Transcriptional enhancers play a key role in the initiation and maintenance of gene expression programmes, particularly in metazoa. How these elements control their target genes in the right place and time is one of the most pertinent questions in functional genomics, with wide implications for most areas of biology. Here, we synthesise classic and recent evidence on the regulatory logic of enhancers, including the principles of enhancer organisation, factors that facilitate and delimit enhancer–promoter communication, and the joint effects of multiple enhancers. We show how modern approaches building on classic insights have begun to unravel the complexity of enhancer–promoter relationships, paving the way towards a quantitative understanding of gene control. The first transcriptional enhancers were identified about 40 years ago, and their critical role in development has been clear for at least two decades. Advances in recent years have led to the identification of millions of enhancers active in an ever-expanding array of cell types. Meanwhile, population genetics studies have revealed the enrichment of these elements for genetic variants associated with common diseases. Most recently, targeted perturbation and imaging techniques have taken functional analyses of enhancers to a new level of scale and resolution. As the quest for a mechanistic understanding of enhancers continues, the function of these elements emerges as a complex phenomenon that integrates multiple levels of nuclear organisation, from primary DNA sequence and sequence-specific transcription factors to higher-order chromatin architecture through chromatin remodelling complexes, chromosomal loops and potentially phase-separated condensates. In this review, we synthesise classic and recent evidence on the organisation and function of enhancers, focusing in particular on the principles governing their communication with target gene promoters.In complex organisms, the vast majority of genes are not controlled by promoters alone, but additionally receive input from one or more non-coding DNA β-globin genes from varying distances from their promoters, regardless of its orientation [Enhancers were first described around 20 years after the discovery of the gene promoter –7. In 19entation , 5. At aentation . We now entation , 12.Drosophila [Drosophila, newly discovered enhancers orchestrated the regulation of developmental genes that were crucial for the correct formation of the embryo [Enhancers were soon discovered in the genomes of other model organisms in a range of complexities from yeast  to Drosoosophila  and mousosophila –17; enhaosophila –22. In De embryo , 24. It e embryo –28.Drosophila, the Zinc Finger Early Drosophila Activator (Zelda) is critical for lowering the nucleosome barrier at enhancers for genes driving zygotic gene activation [.At the DNA level, enhancers represent clusters of binding sites for sequence-specific transcription factors (TFs) Fig. A  proteins and the Mediator complex , 57. TheRecent experiments degrading Mediator and BRD4 have shown that these proteins are critical for gene expression . In partThe majority of active enhancers are themselves transcribed, producing enhancer RNAs (eRNAs) in a bi-directional manner –77. ThespS2 [The chain of molecular events following the binding of a pioneer TF to its target sequence  has been elegantly documented in a fine-grained time-course experiment following oestrogen receptor activation in a cancer cell line . This stpS2 .). The various proposed models for enhancer grammar range from very rigid to flexible, largely dependent on the species, cell types and loci in which they were formulated . At theiewed in . The modiewed in , 89. In nisation , 91. At function , 92. Enhfunction , 93. An function , 91. TF t of TFs . Similart of TFs . Flexibit of TFs . Biophyst of TFs –98. For t of TFs . AdditioThe prevalence of flexible enhancer organisation is also supported by rapid evolutionary turnover of enhancers, which exceeds that of proximal promoter regions , 99, 100Drosophila, SMF can detect the binding of multiple TFs on a single DNA molecule [Drosophila where co-occupied DNA-bound TFs were often spaced quite widely apart (> 50 bp) [While it is clear that enhancers typically favour the more flexible models of sequence organisation, the degree of necessary cooperation between TFs is less obvious, particularly since TF binding to DNA appears to be very transient –117. Recmolecule . SMF shomolecule . SMF hasmolecule . Notablymolecule . Interesmolecule , consist> 50 bp) . JointlyDrosophila embryonic development, which is driven by the expression of pair-rule genes such as even-skipped (eve) [eve expression in early embryogenesis via binding of tissue-specific TFs [How does the flexibility in enhancer organisation result in the robust control of gene expression? In part, this is achieved through tight regulation of the expression of the TFs themselves. One of the best-characterised examples of this is the development of gene expression “stripes” in ed (eve) . The strific TFs –123. Thiific TFs , 124. Anific TFs –128. Conific TFs . FinallyActive enhancers reside in open chromatin and occupy regions away from gene TSSs. However, open chromatin regions are not limited to active enhancers, and also harbour other regions including active gene promoters, regulatory elements in a ‘poised’ chromatin configuration (discussed below), as well as some structural regions such as tissue-invariant binding sites for the architectural protein CCCTC-binding factor (CTCF) –132. PosDrosophila and Caenorhabditis species (through modENCODE) [cis regulatory elements in more than 500 cell or tissue types in human and mouse [The epigenetic hallmarks of enhancers, including histone modifications and the binding of cofactors such as P300, have enabled their detection at a global scale. Following pioneering studies in a small number of cell types –148, lardENCODE) , 153. Whnd mouse . Finallynd mouse . The recnd mouse  and CUT&nd mouse  methods nd mouse , 158. Cond mouse , 159–161While enhancer detection based on chromatin profiling has become standard, this approach may falsely detect regions without appreciable regulatory activity, as well as miss some functional elements , 162. FoDrosophila, for example, point mutations in the binding site for the Kruppel TF within an enhancer controlling the abdominal fate specifier abd-A results in its misexpression in a thoracic segment, leading to its conversion to an abdominal one [Drosophila homolog of Pax2 affected mechanosensory bristle development [PAX6 expression causes the congenital eye malformation aniridia [SHH) gene are associated with several types of congenital limb malformation and skeletal abnormalities [SHH and within an intron of another gene, as will be further discussed below). Functional analysis of the ZRS enhancer revealed that these mutations either create gain-of-function binding sites for activating TFs (ETS1 and GABPα) [SHH expression. Other examples of genes mis-regulated by point mutations in enhancers leading to human disease include TBX5  [PTF1A (pancreatic agenesis) [IRF6 (Van der Woude syndrome) [While much of the evidence for enhancer function was obtained through perturbing these regions artificially, natural genetic variation at enhancers is commonly observed in human populations, consistent with the relatively low sequence constraint of these regions , 192. Muinal one . Likewiselopment . In humaaniridia . This muaniridia . Point mmalities , 198 (nod GABPα) , or abold GABPα) , leadingdisease) , PTF1A (genesis)  and IRF6yndrome) . Capitalyndrome) –207.FTO gene, but lead to the dis-regulation of the distal IRX3 gene [TNFAIP3 has been shown to abrogate NF-kB binding, affecting TNFAIP3 expression [The importance of enhancer variation in human pathology has been further highlighted by genome-wide association studies (GWAS), since GWAS-detected genetic variants associated with complex traits and diseases are typically non-coding, non-promoter associated and highly enriched in enhancers , 209. A RX3 gene . In anotpression , 212. Supression –215. Beypression –219, DNApression –223 and pression , 225 canNotably, some enhancer variants that appear functionally neutral when tested under normal steady-state conditions may show “cryptic” effects under stress or upon stimulus response , 227. Foin cis (as promoters), they may regulate lineage-specific or inducible genes distally (as enhancers) [in cis and in trans may not be fully equivalent.Contrary to the initial assumption that enhancers and promoters were biologically distinguishable elements, it is becoming clear that they share many properties (reviewed in ). The ENhancers) , 247. Inhancers) . TherefoDrosophila assay, unidirectionally transcribing elements were more likely to drive strong promoter and limited enhancer activity, whilst bi-directionally transcribing elements had strong potential for enhancer activity and could also function as weak promoters [Recently, it has been proposed that cis-regulatory elements exist on a spectrum of enhancer/promoter ability , 248. Onromoters . Promoteromoters . In termromoters , 249. Thromoters .The potential interchangeability of enhancers and promoters is relevant for functionally interpreting promoter–promoter contacts that are abundantly detected in the genome, as will be discussed later in the review.Shh ZRS enhancer, which is located within the intron of the Lmbr1 gene approximately 1 megabase distant from Shh [Whilst enhancers were classically tested by cloning them upstream of a core promoter, it quickly became clear that in their native context they are often located large distances away from the genes they control. The classic example of this is the from Shh . In thisfrom Shh , 251, hefrom Shh , and canfrom Shh .β-globin locus, in which the developmental β-globin genes are regulated by a powerful tissue-specific enhancer known as a locus control region (LCR) [Historically, several conceptual models were proposed for enhancer–promoter communication. These models were initially applied to the mammalian on (LCR) –257. It on (LCR) . Loopingon (LCR) . Therefoon (LCR) –261. In on (LCR) . Accordion (LCR) , 264. Aton (LCR) .β-globin LCR forms chromosomal loops with the active β-globin genes in vivo. Peter Fraser’s team used a method called RNA TRAP (“Tagging and Recovery of Associated Proteins”), in which horseradish peroxidase (HRP)-labelled probes bind to mRNA of specific genes and catalyse the deposition of biotin on chromatin proteins in close proximity with the transcript [β-globin locus [β-globin genes, whilst the intervening inactive genes are looped out [β-globin genes are expressed, but not in brain cells, in which they are not [β-globin genes depending on the stage of erythroid cell development [The development of novel techniques provided evidence that looping mechanisms are in fact widespread, even though the picture that is currently emerging is more complex, as will be discussed later. Two papers published in 2002 used different novel biochemical techniques to demonstrate that the anscript . Loci thanscript . In parain locus , after iin locus . In 3C, in locus . 