Regulatory Landscaping: How Enhancer-Promoter Communication Is Sculpted in 3D
2019; Elsevier BV; Volume: 74; Issue: 6 Linguagem: Inglês
10.1016/j.molcel.2019.05.032
ISSN1097-4164
AutoresMichael I. Robson, Alessa R. Ringel, Stefan Mundlos,
Tópico(s)Child Development and Digital Technology
ResumoDuring embryogenesis, precise gene transcription in space and time requires that distal enhancers and promoters communicate by physical proximity within gene regulatory landscapes. To achieve this, regulatory landscapes fold in nuclear space, creating complex 3D structures that influence enhancer-promoter communication and gene expression and that, when disrupted, can cause disease. Here, we provide an overview of how enhancers and promoters construct regulatory landscapes and how multiple scales of 3D chromatin structure sculpt their communication. We focus on emerging views of what enhancer-promoter contacts and chromatin domains physically represent and how two antagonistic fundamental forces—loop extrusion and homotypic attraction—likely form them. We also examine how these same forces spatially separate regulatory landscapes by functional state, thereby creating higher-order compartments that reconfigure during development to enable proper enhancer-promoter communication. During embryogenesis, precise gene transcription in space and time requires that distal enhancers and promoters communicate by physical proximity within gene regulatory landscapes. To achieve this, regulatory landscapes fold in nuclear space, creating complex 3D structures that influence enhancer-promoter communication and gene expression and that, when disrupted, can cause disease. Here, we provide an overview of how enhancers and promoters construct regulatory landscapes and how multiple scales of 3D chromatin structure sculpt their communication. We focus on emerging views of what enhancer-promoter contacts and chromatin domains physically represent and how two antagonistic fundamental forces—loop extrusion and homotypic attraction—likely form them. We also examine how these same forces spatially separate regulatory landscapes by functional state, thereby creating higher-order compartments that reconfigure during development to enable proper enhancer-promoter communication. During development, intricate changes to gene expression transition single-celled embryos to complex organisms with hundreds of cell types. Robustly regulated transcription in time and space is essential for such precision and is thus critical for embryogenesis. However, in metazoans, many core promoters proximal to transcription start sites (TSSs) do not drive such robust and precise gene expression alone. Rather, regulatory information is distributed throughout a promoter’s genomic surroundings in non-coding elements with diverse spatiotemporal activities, termed enhancers. In this way, promoters and enhancers together create gene regulatory landscapes that drive the complex and flexible patterns of transcriptional activity necessary for metazoan life. Remarkably, enhancers can communicate their defined activities across large genomic distances by physically contacting distal promoters via chromatin folding. To achieve this, regulatory landscapes are highly organized in 3D nuclear space at a number of scales, each of which differently influences enhancer-promoter communication (Figure 1). Interactions within regulatory landscapes create enhancer-promoter contacts that support and modulate cell-type-specific gene expression (Andrey et al., 2017Andrey G. Schöpflin R. Jerković I. Heinrich V. Ibrahim D.M. Paliou C. Hochradel M. Timmermann B. Haas S. Vingron M. Mundlos S. Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding.Genome Res. 2017; 27: 223-233Crossref Scopus (78) Google Scholar, Javierre et al., 2016Javierre B.M. Burren O.S. Wilder S.P. Kreuzhuber R. Hill S.M. Sewitz S. Cairns J. Wingett S.W. Várnai C. Thiecke M.J. et al.BLUEPRINT ConsortiumLineage-Specific Genome Architecture Links Enhancers and Non-coding Disease Variants to Target Gene Promoters.Cell. 2016; 167: 1369-1384.e19Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar). Higher-order chromatin folding constrains these contacts within self-interacting topological-associated domains (TADs) separated by insulating boundaries, thereby partitioning the genome into discrete functional blocks (Dixon et al., 2012Dixon J.R. Selvaraj S. Yue F. Kim A. Li Y. Shen Y. Hu M. Liu J.S. Ren B. Topological domains in mammalian genomes identified by analysis of chromatin interactions.Nature. 