are actually functional, with an annotated neighboring super-enhancer being dispensable.Crossref, Medline, CAS, Google Scholar
84 Engreitz JM, Haines JE, Perez EM et al. Local regulation of gene expression by lncRNA promoters, transcription and splicing. Nature 539(7629), 452–455 (2016).Crossref, Medline, CAS, Google Scholar
85 Moorthy SD, Davidson S, Shchuka VM et al. Enhancers and super-enhancers have an equivalent regulatory role in embryonic stem cells through regulation of single or multiple genes. Genome Res. 27(2), 246–258 (2017).Crossref, Medline, CAS, Google Scholar
86 Patwardhan RP, Hiatt JB, Witten DM et al. Massively parallel functional dissection of mammalian enhancers in vivo. Nat. Biotechnol. 30(3), 265–270 (2012).Crossref, Medline, CAS, Google Scholar
87 Arnold CD, Gerlach D, Stelzer C, Boryn LM, Rath M, Stark A. Genome-wide quantitative enhancer activity maps identified by STARR-seq. Science 339(6123), 1074–1077 (2013).Crossref, Medline, CAS, Google Scholar
88 Dickel DE, Zhu Y, Nord AS et al. Function-based identification of mammalian enhancers using site-specific integration. Nat. Methods 11(5), 566–571 (2014).Crossref, Medline, CAS, Google Scholar
89 Canver MC, Smith EC, Sher F et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527(7577), 192–197 (2015).Crossref, Medline, CAS, Google Scholar
90 Sanjana NE, Wright J, Zheng K et al. High-resolution interrogation of functional elements in the noncoding genome. Science 353(6307), 1545–1549 (2016). •• Uses a high-throughput CRISPR-based mutagenesis screen to identify noncoding regulatory elements that control expression of genes responsible for tumor survival.Crossref, Medline, CAS, Google Scholar
91 Diao Y, Fang R, Li B et al. A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells. Nat. Methods 14(6), 629–635 (2017).Crossref, Medline, CAS, Google Scholar
92 Sen DR, Kaminski J, Barnitz RA et al. The epigenetic landscape of T cell exhaustion. Science 354(6316), 1165–1169 (2016).Crossref, Medline, CAS, Google Scholar
93 Fulco CP, Munschauer M, Anyoha R et al. Systematic mapping of functional enhancer–promoter connections with CRISPR interference. Science 354(6313), 769–773 (2016).Crossref, Medline, CAS, Google Scholar
94 Gasperini M, Findlay GM, McKenna A 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(2), 192–205 (2017).Crossref, Medline, CAS, Google Scholar

Vol. 10, No. 4

Metrics

Downloaded 495 times

History

Received 27 November 2017

Accepted 29 January 2018

Published online 27 March 2018

Published in print April 2018

Information

Keywords

Acknowledgements

We would like to sincerely thank members of the Skok lab for their input and especially P Rocha for insightful comments and suggestions.

Financial & competing interests disclosure

JA Skok is supported by NIH grant R35GM122515, 4P30CA016087-36 Cancer Center Support Grant NIH/NCI (Neel) and 2R01CA140729-06A1 NIH/NCI (Carroll). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

PDF download