3C confoped out . Enhance are not . Furtherelopment . These f3C technology, which probes the interactions between a single “anchor” with multiple “other end” fragments, was soon expanded to increase throughput. For example, 4C (“Circularized Chromosome Conformation Capture” or “Chromosome Conformation Capture-on-Chip”) employs a secondary restriction digest and inverse PCR to profile all loci interacting with the anchor fragment , 270. InThe high complexity of libraries generated by 3C-derived methods necessitates very deep sequencing for identification of individual enhancer–promoter loops. This has nevertheless been achieved in a number of cell lines, providing further evidence for looping in transcriptional regulation . TechnolShh and HoxD [Imaging techniques provide a complementary way to ascertain the spatial relationships in the genome. The ‘classic’ DNA fluorescence in situ hybridisation (FISH)  enables and HoxD , 304. Unand HoxD . Single-and HoxD ). Howeveand HoxD . Therefoand HoxD . At presand HoxD , 309. Fiand HoxD –312.β-globin and its LCR was shown to rescue β-globin expression in K562 erythroid cells (which instead aberrantly express γ-globin) in a reversible manner [β-globin derepression was not achieved [The recently developed “forced looping” methodologies have made it possible to probe the causal relationship between chromatin looping and gene expression , 314. The manner . Forced achieved . Hence, β-globin locus, it was ascertained that several TFs were required for looping between the LCR and the distal genes, including GATA-1, FOG-1, EKLF and NLI/Ldb1 [Following the discovery of chromatin looping in the NLI/Ldb1 –317. Howβ-globin and H19/Igf2 loci [β-globin locus then showed that two insulator sites binding CTCF could form a chromatin loop [β-globin locus were found to physically interact in erythroid progenitor cells, forming a large domain encompassing the LCR and its target genes [CTCF is a remarkably versatile protein that contains eleven highly conserved zinc fingers with both DNA-binding and protein-binding capacity . Reportegf2 loci –326. Dozgf2 loci  and in vgf2 loci –330, andgf2 loci . Accordigf2 loci . This wagf2 loci . Transgetin loop . Indeed,et genes , 333.in trans (in sister chromatid cohesion) and in cis . CTCF is not directly required for cohesin loading and unloading from chromatin, which is mediated by other factors . Insteaiewed in , 342. Coiewed in .Vcan) promoter and Xrcc4 epromoter spanning over 350 kb is significantly reduced, and Vcan expression diminished, upon deletion of the CTCF motif at the Vcan promoter. Artificial tethering of CTCF to the Vcan promoter using a dCas9-CTCF construct effectively rescues the loop and, partially, the expression of Vcan [Beyond mediating specific enhancer–promoter loops, the cooperative action of cohesin and CTCF has emerged as a central organiser of the global chromosomal architecture through a mechanism termed loop extrusion, which will be discussed later in the text , which has E3 ligase activity causing the monoubiquitination of histone H2A lysine 118 (Drosophila) or 119 (vertebrates), and Polycomb Repressive Complex 2 (PRC2), which catalyses the di- or tri- methylation of histone H3 lysine 27 [Drosophila, PcG complexes aggregate into foci known as Polycomb bodies, which contain co-repressed genes such as Hox gene clusters  complexes have long been known to orchestrate the epigenetic silencing of chromatin throughout development. Initially discovered in ysine 27 . These hysine 27 ). In Droiewed in ). Withinilencing . These fla cells , 356. Inla cells , 357. Mala cells .Polycomb-mediated looping is likely to work largely independently of CTCF and cohesin and is potentially even counteracted by the looping activities of these factors in ESCs . Whilst Drosophila [Notably, some contacts between PcG-associated regions, including poised promoters and enhancers in ESCs, are retained after these regions are activated during cell differentiation , 143. Moosophila . JointlyYY1 and ZNF143 are both zinc finger TFs with DNA binding domains that have recently been implicated as looping factors mediating interactions between active regulatory elements , 362–365The mechanisms by which YY1 or ZNF143 mediate regulatory chromatin contacts are still under investigation. YY1 does not seem to co-localise with CTCF  and instβ-globin locus [LDB1 significantly disrupted the expression of the gene programs underpinning appropriate cell differentiation [LIM domain-binding protein 1 (LDB1) is a dimeric cofactor that does not have direct DNA-binding capacity, but interacts with the LIM domain of other TFs to form multi-protein complexes . LDB1 wain locus , 371. Lain locus , 373 andntiation , 373. Ecntiation , 374. Jontiation . Consistntiation . LikewisThe multimeric Mediator complex is recruited to active enhancers, where it initiates the assembly of the pre-initiation complex and recruitment of RNAP . MediatoMediator recruitment to active enhancers is facilitated by the bromodomain and extraterminal domain (BET) protein BRD4 , 71 thatThus, despite their importance for transcription, Mediator and BRD4 are likely not essential for looping between regulatory elements. Instead, it was suggested that Mediator can aid transcription by supplying TFs and transcriptional machinery to the promoter by diffusion over a small distance, which would not require a direct “bridging” interaction . Whilst The transcriptional process and associated RNA molecules can also influence DNA architecture. Whilst it is established that transcription helps shape up higher-level genome conformation (reviewed in ), there eve locus in Drosophila detected a causal association between transcription and the enhanced compaction of the locus, bringing the eve enhancers into close proximity with the promoter [There is also evidence suggesting that transcription stabilises enhancer–promoter loops , 311. Lipromoter ), potentpromoter , 390. Knpromoter , 390. Otpromoter . Howeverpromoter , 380.In conclusion, while cohesin and CTCF remain the established key organisers of chromosomal loops, there is evidence for loops independent of these proteins and of additional looping factors, including potentially RNAP and/or transcription itself. To what extent this explains cohesin-independent contacts and the small effects of cohesin/CTCF deletion on gene expression still remains unclear, prompting the question of whether mechanisms other than direct looping can mediate enhancer–promoter communication, as discussed below.Drosophila, pronounced changes in enhancer activity during embryonic development seem to occur without prominent changes in their 3D conformation [Drosophila and mammalian models. Nonetheless, a significant minority of enhancers in human ESCs also show pre-existing “permissive” promoter contacts prior to activation [A long-standing question in the field has been whether enhancer–promoter contacts form concomitantly with the activation of these regions (“instructive”) or, alternatively, precede it (“permissive”) . There aormation , 393. Inormation , 394–397ormation . One postivation , 394. Intivation .The presence of both instructive and permissive contacts reflects the likely diversity of mechanisms facilitating enhancer–promoter connectivity. One proposed mechanism linking enhancer activity with connectivity is based on the observation that cohesins can be recruited to H3K4me1-marked chromatin, which is associated with the “primed” state of enhancers and the binding of MLL/Trithorax transcriptional coactivators . AdditioWhilst there is now a definitive bulk of evidence in support of physical looping between enhancers and promoters, several recently described phenomena suggest that looping is not the only mechanism of how these regions can communicate, as will be discussed in this section.Sox2 gene has an essential enhancer, the Sox2 Control Region (SCR), which was found by 3C-derived techniques to loop to the Sox2 promoter . Hcription . Moreoveechanism . While tch other , as woulch other . Finallych other , 406. ThShh gene moves further away from the Shh promoter upon activation, according to both 3C-derived methods and imaging [Shh expression [Shh promoter, bypassing the need for enhancer activation and causing increased transcription, did not lead to increased distance [Shh promoter is well-established, this suggests that multiple enhancers may control the expression of their shared target gene through a combination of looping and non-looping mechanisms.In contrast, a proximal enhancer of the  imaging . Recruitpression . It has pression  whereby pression . Howeverpression . Notablydistance . CombineDrosophila development  [Drosophila embryos irrespective of the activity of these elements [Finally, multiple lines of recent evidence suggest that many functional enhancers likely do not come into direct contact with their target promoters upon d above) . For exaelements . In addielements .It has been proposed that chromatin can compartmentalise into membrane-less biomolecular condensates in the phenomenon known as liquid–liquid phase separation (LLPS) . SeveralSox2 promoter observed in live imaging data by a combination of looping and phase separation [The engagement of cofactors Mediator and BRD4 in LLPS could explain why their depletion results in transcriptional changes without significantly affecting chromatin loops , 380. Hoparation .Whilst LLPS in general has been demonstrated both in vitro and in vivo , 422, evThe recent interest in LLPS has prompted calls for more rigorous experimental guidelines on its identification and characterisation in the cellular environment , 424. FoNotably, phase-separated condensates might form and dissolve in accordance with local chromatin features, such as histone tail acetylation, the presence of linker histone H1 presence and inter-nucleosome spacing, as well as the concentrations of recruited TFs , 415. MoIt is clear from both genome-wide profiling and perturbation studies that, of the multitudes of enhancers in a locus, not all of them provide input to every promoter , 431. ThIt has become evident from early analyses of Hi-C data that metazoan genomes are organised into megabase-scale segregated domains that are often invariant between cell types, termed topologically associating domains (TADs) –434. WhiEPHA4 expression and a set of adjacent genes, including WNT6 and PAX3, results in their misexpression and abnormal limb development in both mice and humans [SOX9 locus [Drosophila balancer chromosomes compared with their wild-type counterparts result in changes in the expression of only ~ 10% of genes [Shh locus did not entirely disrupt the developmental regulation of Shh expression [TAD boundaries are classically defined through a change in the predominant direction of chromosomal contacts observed in Hi-C data . The majd humans . More reX9 locus . HoweverX9 locus –347, 448of genes . Likewispression , 451.Drosophila embryo, enhancer–promoter loops are formed before TADs, and likely also precede the onset of zygotic transcription [Cis-regulatory contacts were also shown to emerge prior to the formation of TADs in murine cells exiting mitosis [The less pronounced than expected role of TADs in gene control can be partially explained by the ability of some enhancer–promoter contacts to form and/or be maintained independently of TAD constraints. For example, Capture Hi-C analyses in multiple cell types consistently detect enhancer–promoter contacts that cross TAD boundaries , 243. Recription , 454. Ci mitosis .Another layer of complexity in the