2012; 485: 376-380Crossref PubMed Scopus (3955) Google Scholar, Nora et al., 2012Nora E.P. Lajoie B.R. Schulz E.G. Giorgetti L. Okamoto I. Servant N. Piolot T. van Berkum N.L. Meisig J. Sedat J. et al.Spatial partitioning of the regulatory landscape of the X-inactivation centre.Nature. 2012; 485: 381-385Crossref PubMed Scopus (1807) Google Scholar). At a chromosomal scale, multi-megabase interactions between TADs with similar epigenetic states further spatially segregate chromatin according to activity, creating the structurally and functionally distinct active A and inactive B compartments (Lieberman-Aiden et al., 2009Lieberman-Aiden E. van Berkum N.L. Williams L. Imakaev M. Ragoczy T. Telling A. Amit I. Lajoie B.R. Sabo P.J. Dorschner M.O. et al.Comprehensive mapping of long-range interactions reveals folding principles of the human genome.Science. 2009; 326: 289-293Crossref PubMed Scopus (4868) Google Scholar). Subsequent positioning of A compartments near nuclear speckles and B compartments at the nuclear envelope/nucleolus localizes regulatory landscapes at sites conducive or intolerant to transcription, respectively (Chen et al., 2018bChen Y. Zhang Y. Wang Y. Zhang L. Brinkman E.K. Adam S.A. Goldman R. van Steensel B. Ma J. Belmont A.S. Mapping 3D genome organization relative to nuclear compartments using TSA-Seq as a cytological ruler.J. Cell Biol. 2018; 217: 4025-4048Crossref Scopus (156) Google Scholar, Kim et al., 2019Kim J. Khanna N. Belmont A.S. Transcription amplification by nuclear speckle association.bioRxiv. 2019; https://doi.org/10.1101/604298Crossref Google Scholar, Rao et al., 2014Rao S.S. Huntley M.H. Durand N.C. Stamenova E.K. Bochkov I.D. Robinson J.T. Sanborn A.L. Machol I. Omer A.D. Lander E.S. Aiden E.L. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping.Cell. 2014; 159: 1665-1680Abstract Full Text Full Text PDF PubMed Scopus (3598) Google Scholar). Together, these organizational scales sculpt regulatory landscapes. Enhancers define their information content, promoter contacts transmit that information, TADs determine their limits, and compartments reflect their functional state. (For a complete glossary, see Box 1.)Box 1Glossary•Topologically associated domains (TADs): chromatin domains with high self-association that are insulated from the wider genome by boundary elements.•Structural Variant (SV): large chromosomal rearrangements, including duplications, deletions, and translocations, that can restructure the genome and TADs.•Lamina-associated domains (LADs): chromatin domains detected by DamID to interact with the nuclear envelope.•Nucleolar-associated domains (NADs): chromatin domains that interact with the nucleolus.•Replication-timing domain: chromatin domains that engage in DNA replication at different times in S phase.•A/B compartments: higher-order structures formed by preferential inter-TAD interactions between chromatin of the same A or B type.•Loop extrusion: postulated mechanism of TAD formation whereby loops are produced by progressive extrusion of chromatin by a loop extrusion factor.•Homotypic attraction: postulated force driving chromatin with similar epigenetic properties or DNA binding proteins to preferentially self-associate, for example, into compartments.•Liquid-liquid phase separation (LLPS): process in which molecules separate in the absence of membranes into discrete liquid condensates with distinct compositions.•Intrinsically disordered region (IDR): portion of a protein that lacks a fixed or ordered structure and can induce LLPS. •Topologically associated domains (TADs): chromatin domains with high self-association that are insulated from the wider genome by boundary elements.•Structural Variant (SV): large chromosomal rearrangements, including duplications, deletions, and translocations, that can restructure the genome and TADs.•Lamina-associated domains (LADs): chromatin domains detected by DamID to interact with the nuclear envelope.•Nucleolar-associated domains (NADs): chromatin domains that interact with the nucleolus.•Replication-timing domain: chromatin domains that engage in DNA replication at different times in S phase.•A/B compartments: higher-order structures formed by preferential inter-TAD interactions between chromatin of the same A or B type.•Loop extrusion: postulated mechanism of TAD formation whereby loops are produced by progressive extrusion of chromatin by a loop extrusion factor.•Homotypic attraction: postulated force driving chromatin with similar epigenetic properties or DNA binding proteins to preferentially self-associate, for example, into compartments.•Liquid-liquid phase separation (LLPS): process in which molecules separate in the absence of membranes into discrete liquid condensates with distinct compositions.•Intrinsically disordered region (IDR): portion of a protein that lacks a fixed or ordered structure and can induce LLPS. Although such details are increasingly clear, precisely what mechanisms drive these organizational scales or enable them to influence enhancer-promoter communication have remained elusive. Here, we discuss recent advances addressing these questions, driven by developments in genome engineering, protein-depletion technologies, and single-locus structural mapping. We describe how enhancers combinatorially coordinate promoter transcription and how invariant and tissue-specific chromatin interactions influence their spatial proximity. We evaluate how TADs define regulatory landscapes, what they physically represent at single loci over time, and which mechanisms drive their formation. Finally, we will examine how regulatory landscapes physically transition between active and inactive compartments, thereby providing a unified model of enhancer-promoter communication throughout all organizational scales. In metazoans, regulatory information is uncoupled from the proximity of TSSs and transmitted to promoters from distal enhancers (Figure 2). Though enhancers are defined as blocks of non-coding sequences that induce spatiotemporally precise transcription in even distal promoters, how they achieve this is still fundamentally unknown (Furlong and Levine, 2018Furlong E.E.M. Levine M. Developmental enhancers and chromosome topology.Science. 2018; 361: 1341-1345Crossref PubMed Scopus (245) Google Scholar). What is clear is that enhancer activities stem from their recruitment of distinct combinations of sequence-specific transcription factors (TFs). Once bound, these TFs recruit coactivator proteins that promote RNA polymerase II (Pol II) recruitment and processivity at target genes (Haberle and Stark, 2018Haberle V. Stark A. Eukaryotic core promoters and the functional basis of transcription initiation.Nat. Rev. Mol. Cell Biol. 2018; 19: 621-637Crossref PubMed Scopus (247) Google Scholar, Long et al., 2016Long H.K. Prescott S.L. Wysocka J. Ever-Changing Landscapes: Transcriptional Enhancers in Development and Evolution.Cell. 2016; 167: 1170-1187Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). Nevertheless, not all enhancers can activate all promoters. Thus, different enhancers likely utilize diverse mechanisms to stimulate transcription of only compatible promoters, thereby refining the potential targets they can regulate (Haberle and Stark, 2018Haberle V. Stark A. Eukaryotic core promoters and the functional basis of transcription initiation.Nat. Rev. Mol. Cell Biol. 2018; 19: 621-637Crossref PubMed Scopus (247) Google Scholar). Though candidate enhancers can be identified from a distinct signature of accessible chromatin, H3K4me1, H3K27ac, and transcribing Pol II, these features do not guarantee an ability to function as one (Catarino and Stark, 2018Catarino R.R. Stark A. Assessing sufficiency and necessity of enhancer activities for gene expression and the mechanisms of transcription activation.Genes Dev. 2018; 32: 202-223Crossref PubMed Scopus (107) Google Scholar). Consequently, putative elements must be experimentally validated through, for example, enhancer-reporter assays performed on single elements in vivo or many elements in parallel in vitro (Figures 2A and 2B) (for review, see Catarino and Stark, 2018Catarino R.R. Stark A. Assessing sufficiency and necessity of enhancer activities for