relationship between chromosomal domains and specific enhancer–promoter contacts arises from the fact that active promoters and enhancers can likely give rise to TAD boundaries even in the absence of convergent CTCF sites . DisentaIn conclusion, whilst there is clear evidence for a role of some TADs in gene control, the emerging view is more nuanced than originally envisaged. Rather than being fully insulated structures that predetermine enhancer–promoter contacts, TADs and related loop domains can be seen as flexible hierarchical features of genomic organisation that loosely contain, but also can potentially be generated by, enhancer–promoter contacts.th of the genome undergoes compartment changes during the 24 h circadian cycle in mouse adult liver [cis-regulatory elements (particularly those independent of cohesin/CTCF) also participate in compartment formation currently remains an open question.Beyond TADs, other aspects of the global chromatin structure may also play a role in enhancer–promoter communication. Most prominently, contacts from active chromatin and heterochromatin regions appear largely segregated into separate compartments  . These “lt liver . The A/Blt liver , 346–348lt liver . It has lt liver , 461. WhA subset of B compartments corresponds to lamina-associated domains (LADs) . LADs reChromosomal contacts follow the law of constrained Brownian motion (reviewed in ), with tShh locus suggests that within-TAD chromosomal contacts there do not show a pronounced distance dependence [Sox2 promoter. Consistent with this, in the ectopic context this enhancer–promoter pair did not show prominent specific interactions, despite the clearly detectable transcriptional effects of SCR [The effects of distance, however, are likely not absolute, even within the same TAD. For example, analysis in the pendence . In addipendence , 468–471pendence ). In conpendence  discusses of SCR . TherefoDrosophila that used randomly integrated reporter genes driven by a TATA- and a DPE-containing promoter, respectively, showed that some endogenous enhancers had a preference for either of the two promoters [Drosophila cells revealed distinct groups of enhancers that preferred one of two types of core promoter, derived from either a housekeeping or a developmental gene and enriched for different TF binding motifs [Even within the same TAD, enhancers are not always fully promiscuous and can sometimes “skip” nearby genes, in favour of more distal targets , 473. Seromoters . More reg motifs . Notablyg motifs .Drosophila model system, there is currently little evidence for inherent enhancer–promoter compatibility. Further insights on potential selectivity may yet come from machine learning methods that are gaining the ability to predict enhancer–promoter pairs from sequence alone in mammalian cells [Beyond the an cells , 477. Whan cells . In addian cells .in cis by TFs, following which their contacts with target promoters are then “facilitated” by the underlying chromosomal topology and then further “specified” by parameters of enhancer–promoter compatibility [The findings discussed in this chapter show a diversity of the aspects of nuclear organisation that regulate enhancer–promoter communication. The outstanding question is how exactly these multiple aspects work together to establish appropriate enhancer–promoter relationships. One way to conceptualise this complexity at the current level of understanding is to assume enhancer–promoter pairing to be a largely stepwise process, as proposed recently by Schoenfelder and Fraser . In thistibility . FurtherIt follows from the rather flexible requirements for enhancer–promoter selectivity (discussed above) and the very large number of enhancers active in a given cell at a time that genes often receive output from multiple enhancers. It is also currently clear that the same enhancer can control multiple genes. Here, we discuss the functional and structural considerations around this multiplicity of enhancer–promoter relationships.Hox gene cluster, activation of genes with crucial roles in development is tightly controlled at each developmental stage by complex enhancer networks [Drosophila, eve expression is controlled by five separate enhancers to produce stripe patterning in the embryo [Enhancers vastly outnumber promoters in human and mouse genomes , and connetworks , 482. Ine embryo .cis-regulatory units (CRUs) [Multiple enhancers associated with a single promoter in a given cell type may be conceptualised as s (CRUs) . Analyses (CRUs) . Howevers (CRUs) . Upon eas (CRUs) . Changess (CRUs) . NotablyDrosophila Hox locus, where one enhancer regulates either pb or zen2 depending on the developmental stage via distinct chromatin loops [β-globin locus can simultaneously engage two adult globin genes, Hbb-b1 and Hbb-b2, while skipping over embryonic globin genes, in mouse fetal liver cells [The same enhancer can also regulate more than one target gene—either in different spatiotemporal settings or in the same one. An example of the former is found in the in loops . Exampleer cells . Likewiser cells .INK4a/ARF locus revealed that perturbing any promoter-interacting enhancer within the chain abolished the functionality of the entire network, but deleting the enhancers further down the chain had more moderate effects [In addition to multiple direct enhancer–promoter contacts, there is also evidence for enhancer–enhancer interactions that create an enhancer “chain” or network –489. In  effects . Further effects . CombineHox genes in ESCs (in which they are inactive) that were shown to depend on Polycomb Repressive complex 1 [3C-derived methods commonly detect multitudes of promoter-promoter contacts, typically in addition to promoter-enhancer contacts of the same genes. In some well-characterised cases, multiple promoters engage in combinations of pairwise interactions with each other, suggestive of promoter-promoter “networks”. These include contacts between the promoters of omplex 1 . A similomplex 1 , 492. Thomplex 1 . In addiomplex 1 , as wellomplex 1 .The subsections immediately above provide an empirical view on the multiplicity of enhancer–promoter and promoter-promoter relationships. How can such configurations be achieved structurally? Whilst 3C-derived data have indicated that interactomes comprising multiple interacting elements may be common in mammalian genomes , 494–496α- and β- globin loci respectively, provided evidence for higher-order hubs, containing three and more regulatory elements [Drosophila embryo to show that cis-regulatory elements cooperate to form hubs early in development prior to transcription, facilitated by the pioneer factor Zelda [Genome Architecture Mapping (GAM) is one technique that enables probing multi-way chromosomal contacts . GAM worelements , 485. Fielements ) now enaelements , 501–503elements . Hi-M waor Zelda . Notablyor Zelda , 498.β-globin locus, interactions between the LCR and the γ-globin or the β-globin genes can switch very rapidly on the same DNA strand [Evidence from live-imaging studies at candidate loci suggests that physical contacts between enhancers and promoters are often highly dynamic , 448. FoA strand . NonetheA strand , 507. ThHox cluster, a strong enhancer situated outside of the gene locus could prime the region, allowing for the emergence of novel enhancers [Sox9 sex determination locus leads to sex reversal, while the phenotypic effects of deleting stronger and more proximal Sox9 enhancers are much less pronounced [So far we have been considering multiple active enhancers in a unit as generally serving the same role. However, there is a possibility that some enhancers may also help others to associate with promoters, possibly in addition to their direct activity on those same promoters. For example, in the vertebrate nhancers , leadingnhancers  in an anonounced . Analysionounced . One posonounced , 512.Theoretically, cohesin/CTCF-associated enhancers are good candidates for pioneer enhancers for a number of reasons, most important of which is the selective requirement of cohesin for inducible gene control . CohesinWhat are the consequences of enhancer action on promoters? Conceptually, the simplest model would suggest that when an enhancer comes into proximity with a compatible promoter, it transmits to it a “dose of activation” that results in a certain level of transcriptional output .  [However, it has long been known that additivity in enhancer action is not absolute, and some enhancers work together either synergistically or sub-additively. For example, a mixture of additive, synergistic and sub-additive effects was demonstrated in a live imaging analysis of candidate developmental enhancers in  embryos . Sub-addutations –522. In (EpiLCs)  and at u(EpiLCs) .Some deviations from enhancer additivity could potentially be explained by steric constraints arising from enhancer competition  or involIn theory, the functional “decoupling” of enhancer–promoter contacts from transcriptional bursting may accommodate the existence of multi-enhancer hubs and non-looping mechanisms of enhancer–promoter communication. For example, such hubs formed through phase separation may help to cluster RNAP at gene promoters, prior to RNAP pause release that directly results in a transcriptional burst , 529. Hoin cis and in trans. However, the bulk of evidence of enhancer–promoter communication to date is based on 3C-derived methods that presume physical looping between these elements and necessitate a largely pairwise view on their relationships. Modern technologies gradually make it possible to transcend these limitations, enabling more direct investigations of non-looping mechanisms and multi-enhancer regulatory logic. We are also beginning to expand our conceptual understanding of enhancer types and states beyond a simple “on” and “off”, with emerging notions such as poised and pioneer enhancers. Single cell methods, particularly those based on high-resolution live imaging, combined with powerful genetic and epigenetic perturbation tools and state-of-the-art computational analysis paradigms, have the potential to significantly advance our ability to probe these concepts, paving the way towards comprehensive quantitative models of cis-regulatory gene control.Research over the last few decades has provided ample evidence for the key role of enhancers in metazoan gene regulation and the importance of their aberrations in disease. We now also have efficient tools for detection of enhancers and their target genes genome-wide in cell populations and, increasingly, in single cells. Jointly, these advances have improved our understanding of the regulatory logic and molecular mechanisms underpinning enhancer activity


Epigenetic regulators play a crucial role in establishing and maintaining gene expression states. To date, the main efforts to study cellular heterogeneity have focused on elucidating the variable nature of the chromatin landscape. Specific chromatin organisation is fundamental for normal organogenesis and developmental homeostasis and can be affected by different environmental factors. The latter can lead to detrimental alterations in gene transcription, as well as pathological conditions such as cancer. Epigenetic marks regulate the transcriptional output of cells. Centromeres are chromosome structures that are epigenetically regulated and are crucial for accurate segregation. The advent of single-cell epigenetic profiling has provided finer analytical resolution, exposing the intrinsic peculiarities of different cells within an apparently homogenous population. In this review, we discuss recent advances in methodologies applied to epigenetics, such as CUT&RUN and CUT&TAG. Then, we compare standard and emerging single-cell techniques and their relevance for investigating human diseases. Finally, we describe emerging methodologies that investigate centromeric chromatin specification and neocentromere formation. In 1942, for the first time, Waddington coined the term ‘epigenetic’ as the branch of biology that studies the causal interactions between genes and their cellular products and implements the phenotype [In past decades, the concept of epigenetic regulation has evolved, thanks to technological advances that have revolutionised the investigation of biological phenomena. Currently, epigenetics is defined as the science that studies stable and potentially heritable changes in gene expression and the phenotype occurring without alterations in the DNA sequence .The main epigenetic mechanisms include DNA methylation , histoneMost often, DNA methylation is found in association with silent chromatin states and typically takes the form of methylation of cytosine; such a modification mainly occurs on CpG islands, which are typically found in regulatory regions such as enhancers and promoters of genes or in repetitive DNA sequences .Histone modifications  are key regulatory modifications resulting in changes in transcription, DNA replication and chromosome condensation ,10,11. GNon-coding RNAs (ncRNAs) are a heterogeneous class of regulatory molecules derived from genes that are transcribed but not translated into proteins. They can be further classified in small-interfering RNAs (siRNAs), microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and long non-coding RNAs (lncRNAs) which have key roles in many biological processes ,14.Epigenetic modifications are continuously and dynamically modulated, especially by environmental factors. Indeed, the main benefit they provide lies in the ability to ‘fine-tune’ gene expression in line with progressive ontogenic and environmental changes. Thus, epigenetic markers are preferred developmental indicators ,16,17, bHuman health is impacted by epigenetics, and disruption in the correct balance of open and closed chromatin states can result in the onset of epigenetic machinery disorders. Growing evidence support that epigenetic machinery may profoundly affect human health.Mendelian disorders of the epigenetic machinery (MDEMs) are genetic diseases caused by mutations in genes coding for epigenetic factors and are often associated with intellectual disability, revealing that epigenetic mechanisms are very crucial for normal neurological development .In addition to monogenic diseases, epigenetic mechanisms are significantly involved in multifactorial diseases such as neurodegenerative disorders , metabolChromosomal instability (CIN) is a hallmark of many types of cancer. However, how CIN contributes to cancer progression remains to be elucidated. CIN is characterised by loss or rearrangement of genetic material during cell division, resulting in aneuploidy, chromosome structural alterations and segregation errors. Thus, CIN can increase genetic heterogeneity between tumour cells. A key structure for chromosome segregation is a chromatin region called the ‘centromere’. Centromeres recruit the kinetochore complex, a proteinaceous structure that provides the physical attachment to the microtubules of the mitotic spindle. Any alteration in the architecture of the centromere can result in cell cycle defects, leading to chromosomal instability. Indeed, the overexpression of CENP-A, the main epigenetic centromere marker, has been reported in several types of tumours, and it correlates with poor prognosis for patients. The molecular consequences of CENP-A overexpression in cancer cells are still unclear ,26,27. HEpigenetic marks can affect the cells’ transcriptional output, and consequently, their function. Therefore, understanding their effects on single-cell resolution can determine variations that occur in cell fate and function. Owing to this need, new techniques for studying single-cell epigenomics have been developed and previously deepened in some respects ,32,33,34In this review, we discuss single-cell methodologies that have recently emerged and that may provide new impetus in achieving the goals of ‘personalised medicine’. We present a critical review of such techniques examining strengths, challenges and limitations and offering insight in the epigenetic field into molecular components of the genome and its functional output.Over the last few years, single-cell sequencing (SCS) has emerged as a powerful set of technologies applied to a multitude of biological questions. Before the widespread adoption of single-cell genomics, RNA (in the form of cDNA) or (genomic) DNA sequencing was performed on nucleic acids isolated from whole tissue. Tissues are made of different cell types that are difficult to separate. Moreover, even within a single cell type, the population is heterogeneous, which in large part is due to the stochastic accumulation of mutations introduced during DNA replication . In thisIn light of the potential impact on several diseases’ development, epigenetics is a powerful field to study all mechanisms and risk factors contributing to their progression. Indeed, in an organism, the epigenome is normally established at the end of embryonic development and subsequently subjected to several modifications, which in not common cases, can unbalance the activation state of a gene, resulting in the onset of cancer . Even ifIn recent years, more attention has been paid to the phenomenon of cellular heterogeneity in which metabolism can play a role. The complexity of the mechanisms underlying the functioning of the metabolome, both in pathological and non-pathological conditions, is a topic of high interest to which transcriptomic and proteomics single-cell analysis have contributed enormously . In thisAdditionally, in cancer cells, many global epigenetic alterations have been observed, suggesting that epigenetic aberrations play a key role in cancer initiation and progression. In order to ensure effective diagnosis and treatment for cancer, scRNA-seq helps in the detection of genetic information, in the control of genes and in the difference between gene expressions in individual cells. Recently, Park et al. using scRNA-seq, revealed that the major contribution in pathogenicity in several mouse kidney diseases could be found in cell-type-specific markers, conversely to that which has previously been shown by transcriptomic studies studying the global influence of different cell groups .It has also been shown that obesity and the alteration of genes related to fat metabolism are important negative regulators of histone methylation and autophagic processes, as demonstrated by Pan et al. with single-cell RNA transcriptome sequencing in mouse embryos . AdditioDrosophila melanogaster have significantly contributed to deepening our knowledge of human diseases, unlocking molecular pathogenesis pathways in different fields, such as neurodegenerative disease, cardiovascular, metabolic and immunological disorders [Drosophila eye-disc development process, in which the authors have identified an altered glycolytic activity in Rbf (retinoblastoma family protein) mutant cells compared to wild-type, using a scRNA-seq study [Of note, studies in isorders . In an ieq study .Drosophila’s blood system has also been used as a useful tool for identifying new cell types that have not yet been discovered and potentially expressed even in the human immune system, acknowledging the analysis of single subtypes of cellular populations that with a common sequencing would not be recognisable [gnisable .In this regard, considering the importance as common epigenetic markers of miRNAs detection, histone variants and PMTs, and also histone detection in patient’s blood, could be a non-invasive system to research epigenetic markers that distinguish health from pathological conditions. Furthermore, in the last decade, new technological approaches have been emerged to develop strategies to employ epigenetic biomarkers in diagnostics for human health. Moreover, epigenetic compound development could be advantageous to heal several disorders that display resistance to standard treatment, leading to solutions for biomedical problems.Since diverse mutations can be encountered during the early cell divisions and sometimes cause genetic disorders and the onset of genetic variations, single-cell sequencing has allowed more clues on the cell type involved and on the disease progression. Indeed, single-cell genomics has made it possible to carry out wide-ranging studies of action in an organism’s life cycle, highlighting the effects of increased vulnerability during the early stages of development. The methods developed so far can allow the dissection of the single-cell genetic content in normal organs, pre-malignant states and established tumours, providing insights into the functioning of maintaining the genome in health and disease.Cellular differentiation is a highly dynamic process that involves continuous interactions between DNA and proteins such as transcription factors, histones and histone-modifying enzymes. Years ago, a lack of information on the genomic distribution of histone modifications prevented the full understanding of the ‘histone code’. This refers to the instructive role conferred by specific histone modifications and their combinations to the structural organisation and expression of the genome . Major aThe aim of ChIP techniques is to identify which DNA sequences are bound to variants of structural or functional proteins in the chromatin. ChIP-PCR is limited to investigating candidate sequences, whereas the development of ChIP-on-chip has opened the door to unbiased genome-wide studies. However, ChIP-on-chip approaches suffer substantial technical limitations. A microarray chip is a solid substrate on which thousands of DNA sequences called ‘probes’ are arranged in rows and columns such that position reflects identity. Thus, the identification of the DNA sequences part of the immune-precipitated chromatin relies on their hybridisation to the probes after an amplification step . Biases For many years ChIP-seq has been the method of choice for genome-wide mapping of protein–DNA interactions. However, some concerns have been raised recently ,77. In aE. coli, which can only access DNA regions not wrapped around histones. After, lysis DNA fragments are generated by DNase I restriction and identified by Next Generation sequencing (NGS) [Recent advances on the traditional ChIP-seq method include the ChIP-exo assay that introduces a significant improvement in mapping protein–DNA interaction sites. It achieves up to a single base-pair resolution by employing an endo and exonuclease combination to cleave and remove all exposed DNA sequences but preserving those engaged in interactions with proteins . Additiong (NGS) . FAIRE-Sng (NGS) . Howeverng (NGS) . Furtherng (NGS) . This asng (NGS) . CompareThe techniques described above have been