gene expression and the mechanisms of transcription activation.Genes Dev. 2018; 32: 202-223Crossref PubMed Scopus (107) Google Scholar). Through these approaches, thousands of putative enhancer activities have been mapped in space and time in vivo across different cell types of developing embryos (Manning et al., 2012Manning L. Heckscher E.S. Purice M.D. Roberts J. Bennett A.L. Kroll J.R. Pollard J.L. Strader M.E. Lupton J.R. Dyukareva A.V. et al.A resource for manipulating gene expression and analyzing cis-regulatory modules in the Drosophila CNS.Cell Rep. 2012; 2: 1002-1013Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, Pennacchio et al., 2006Pennacchio L.A. Ahituv N. Moses A.M. Prabhakar S. Nobrega M.A. Shoukry M. Minovitsky S. Dubchak I. Holt A. Lewis K.D. et al.In vivo enhancer analysis of human conserved non-coding sequences.Nature. 2006; 444: 499-502Crossref PubMed Scopus (875) Google Scholar). As such, enhancers are significant contributors to the diversity of gene expression patterns. In metazoans, many promoters are controlled by multiple enhancers with differing spatiotemporal activities, each of which regulates a distinct subset of a gene’s overall pattern of activity (Figure 2A) (Andrey and Mundlos, 2017Andrey G. Mundlos S. The three-dimensional genome: regulating gene expression during pluripotency and development.Development. 2017; 144: 3646-3658Crossref PubMed Scopus (65) Google Scholar). For instance, the composite activities of at least 11 enhancers drive Sonic hedgehog (Shh) in multiple tissues, including central nervous system, epithelial linings, and limbs, during mouse embryogenesis (Anderson et al., 2014Anderson E. Devenney P.S. Hill R.E. Lettice L.A. Mapping the Shh long-range regulatory domain.Development. 2014; 141: 3934-3943Crossref PubMed Scopus (48) Google Scholar). Consequently, loss of a single element is thought to exclusively eliminate only its corresponding portion of a gene’s expression pattern (Figure 2A). Supporting this, removal of the limb-specific ZRS enhancer specifically eliminates Shh expression only in that tissue, thereby disrupting limb outgrowth (Anderson et al., 2014Anderson E. Devenney P.S. Hill R.E. Lettice L.A. Mapping the Shh long-range regulatory domain.Development. 2014; 141: 3934-3943Crossref PubMed Scopus (48) Google Scholar). However, such situations are rare. More commonly, complex expression patterns are generated by multiple redundant enhancers with overlapping activities that resist genetic variation. Indeed, the overlapping activities of multiple ultra-conserved enhancers at the Gli3 and Shox2 genes require that several elements are deleted before expression is pathogenically disrupted in developing mouse limbs (Osterwalder et al., 2018Osterwalder M. Barozzi I. Tissières V. Fukuda-Yuzawa Y. Mannion B.J. Afzal S.Y. Lee E.A. Zhu Y. Plajzer-Frick I. Pickle C.S. et al.Enhancer redundancy provides phenotypic robustness in mammalian development.Nature. 2018; 554: 239-243Crossref PubMed Scopus (283) Google Scholar). Nevertheless, such redundant enhancers are frequently not completely interchangeable (Will et al., 2017Will A.J. Cova G. Osterwalder M. Chan W.L. Wittler L. Brieske N. Heinrich V. de Villartay J.P. Vingron M. Klopocki E. et al.Composition and dosage of a multipartite enhancer cluster control developmental expression of Ihh (Indian hedgehog).Nat. Genet. 2017; 49: 1539-1545Crossref PubMed Scopus (68) Google Scholar). Thus, regulatory landscapes assemble combinatorially complex and genetically resistant expression patterns through multiple enhancers, each of which can contain both distinct and redundant overlapping activities (Figure 2B). Further, by allowing mutations in enhancers to accumulate without total gene loss of function, such redundancy likely also provides a rich template from which to generate new regulatory activities during evolution (Long et al., 2016Long H.K. Prescott S.L. Wysocka J. Ever-Changing Landscapes: Transcriptional Enhancers in Development and Evolution.Cell. 2016; 167: 1170-1187Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). Beyond complexity and genetic resistance, overlapping activities of enhancers also provide transcriptional robustness under sub-optimal conditions. In Drosophila melanogaster, redundant shavenbaby enhancers can be deleted without regulatory or phenotypic effects when the embryos develop at 25°C. However, when developing at 17°C or 32°C, shavenbaby gene expression is disrupted in individual enhancer deletions, ultimately perturbing trichome formation (Frankel et al., 2010Frankel N. Davis G.K. Vargas D. Wang S. Payre F. Stern D.L. Phenotypic robustness conferred by apparently redundant transcriptional enhancers.Nature. 