applied to the study of epigenetics in several contexts, including development, cell differentiation and cancer.Enzyme-tethering strategies such as DamID (DNA Adenine Methyltransferase Identification) , ChEC C.Escherichia coli and a chromatin protein of interest to map the protein contact sites on the DNA. Dam methylates the adenines of adjacent GATC sites in vivo. After DNA extraction, methylated GATC motifs are first enriched using a methylation-sensitive endonuclease (DpnI) that restricts unmethylated sites and then are mapped by sequencing to produce a genome-wide binding profile [In 2000, Bas van Steensel and Steven Henikoff introduced a method based on the fusion between the DNA adenine methyltransferase (Dam) enzyme of  profile . Unlike  profile . Thus, D profile  for stud profile . Indeed, profile , which h profile ,97,98,99Saccharomyces cerevisiae [Subsequently, two complementary methods, ChEC and ChIC, were developed to avoid the bias introduced by the chromatin fragmentation–solubilisation step in ChIP. The two approaches were first used to map the DNA binding sites of CBPs in the baker yeast revisiae . In ChECrevisiae . In ChICrevisiae . Additiorevisiae ,100. Therevisiae , and by revisiae . In summrevisiae .Candida albicans, which is implicated in fungal fitness and drug resistance [More recently, Donczew et al. used these methods to map the specific transcription coactivator’s localisation both in normal and pathological events . Furthersistance . Additiosistance . The manChromatin is crucial in regulating gene expression. Features such as occupancy by transcription factors (TFs), regions of DNA accessibility and histone modifications can vary between cell types, in response to stimuli and in pathological versus healthy conditions.Thus, a genome-wide understanding of chromatin profiling is needed to characterise pathological changes that occur in disease.Generally, from its first application, genome-wide chromatin profiling has been performed using the ChIP-seq method , but in 2+ results in DNA cleavage. The DNA fragments that have been generated are then used for library preparation. In contrast to other strategies, CUT&RUN has low backgrounds, resulting in a high-throughput genome-wide mapping. It is an attractive strategy that has overcome some methodological limitations such as crosslink and solubilisation issues [Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an efficient chromatin profiling technique similar to ChIC ,107. It n issues .In addition to this advantage and in contrast to standard ChIP-seq methodology, CUT&RUN can be carried out in few hours, beginning from only 100 cells for a histone modification profiling and starting from 1000 cells for a transcription factor .Arabidopsis, it has been used to generate a genome-wide map of the histone modification H3K27me3 (tri-methylation at lysine 27 of histone 3) in nuclei of the endosperm [CUT&RUN has been employed successfully in several model systems. For instance, in ndosperm , in the ndosperm  and in andosperm .Furthermore, an automated CUT-RUN procedure has been reported (AutoCUT&RUN), providing a high-throughput chromatin profiling of patients for clinical use in the biomedicine field .Derived from CUT&RUN, a modification called Cleavage Under Targets and Tagmentation (CUT &TAG) has emerged in the last few years ,109,110.CUT&TAG is quite inexpensive, fast and suitable for single-cell sequencing applications. The main limitation is that it requires access to specific antibodies whose affinity determines the overall efficacy of the reaction ,109,110.Compared to ChIP-seq that requires a high number of starting materials, making it not suitable for diagnostic applications, CUT&TAG is a higher-sensitivity technique that can be performed starting from a low number of cells. In addition, CUT&RUN and CUT&TAG have been carried out on unfixed and permeabilised cells, avoiding epitope masking generally associated with the ChIP method, in which DNA and proteins have cross-linked with formaldehyde or UV lights.CUT&TAG is a low-cost genome-wide chromatin profiling and show crucial advantageous features that allow it to be employed for clinical and diagnostic application in the biomedicine field. In fact, it is not so expensive, can give results in only a day and it does not need a great number of cells, resulting in a single-cell methodology. Indeed, it could be suitable to explore cell-to-cell heterogeneity, a hallmark of cancer cells.Despite being a very new technique, there are several publications describing CUT&TAG applications. For instance, the genomic profiling of native R loops, which plays a pivotal function in many biological processes with consequences in human disease . Yu et aBartosovic et al. used CUT&TAG to profile histone modifications and transcription factors occupancy at the single-cell level in the mouse brain .In addition to mammalian cells, the CUT&TAG technique has been recently used for epigenetic profiling in cotton cells, leading to improvement in plant epigenetic research and giving the opportunity to widely employ this methodology in other model systems ,120.CUT&TAG can be regarded as an ideal method to investigate cellular heterogeneity in physiological conditions and in human disorders such as in a patient tumour mass.In particular, we have already mentioned that the cancer cells population is characterised by a high heterogeneity that drives crucial issues in diagnosis and mostly in therapy and drug resistance. For this reason, single-cell omics techniques appear as optimal methodologies to investigate alterations at the single-cell level in cancer mass.Of note, Wu and colleagues developed the CUT&TAG technique for single-cell chromatin profile of brain tumour cells from an autopsy of a patient before and after cancer therapy, giving the opportunity to explore tumour heterogeneity .Therefore, the improvement of methodologies helps us to understand better physiological epigenetic mechanisms, clarifying alterations involved in diseases.The centromere is a typical example of epigenetic regulation. Indeed, it is an epigenetically regulated chromosome structure critical for fidelity in cell divisions. Alterations in centromere regulation can induce aberrant mitosis and chromosome instability observed in cancer and many pathologies.Therefore, it is crucial to gain insight into mechanisms that control epigenetic regulation of the centromere to control aneuploidy in cancer cells since several evidences show a correlation between centromere dysfunction and neoplasia ,123.These emerging single-cell techniques could be used in the next few years to elucidate more in-detail epigenetic regulation of the centromere, contributing to understanding pathological mechanisms in diseases.Finally, these enzyme-tethering techniques, and in particular CUT&TAG, are efficient methods for epigenetic factors profiling in a high-resolution manner and low background, using low cell numbers or single cells, opening the way to epigenetic studies in any field of biological research.Centromeres are regions of the chromatin that are crucial for the faithful segregation of chromosomes during mitosis. They are the site of assembly of the kinetochore complex, a proteinaceous structure that binds to the microtubules of the mitotic spindle driving chromosome segregation. Their disfunction can lead to genome instability or to an aberrant cell cycle resulting in tumorigenesis .Centromeres are characterised by repetitive DNA sequences ,126 and A specific type of centromeric chromatin, named ‘centrochromatin’ is found in addition to CENP-A. This is characterised by a specific pattern of histone modifications that is crucial for CENP-A recruitment and centromere function ,135,136.A correct balance between specific epigenetic modifications is required to promote the loading of CENP-A to centromeric regions and to prevent the spreading of heterochromatin, avoiding centromere and kinetochore inactivation .ChIP experiments have enabled most studies on the epigenetic regulation of the centromere. Notably, it has been difficult to map histone modifications on centromeric repetitive DNA sequences employing ChIP-seq methodologies  as they As with other histones, CENP-A is also modified by post-translational modifications, including phosphorylation, methylation, acetylation and ubiquitination, which are critical for centromere specification ,142,143.The characterisation of CENP-A post-translational modifications can have a pivotal role in cancer research. Because they may lead to chromosome segregation defects and chromosomal instability, they could be helpful diagnostic markers or therapeutic targets . MoreoveEmploying emerging techniques, it would be interesting to analyse CENP-A modifications at the single-cell level in cancer cells to better evaluate their contribution to cancer initiation and progression, which could become a helpful diagnostic marker or therapeutic target.Thus, investigating specific histone modifications, including CENP-A modifications, could be an exciting topic for future perspectives in order to further explore epigenetic mechanisms in centromere regulation.Indeed, innovative genome-wide approaches to study epigenetic features as chromatin modifications are recently developed, revealing new aspects of biology. The application of these techniques has greatly advanced our knowledge of epigenetic processes and how they are regulated and could be used for centromere regulation exploration.C. elegans centromeres have been analysed using ChIP-on-chip with CENP-A antibodies. These investigations have confirmed CENP-A presence along the chromosome arms. In addition, a link has emerged between transcription and CENP-A deposition in nematodes. In fact, CENP-A is found in chromosome regions that are not transcribed in the germline and in the first stages of embryonic development [In the last decade, elopment .Usually, centromeres occupy specific chromosome regions, but sporadicallym they shift to ectopic sites driving neocentromere formation. Neocentromeres have been identified in different organisms. Generally, they are formed following a chromosomal rearrangement to stabilise an acentric chromosome. Such events are still poorly understood, although some models have been proposed ,151,152.Different methodologies provide an opportunity to investigate such rare events. For instance, ChIP-seq has been instrumental in exploring the epigenetic environment surrounding neocentromeres. Shang et al. have described a chromosome-engineering system to experimentally generate neocentromere in chicken DT40 cells. Using a specific CENP-A antibody and ChIP-seq, they demonstrated the presence of CENP-A in ectopic centromeres and clarified the epigenetic features occurring in those regions .Recently, Pineda and colleagues described for the first time a system to characterise a ‘spontaneous’ neocentromere in human culture cells, opening a new era for centromere research.In this work, they could observe early driven events and subsequently step-by-step neocentromere maturation in cellular generations. First, using CENP-A immunoprecipitation followed by long-read DNA sequencing, they identify genomic features of neocentromere confirming the epigenetic nature of the event supposed previously ,155.In some cases, new centromeres have been observed in patients with developmental