2010; 466: 490-493Crossref PubMed Scopus (342) Google Scholar). Such redundant enhancers may mediate this robustness by maintaining target expression in excess of minimal requirements within each cell, thereby buffering against adverse conditions. Indeed, deletion of individual redundant enhancers at the Gli3 or Shox2 loci normally yields no phenotype. However, when Gli3 or Shox2 baseline expression is reduced by 50%, loss of single enhancers lowers their expression beyond a critical level and disrupts limb development (Osterwalder et al., 2018Osterwalder M. Barozzi I. Tissières V. Fukuda-Yuzawa Y. Mannion B.J. Afzal S.Y. Lee E.A. Zhu Y. Plajzer-Frick I. Pickle C.S. et al.Enhancer redundancy provides phenotypic robustness in mammalian development.Nature. 2018; 554: 239-243Crossref PubMed Scopus (283) Google Scholar). Alternatively, redundancy may overcome the inherent probability that each enhancer fails to activate at least one copy of its target gene per cell, thereby achieving consistent transcription across cell populations (Perry et al., 2011Perry M.W. Boettiger A.N. Levine M. Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo.Proc. Natl. Acad. Sci. USA. 2011; 108: 13570-13575Crossref PubMed Scopus (159) Google Scholar). Regardless, additive cooperativity between redundant enhancers ensures robust and consistent expression, both within single cells and across cell populations. Taken together, these examples demonstrate how complex and robust expression patterns are assembled from ensembles of enhancers and promoters within modular regulatory landscapes. However, with regulatory landscapes spanning hundreds to thousands of kilobases, the question remains: how does chromatin’s spatial organization influence the transmission of enhancer activities to target genes? Enhancers utilize chromatin folding to bypass up to megabase (Mb) genomic distances and transmit their regulatory outputs to promoters by physical proximity. Supporting this, artificial enhancer-promoter contacts are sufficient to induce robust gene transcription (Deng et al., 2014Deng W. Rupon J.W. Krivega I. Breda L. Motta I. Jahn K.S. Reik A. Gregory P.D. Rivella S. Dean A. Blobel G.A. Reactivation of developmentally silenced globin genes by forced chromatin looping.Cell. 2014; 158: 849-860Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Similarly, live-cell imaging of labeled enhancers, promoters, and nascent RNA products has unequivocally visualized that their direct proximity corresponds with bursts of target gene transcription in Drosophila (Chen et al., 2018aChen H. Levo M. Barinov L. Fujioka M. Jaynes J.B. Gregor T. Dynamic interplay between enhancer-promoter topology and gene activity.Nat. Genet. 2018; 50: 1296-1303Crossref PubMed Scopus (195) Google Scholar, Lim et al., 2018Lim B. Heist T. Levine M. Fukaya T. Visualization of Transvection in Living Drosophila Embryos.Mol. Cell. 2018; 70: 287-296.e6Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Within endogenous regulatory landscapes, many naturally occurring enhancer-promoter contacts have been identified through derivatives of chromosome conformation capture (see Box 2) and other structural mapping approaches (for review, see Bonev and Cavalli, 2016Bonev B. Cavalli G. Organization and function of the 3D genome.Nat. Rev. Genet. 2016; 17: 772Crossref PubMed Scopus (2) Google Scholar). However, the relative strengths of these contacts vary significantly. Indeed, numerous enhancer-promoter contacts were only observed when averaged together in recent ultra-high-resolution Hi-C maps or when selectively enriched through Promoter-CaptureC or H3K27ac-HiChIP (Andrey et al., 2017Andrey G. Schöpflin R. Jerković I. Heinrich V. Ibrahim D.M. Paliou C. Hochradel M. Timmermann B. Haas S. Vingron M. Mundlos S. Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding.Genome Res. 2017; 27: 223-233Crossref Scopus (78) Google Scholar, Bonev et al., 2017Bonev B. Mendelson Cohen N. Szabo Q. Fritsch L. Papadopoulos G.L. Lubling Y. Xu X. Lv X. Hugnot J.P. Tanay A. Cavalli G. Multiscale 3D Genome Rewiring during Mouse Neural Development.Cell. 2017; 171: 557-572.e24Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, Mumbach et al., 2016Mumbach M.R. Rubin A.J. Flynn R.A. Dai C. Khavari P.A. Greenleaf W.J. Chang H.Y. HiChIP: efficient and sensitive analysis of protein-directed genome architecture.Nat. Methods. 2016; 13: 919-922Crossref PubMed Scopus (526) Google Scholar). Thus, enhancer-promoter contacts appear to represent genuine, albeit variable, structural features of regulatory landscapes.Box 2Mapping Chromatin Structure by Chromosome Conformation Capture (3C)C-technologies employ digestion of crosslinked chromatin to generate complexes of DNA fragments and their bound proteins. Proximity between loci (contact frequency) is then determined by the incidence that fragments are ligated together. Many 3C derivatives exist (for review, see Bonev and Cavalli, 2016Bonev B. Cavalli G. Organization and function of the 3D genome.Nat. Rev. Genet. 2016; 17: 772Crossref PubMed Scopus (2) Google Scholar). Hi-C enables genome-wide identification of contact frequencies, though resolution is often limited by cost. However, selected interactions can be enriched through immunoprecipitation or complimentary oligonucleotide probes. This enables interactions from selected proteins (HiChIP), specific viewpoints (CaptureC), or entire genomic regions (Capture-Hi-C) to be affordably analyzed at high resolution. C-technologies employ digestion of crosslinked chromatin to generate complexes of DNA fragments and their bound proteins. Proximity between loci (contact frequency) is then determined by the incidence that fragments are ligated together. Many 3C derivatives exist (for review, see Bonev and Cavalli, 2016Bonev B. Cavalli G. Organization and function of the 3D genome.Nat. Rev. Genet. 2016; 17: 772Crossref PubMed Scopus (2) Google Scholar). Hi-C enables genome-wide identification of contact frequencies, though resolution is often limited by cost. However, selected interactions can be enriched through immunoprecipitation or complimentary oligonucleotide probes. This enables interactions from selected proteins (HiChIP), specific viewpoints (CaptureC), or entire genomic regions (Capture-Hi-C) to be affordably analyzed at high resolution. Interestingly, systematic analysis of different developmental time points and cell types by high-resolution Promoter-CaptureC have demonstrated both preformed (invariant) and facultative (activity-dependent) enhancer-promoter contacts, each of which influences gene regulation differently (Andrey and Mundlos, 2017Andrey G. Mundlos S. The three-dimensional genome: regulating gene expression during pluripotency and development.Development. 2017; 144: 3646-3658Crossref PubMed Scopus (65) Google Scholar). At invariant contacts, multiple proteins maintain enhancers and target promoters in close proximity independently of their activity. In this way, pre-established proximities can poise enhancer-promoter communication, allowing enhancers to immediately activate target genes. Indeed, during development, the PRC1 and PRC2 complexes generate Polycomb-repressed enhancer-promoter contacts, maintaining them in the close proximity necessary for robust gene induction during subsequent differentiation (Cruz-Molina et al., 2017Cruz-Molina S. Respuela P. Tebartz C. Kolovos P. Nikolic M. Fueyo R. van Ijcken W.F.J. Grosveld F. Frommolt P. Bazzi H. Rada-Iglesias A. PRC2 Facilitates the Regulatory Topology Required for Poised Enhancer Function during Pluripotent Stem Cell Differentiation.Cell Stem Cell. 2017; 20: 689-705.e9Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, Ogiyama et al., 2018Ogiyama Y. Schuettengruber B. Papadopoulos G.L. Chang J.M. Cavalli G. Polycomb-Dependent Chromatin Looping Contributes to Gene Silencing during Drosophila Development.Mol. Cell. 2018; 71: 73-88.e5Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Similarly, the zinc-finger protein CTCF and cohesin DNA-bridging complex generate a 1-Mb-spanning contact