disorders during karyotyping analysis  and in hIndeed, chromosomal aberrations are a common feature in cancer pathogenesis, and genomic anomalies can contribute to neoplastic progression and metastasis ,157.CENP-A is often overexpressed in some type of tumors, and for this reason, its detection could have diagnostic and prognostic values ,26,27. IFor instance, CENP-A deposition in ectopic sites can generate a neocentromere, leading to a dicentric chromosome and chromosome rearrangements. Moreover, CENP-A can change endogenous transcription and alter gene expression.Recent studies have shown that CENP-A plays a protective role in centromere integrity, preventing centromeric satellite DNA from rearrangements. This suggests that centromere aberrations in cancer cells could be associated with CENP-A dysfunction .Chromosomal instability is a hallmark of human cancer. Tumorigenesis is a stepwise process that leads to heterogeneity in cell populations. Therefore, it may be helpful to monitor cell diversity over time to characterise cancer evolution and to inform therapeutic strategies.Recently, Bolhaqueiro et al. reported a system to detect chromosomal instability in human colorectal tumour cells during cell division using microscopy. In addition, they repeatedly performed sc-Karyo-seq, a single-cell DNA sequencing technique, to detect chromosomal changes across cell divisions, revealing karyotype heterogeneity .To study centromeric chromatin in native conditions, Thakur and Henikoff have employed CUT & RUN combined with salt fractionation . CUT and RUN, as previously described, is a powerful method for chromatin profiling. The authors demonstrated that even slight differences in centromeric satellite sequences could cause changes in the architecture of the chromatin and in the binding of proteins to the centromere ,162.Using innovative single-cell techniques such as CUT&TAG, it would be interesting to study in detail if CENP-A islands in non-canonical regions of the genome differ from one cell type to another and investigate whether CENP-A occupancy in ectopic sites occurs at random or if it localises in defined genome regions following some sequence-specificity.Hence, the CUT&TAG methodology could be useful to investigate CENP-A attendance in the genome at the single-cell level.Finally, we have shown how ChIP-seq and other omics techniques have been helpful to investigate epigenetic features on centromeric chromatin such as CENP-A distribution and histone post-translation modifications in endogenous centromere and in ectopic centromeres. Indeed, these techniques have contributed to clarifying epigenetic mechanisms that drive neocentromere formation and characterising genomic features of de novo centromeres.In light of this, we can speculate that innovative technological approaches could be suitable to rather explore epigenetic involvement in centromere specification and useful for epigenetic studies in clinical research and biomedicine.Epigenetic modifications are what determine the specialisation of a cell, and for this reason, epigenetic mechanisms impinge on fundamental biological phenomena such as gene expression and chromosome integrity.Alterations of epigenetic processes have been associated with multiple diseases, for example, behaviour plasticity, memory, cancer, autoimmunity, addiction as well as neurodegenerative and psychological disorders. Thus, epigenetic studies have become crucial for understanding physiological and pathological conditions. Dissecting the regulation of epigenetic pathways and establishing their connection with environmental changes is technically challenging, but novel approaches have become available in the last few years.The most widely used technique for chromatin profiling is ChIP-seq which has been instrumental in describing chromatin features such as protein binding sites, transcription factors occupancy and correlated histone modifications.However, biases have emerged, especially when applying the technique to smaller and smaller samples, leading researchers to develop other methodological approaches to bypass ChIP-seq limitations.Technically, ChIP methods are afflicted by high background due to cross-linking, poor solubility of DNA–proteins complexes and poor affinity of some antibodies. Thus, traditional ChIP assays can provide a snapshot of DNA–protein interactions at any one time but are quite poor at providing kinetic information.A powerful counterpart to ChIP comes from the strategy of using tethered enzymes. ChIC-seq and ChEC-seq allow the investigation of protein–DNA interactions over time, providing an independent means by which to compare the results of ChIP-seq and discover new insights on genomic regulation.Finally, CUT&RUN and CUT&TAG allow producing high-quality chromatin profiling from low cell numbers using a relatively quick and easy protocol.Indeed, we reported how single-cell methodologies play a key role in dissecting epigenetic mechanisms underlying pathologies and chromosomal instability in cancer cells.Recent enhancements in technical methods have offered opportunities to elucidate the epigenetic environment in human disorders. Other single-cell methodologies need to be developed in the future for personalised medicine.


Drosophila melanogaster is organized into the cell nucleus, from small scale histone–DNA interactions to chromosome and lamina interactions in the nuclear space.Eukaryotic genomes are packaged into high-order chromatin structures organized in discrete territories inside the cell nucleus, which is surrounded by the nuclear envelope acting as a barrier. This chromatin organization is complex and dynamic and, thus, determining the spatial and temporal distribution and folding of chromosomes within the nucleus is critical for understanding the role of chromatin topology in genome function. Primarily focusing on the regulation of gene expression, we review here how the genome of  In eukaryotic cells, nuclear organization refers to the spatial distribution of nuclear contents and components. The cell nucleus contains DNA, which is organized as multiple long linear molecules in a complex with a large variety of proteins, such as histones, to form chromatin and chromosomes. Thus, the eukaryotic genome is packaged into higher-order chromatin structures and organized in a manner that accommodates highly dynamic processes such as DNA replication, gene transcription, and DNA repair.Drosophila melanogaster, a pre-eminent animal model system for genetic studies. Starting from basic DNA composition, we review what is currently known about the general structure of chromatin, chromosomes, and nuclear organization.There are many different levels of nuclear organization and whether they affect gene function or just reflect this function is still unclear. Here, we focus on Drosophila was published more than two decades ago, when it was estimated to contain around 13,600 genes , the Boundary Element Associated Factor (BEAF-32), and the Motif-1 binding protein (M1BP) . . 142]. Tlicating . During licating .Drosophila cell lines have demonstrated that knocking down lamin proteins decreases the compactness of inactive chromatin domains, increases the accessibility of the promoters located in heterochromatic regions, enhances the levels of histone H3 acetylation, and increases gene expression [Lamin B receptor, located at the inner nuclear membrane, provides a tethering mechanism for heterochromatin by binding to HP1a, which is preferentially located in the H3K9me3-rich heterochromatic region ,146,147.pression ,154,155.pression . Additiopression ,157,158.pression . Togethepression .Drosophila, the PCH regions from the four chromosomes cluster in the 3D nucleus, forming membraneless structures called chromocenters that were first described in the 1970s [Drosophila embryos [Apart from the heterochromatin contacts with the NL, interactions between different heterochromatic regions, most likely mediated by the affinity between repetitive elements ,162 or hhe 1970s ,170,171. embryos , the mec embryos . However embryos . Since HDrosophila, as well as in mammals, chromatin binding maps show differential binding preferences for different Nup subtypes. For instance, Nup153 and Megator (Mtor) are known to bind to the fly genome in continuous domains enriched in marks of active transcription [Drosophila cells; while Nup107 is mainly found at active genes, Nup93 is located preferentially at repressed chromatin regions, bound by PcG proteins [Besides the nuclear lamina, chromatin can also interact with the nuclear pore complexes (NPCs) located in the nuclear envelope and whose canonical function is to mediate nucleocytoplasmic transport. NPCs are composed of multiple nucleoporins (Nups), for which ~30 different subtypes have been described . Dependicription . In factcription . Howeverproteins . Indeed,proteins . These fDynamic Nups can also interact with the genome within the nucleoplasm, outside the NPC. This is the case of Nup98, which binds genes both at the NPC and within the nucleoplasm ,178. TheDrosophila, as well as in mammals, several Nups are found targeting a subset of promoters and enhancers. These contacts are preferentially located in NPC-bound Nups rather than nucleoplasmic Nups, and occur regardless of the transcriptional state of the genes, suggesting that NPCs can target silent or poised genes and enhancers [NPCs also seem to play a role in genome architecture. Multiple studies have demonstrated that NPC components contribute to the formation of long-range genomic contacts ,181,182.nhancers ,181. Parnhancers . Moreovenhancers . AltogetDrosophila, HP1a undergoes liquid phase separation in vitro and can form condensates suggested to facilitate heterochromatin formation in early embryos [Drosophila embryos, such as a reduced contact frequency within heterochromatic regions and increased crosstalk between active and inactive domains [Functional compartmentalization of the cell nucleus plays an important role in the regulation of genome activity. Recent evidence suggests that liquid–liquid phase separation (LLPS) underlies the formation of membraneless compartments in the nucleus . These m embryos . Indeed, domains . HP1a de domains . The hum domains . However domains .Other proteins have been suggested to facilitate chromatin condensation through LLPS, including histones and transcriptional regulators. The linker histone H1 was recently shown to promote the phase separation of reconstituted chromatin into denser and less dynamic droplets . On the Drosophila melanogaster regarding chromatin composition and 3D organization, mostly related to the regulation of gene expression. As we have discussed, the role that chromatin modifications play in transcription is still unclear, and new studies are now focusing on the effects of local chromatin environment and genome folding. In spite of several open questions, the 3D organization of the genome in nuclear compartments is increasingly recognized as a major feature of gene regulation. How these nuclear compartments are formed and what is their importance regarding gene function remains to be elucidated.In this review, we aimed to highlight what is known about the genome of Drosophila, these studies have revealed that the genome is organized into physical domains of particular chromatin states named TADs. However, Hi-C experiments reflect averaged information coming from cell populations, and thus cannot account for individual heterogeneity between cells, cell-types or tissues. To overcome this limitation, new methodologies have been developed, including scHi-C [Drosophila, CRISPR/Cas9 screens have recently been used to identify factors involved in genome architecture, including chromatin-binding proteins and functional cis-regulatory elements, such as enhancers and boundary elements or insulators [Over the last decade, numerous studies have used chromosome conformation capture techniques to analyze the 3D architecture of genomes . In Drosg scHi-C  and micrg scHi-C ,196,197.g scHi-C ,196,197.g scHi-C . In Drossulators . Furthersulators . In the sulators .Drosophila, the assembly of heterochromatic regions is still incomplete. New technologies based on long-read sequencing will improve the current genome assembly and thus increase the quality of Hi-C maps. In the field of microscopy, one of the major limitations is the number of loci that can be probed simultaneously. Recent advances including ORCA [Sequencing-based technologies rely on the available version of the genome and, in the case of ing ORCA , Hi-M [1ing ORCA , and Oliing ORCA  allow th


The results of these studies will potentially facilitate the discovery of new therapeutic pathways, particularly for personalized medicine, or new biomarkers. This review focuses on the complex relationship between replication timing, replication stress, and genomic instability.The replication-timing program constitutes a key element of the organization and coordination of numerous nuclear processes in eukaryotes. This program is established at a crucial moment in the cell cycle and occurs simultaneously with the organization of the genome, thus indicating the vital significance of this process. With recent technological achievements of high-throughput approaches, a very strong link has been confirmed between replication timing, transcriptional activity, the epigenetic and mutational landscape, and the 3D organization of the genome. There is also a clear relationship between replication stress, replication timing, and genomic instability, but the extent to which they are mutually linked to each other is unclear. Recent evidence has shown that replication timing is affected in cancer cells, although the cause and consequence of this effect remain unknown. However, in-depth studies remain to be performed to characterize the molecular mechanisms of replication-timing regulation and clearly identify different  The process of DNA replication enables the faithful and complete duplication of the cell genome in order to transmit a copy in its entirety to daughter cells after cell division. This molecular process must be finely orchestrated and coordinated with other molecular processes in the nucleus to avoid defects in DNA replication, which can be deleterious to the cell. Although the process of DNA replication takes place during the S-phase of the cell cycle, the regulatory mechanisms, including checkpoints, occur during the G2-phase of the previous cell cycle and throughout the G1-phase [In this review, we focus on the different actors involved in replication timing and its correlations with molecular elements in the genome. We also explore the link between the replication program and the mutational landscape in the genome, together with its evolutionary conservation. We then investigate the potential relationship between replication timing and DNA replication stress. DNA replication stress represents alterations in the velocity of ongoing replication forks, resulting in asymmetry in the fork progression, unreplicated regions, and/or fork collapse. We finally discuss how the deregulation of this temporal program affects chromatin stability, examine the links between them, and explore the causes and consequences of changes in the replication timing in the context of tumorigenesis.For many years, it has been well known that different parts of the metazoan genome are replicated at different time points during the S-phase. Genome replication does not occur randomly and is defined by a very precise sequence of events called the RT program . Given tcis and trans classes of RT regulatory factors have been described. First, a strong origin associated with an active promoter in a given region enables its earlier replication in the S-phase [trans also regulates origin activation at a specific time point during the S-phase. The best known thus far is RIF1, whose role in the control of RT is conserved from yeast to humans [To date, few molecular elements that regulate RT have been identified. Gene invalidation approaches (by knock-out (KO) or knock-in (KI)) have been used to study the effects of these elements, and it appears that the measured percentage of genome-wide RT changes never exceeds a rather low limit. For instance, depletions of some RT regulators such as RIF1, SUV4-20H, and DNA polymerase θ (Polθ) have been shown to induce around 16%, 15%, and 5% of genome-wide RT changes, respectively ,11,12. T S-phase . Then, t S-phase . Knowingo humans ,18,19,20o humans . Finallyo humans .In the last few years, chromatin conformation capture approaches and RT profile studies have shown that there is a strong link between RT, 3D interactions, and the organization of the genome ,23. CTRsSaccharomyces have shown that the RT program is fairly well conserved between different species [Lachancea, covering the continuous evolution of their genomes [The organization and structure of the genome are known to be crucial for the identity and fate of the cell. This is similarly the case for RT, as studies using abortive approaches of different factors that regulate it and treatments with different drugs have never shown more than a limited percentage of RT changes ,28,29,30 species . Further genomes . The res genomes . However genomes ,25. As pTherefore, the RT program appears to be a robust nuclear mechanism. Nonetheless, as we describe below, variations in the type of mutational events appear to correlate with the RT of the associated replication domains.The “primary mission” of replication is to completely duplicate the genome with flawless fidelity. However, there are several constraints. First, the time allowed for this duplication is limited (S-phase only), which necessitates a fine-tuned coordination of this process. Second, the replicative machinery can encounter several obstacles at the DNA and chromatin levels that can slow down or stop the process. There are several types of obstacles: (i) secondary DNA structures or the binding of non-histone proteins to chromatin or RNA–DNA hybrids such as R-loops, (ii) the level of chromatin compaction, and (iii) interference with the transcriptional machinery. This list is not exhaustive and is reviewed elsewhere . All of Mutational events fall into three main categories: (i) base substitutions, (ii) copy number variations, and (iii) chromosomal rearrangements. The influence of RT differs between these types of mutational events .S. cerevisiae and in several species of flies of the genus Drosophila [In 2009, one of the first studies on this topic showed that the number of SNPs (single-nucleotide polymorphisms) was not homogeneous throughout the human genome . Indeed,osophila ,41. Finaosophila ,43,44,45osophila ,46. Otheosophila ,48. Finaosophila . Heterocosophila .Drosophila melanogaster: in other words, losses are associated with early-replicating regions, while amplifications are associated with late-replicating regions [While there is a general consensus about base substitution rates and late replication among different eukaryotes, there are no common rules regarding CNVs and replication timing. A study on iPS cells indicates that amplifications are associated with early-replicating regions, while deletions are associated with later ones ,51. Anot regions .Drosophila. Thus, further research on this topic is required.What could explain these differences between eukaryotes? Unfortunately, it is difficult to answer this question because studies on other model organisms are lacking. CNVs seem to appear following the stalling of the fork and its destabilization . Data frStudies on the positions of chromosomal translocations in mouse and human genomes have shown that these translocations are enriched in regions that undergo early replication . StudiesEach step of the temporal DNA replication program  seems to be associated with replication stress at specific genomic loci in challenging replication conditions. For different reasons, these specific genomic regions are more prone to replication stress than others and strongly contribute to genomic instability.cis-acting AT sequences. Furthermore, the impact of these first two mechanisms has been minimized by Letessier et al., who found no differences in the frequency of slowed or stalled forks between FRA3B (the most active CFS in human lymphocytes) and the bulk genome, with or without APH treatment [The most well-described example is the sensitivity of common fragile sites (CFSs). CFSs are characterized by specific genomic features that are similar to late-replicating regions. They colocalize with very large genes , contain long AT-rich sequences , have a paucity of replication origins and are replicated in the late S-phase . reatment . This woreatment . Thus, tAnother subset of fragile sites has been identified: early-replicating fragile sites (ERFSs). Barlow et al. found that high hydroxyurea (HU) doses (10 mM) induced the slowing and collapse of the replication fork during early replication at specific sensitive genomic sites . In contTaken together, these results implicate replication timing as a key parameter of fragile site instability, particularly via the complex relationship between the timing of origin firing and the transcription dynamics of these regions. These interconnections are very important for the coordination of both replication and transcription processes and are thus critical for replication fidelity. To summarize, the impact of replication timing on CFS fragility seems to arise from a failure to complete replication, largely due to a paucity of origins in an end-phase replication context. ERFS fragility appears to arise from an increase in fork collapses due to the discoordination between high transcriptional activity and numerous replication initiation events that are specific to the beginning of the S-phase.Furthermore, some studies suggest that timing transition regions also make genomic regions more prone to replication stress sensitivity. TTRs are located between early- and late-replicating domains that primarily replicate from the early-mid to late S-phase. These very large areas, which contain no or very few replication origins, are characterized by a progressive change in the replication timing and the GC content . TTRs arThus, specific regions of the genome are more sensitive to replication stress than others due to particular genomic or genetic contexts. Importantly, the time of the replication  plays a role in this sensitivity. Transcription–replication conflicts and secondary DNA structures seem to be the main sources of replication stress in these fragile genomic regions. Other sources of replication stress can occur during the S-phase and disturb replication dynamics. Potential links between