between the Shh promoter and ZRS enhancer (Paliou et al., 2019Paliou C. Guckelberger P. Schöpflin R. Heinrich V. Esposito A. Chiariello A.M.M. Bianco S. Annunziatella C. Helmuth J. Haas S. et al.Preformed chromatin topology assists transcriptional robustness of Shh during limb development.Proc. Natl. Acad. Sci. USA. 2019; (Published online May 30, 2019): 201900672Crossref Scopus (80) Google Scholar). This contact constrains Shh and ZRS within an average distance of ∼400 nm, irrespective of their activation or repression in different parts of the developing limb (Williamson et al., 2016Williamson I. Lettice L.A. Hill R.E. Bickmore W.A. Shh and ZRS enhancer colocalisation is specific to the zone of polarising activity.Development. 2016; 143: 2994-3001Crossref PubMed Scopus (80) Google Scholar) (Figure 3A). However, 80% of ZRS and Shh loci further move to within 200 nm when activated in posterior limb cells. As such, their pre-established proximity appears to support the generation of a closer activity-dependent contact that is necessary for Shh transcriptional activation (Williamson et al., 2016Williamson I. Lettice L.A. Hill R.E. Bickmore W.A. Shh and ZRS enhancer colocalisation is specific to the zone of polarising activity.Development. 2016; 143: 2994-3001Crossref PubMed Scopus (80) Google Scholar). Moreover, similar analogous progressions from a protein-driven contact to an activating proximity have also now been observed directly by live-cell imaging in Drosophila (Chen et al., 2018aChen H. Levo M. Barinov L. Fujioka M. Jaynes J.B. Gregor T. Dynamic interplay between enhancer-promoter topology and gene activity.Nat. Genet. 2018; 50: 1296-1303Crossref PubMed Scopus (195) Google Scholar). Thus, proteins such as CTCF and cohesin or PRC1 and PRC2 can constrain inactive enhancers and promoters in close proximity and enable subsequent activation-associated contacts to robustly form and induce transcription. By contrast, facultative interactions form and disassemble dynamically during development, coinciding with transcription and active chromatin modifications at contacting enhancers and promoters (Andrey et al., 2017Andrey G. Schöpflin R. Jerković I. Heinrich V. Ibrahim D.M. Paliou C. Hochradel M. Timmermann B. Haas S. Vingron M. Mundlos S. Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding.Genome Res. 2017; 27: 223-233Crossref Scopus (78) Google Scholar, Bonev et al., 2017Bonev B. Mendelson Cohen N. Szabo Q. Fritsch L. Papadopoulos G.L. Lubling Y. Xu X. Lv X. Hugnot J.P. Tanay A. Cavalli G. Multiscale 3D Genome Rewiring during Mouse Neural Development.Cell. 2017; 171: 557-572.e24Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, Javierre et al., 2016Javierre B.M. Burren O.S. Wilder S.P. Kreuzhuber R. Hill S.M. Sewitz S. Cairns J. Wingett S.W. Várnai C. Thiecke M.J. et al.BLUEPRINT ConsortiumLineage-Specific Genome Architecture Links Enhancers and Non-coding Disease Variants to Target Gene Promoters.Cell. 2016; 167: 1369-1384.e19Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar). This link to transcription suggests facultative contacts act to physically transmit enhancer activities. However, their dynamic nature also allows facultative interactions to guide enhancer activity in different ways. For instance, though active in both embryonic hind- and forelimbs, the Pen enhancer drives Pitx1 expression only in the hindlimb through a dynamic topological switch. Specifically, a hindlimb-specific locus configuration reduces the large distance normally separating Pen and Pitx1, thereby bringing them into close spatial proximity to drive Pitx1 transcription uniquely in that tissue (Figure 3B) (Kragesteen et al., 2018Kragesteen B.K. Spielmann M. Paliou C. Heinrich V. Schöpflin R. Esposito A. Annunziatella C. Bianco S. Chiariello A.M. Jerković I. et al.Dynamic 3D chromatin architecture contributes to enhancer specificity and limb morphogenesis.Nat. Genet. 2018; 50: 1463-1473Crossref Scopus (93) Google Scholar). Similarly, locus control region (LCR) enhancers switch contacts between embryonic and adult β-globin genes during erythroid differentiation, thereby sequentially activating them at the correct developmental stages (Figure 3C) (Deng
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