these factors and replication timing are discussed in the next section.In a physiological context, the cellular dNTP pool is finely regulated with specific concentrations for each nucleotide . ReductiSML1  induced a 66% increase in fork speed. Interestingly, this deletion caused a 2.5-fold increase in dNTP levels, indicating that replication fork velocity is sensitive to changes in dNTP levels. Studies in human cells have shown similar results [Indeed, an appropriate supply of dNTPs during the S-phase is critical for DNA synthesis dynamics. During replication, the dNTP pool is maintained by the ribonucleotide reductase (RNR) enzyme, which catalyzes the reduction of ribonucleoside diphosphates in deoxyribonucleoside diphosphates ,83. Stud results ,86,87, p results . In a moInterestingly, both studies reported that a reduction in dNTP levels also affected the density of replication initiation events. However, the origin response appears to be different among species. In yeast, the decrease in the dNTP pool induced by HU treatment resulted in a 25-fold reduction in origin firing . ConversRibonucleotides or ribonucleotide-induced DNA breaks can impede replication fork progression and lead to replication stress . RibonucThe presence of ribonucleotides may also have structural implications for eukaryotic genomes. RNA is 100,000 times more susceptible than DNA to spontaneous hydrolysis under physiological conditions, which may end in strand breaks. Furthermore, the chromatin assembly taking place during replication as well as the nucleosome positioning may be affected by the presence of ribonucleotides. The nucleosome assembly on the lagging strand could occur before Okazaki fragment maturation, leading to the incorporation of the RNA primer into nucleosomal DNA , althougParticular proteins on DNA or chromatin form DNA–protein complexes, which have a direct influence on RT in human cells . For exaDifferent histone proteins and chromatin marks may have a more indirect association with the set-up of replication timing. For example, histones H3.3 and H3.1 have been shown to preferentially bind early- and late-replicating chromatin, respectively. H3.3 forms domains in the nucleus that decrease in density during the replication process . Some hiTherefore, the chromatin regions decorated with transcription-permissive marks are replicated during the early stage of the S-phase, and H3K27me3/H3K9me3-decorated chromatin domains are replicated in the late stage of the S-phase . FurtherSome proteins have also been shown to play a role in preventing replication–transcription conflicts. For instance, the depletion of FACT in humans and yeast induced transcription-associated genetic instability and fork progression impairment . FurtherCCNE1 (cyclin E) and MYC oncogenes have been shown to induce the firing of new replication origins within highly transcribed genes in early-replicating domains  or by a scarcity of origins (TTRs and late-replicating regions).  domains . These eGiven their nature, interspersed or tandem repetitive DNA elements present a challenge for the replication machinery and may result in replication stress. Repetitive DNA elements account for more than half of the human genome and are replicated at specific time points of the S-phase . EuchromXenopus have demonstrated that TOP2 resolves topological stress to prevent converging forks from stalling during replication termination [Other secondary DNA structures can be formed. Among these, R-loops are abundant RNA–DNA hybrid structures covering up to 5% of mammalian genomes . They fomination . R-loop mination . In yeasmination . InteresReplication stress and replication timing variations are major changes that can alter the replication process. Each of them has been implicated in the generation of genomic instability and cancer. As described above, replication stress and replication timing seem to be closely related, as the type of replication stress appears to vary depending on the time point of the RT program. Surprisingly, almost none of the available literature has explored the direct relationship between replication stress and changes in the temporal organization of replication, highlighting the complexity of their connections. The activation of new origins and the stalling of replication forks are known consequences of replication stress (reviewed in ). Thus, Alternatively, it is possible that RT changes are induced by the consequences of replication stress and persist throughout several cell cycles because they are not properly resolved, as occurs in cancer cells, for example. It is known that replication stress can lead to genomic instability through events such as DNA breaks (reviewed in ) or chroTP53, RAD51, ATM, PTEN, and cMYC in breast cancer cells [TP53, all of the other genes shifted toward earlier replication. In addition to loci-specific alterations, RT changes at the genome scale have also been identified . Iniewed in , seems t cancers ,144,145.The replication timing and the chromatin state are closely related ,146. IndRIF1 gene is mutated in some breast cancer cell lines [RIF1 deregulation are involved in tumorigenesis. For many years, the RIF1 protein has been identified as the only molecular regulator with such an impact on the temporal DNA replication program. Recently, one study identified the tumor suppressor PREP1 as a new regulator of RT in human cells [PREP1 downregulation in HeLa cells induces a late-to-early shift of 25% of the human genome and is associated with a substantial decrease in the Lamin-B1 level. Some studies have also observed that the lack of PREP1 occurs in a large fraction of human cancers and is involved in tumor development [PREP1 downregulation, through the alteration of Lamin-B1 levels, could be involved in cancer transformation. Misregulation of key regulators of RT can also cause changes in this program. Notably, many studies have identified the RIF1 protein as an important player in the maintenance of accurate RT. Its role has also been established in yeast ,19, micell lines ,160, ovell lines . These san cells . They foelopment ,163. TakMoreover, the relationship between RT and transcription is very complex. In yeast, no correlation between these two processes has been observed . In contcis-acting elements that regulate the RT program of individual chromosomes. Two examples have been described in particular: disruptions of the ASAR6 and XIST genes result in extremely late replication of chromosomes 6 and X, respectively . Severaiewed in ). It hasiewed in ), whose Above, we describe different mechanisms that can lead to RT changes in the cancer context. However, to what extent do replication timing aberrations contribute to cancer progression? Do these RT changes trigger cancer transformation or are they consequences of alterations in counterpart processes? The answers to these questions remain unclear at the moment, as no direct relationship between RT changes and the cancerous phenotype has been confirmed yet. Nonetheless, given the interconnectedness of the replication timing with chromatin, transcription, and genomic stability, it is reasonable to suspect that RT changes induce abnormal variations in these three processes and contribute to or even amplify the development of cancer. Replication timing changes may alter the chromatin structure ,177. An Replication timing may also affect gene expression profiles. We previously described the potential impact of variations in transcriptional activity on RT. Conversely, replication changes may also affect the transcription competence of a region. Some but not all cancer-related genes associated with significant changes in RT were found to also display significant changes in their expression, either increasing or decreasing it . It remaReplication timing changes may eventually lead to replication stress. Local variations in RT may result in discoordination of the replication and transcription machineries, thus promoting collisions. Moreover, an excess of origin licensing at the same time may lead to a lack of factors necessary for the replication process (such as nucleotides or replication proteins) and impede global replication progression, resulting in the induction of replication stress ,179. Expcis rearrangements) at late-replicating regions and inter-chromosomal rearrangements  at early-replicating regions [ETV6/RUNX1 translocation site) [ETV6/RUNX1 translocation, suggesting that the RT change preceded (and may have predisposed) this translocation.Replication timing changes may also induce genomic/chromosomal instability. As described previously, replication timing seems to have direct consequences on mutagenesis and thus may be a contributor to genomic instability, a major hallmark of cancer . In addi regions . For thi regions ,134,181. regions . SimilarAs described in the first paragraphs, RT is one of the main molecular elements of genome organization, and to date, no study has reported more than a rather low threshold of genome-wide RT changes in the studied genomes . SimilarThere are two possible explanations for the lack of such observations. Primarily, the RT program is a highly regulated process that makes it very robust. An alternative hypothesis is that cells cannot survive drastic RT changes, explaining the absence of such observations in studied cell populations. In conclusion, RT is extraordinarily and precisely reproducible, conserved from one species to another and, to some extent, adaptable to different circumstances. To our knowledge, no other molecular mechanisms display these same characteristics. Thus, RT shows very strong links to transcription, the chromatin state, genomic and nuclear organization, cell fate, evolution, etc. However, even if these links are very strong, there are still many remaining questions such as: (i) What are the molecular actors that control and regulate RT? and (ii) How can the “chicken-and-egg” paradox between these different processes be resolved? In the future, the molecular processes that facilitate the regulation of RT and its coordination with other nuclear processes must be unraveled and understood. This is one of the main challenges that must be carried out in the forthcoming years by different research teams. The combination of genomic, genetic, and biochemical approaches will help to achieve this task. However, new avenues of research can be explored such as the importance of nuclear pores and their components in the coordination of these processes, the identification of new post-translational modifications of different factors, and a more detailed characterization of nuclear territories that can be assimilated to “mini-factories” with “mini-stocks”.The study of RT in the context of cancer is promising because, once again, strong links have been identified, and some results may lead to new therapeutic pathways. Indeed, a new treatment that results from an understanding of the link between RT and cancer and that targets an RT regulator could enable the specific inhibition of the proliferation of affected cells. However, much research is still needed to better understand the involvement of RT in the kinetics of oncogenesis. In cancer cells, RT variations have been shown to correlate with alterations in transcriptional activity, the chromatin state and genomic/chromosomal stability. The interconnectedness of the replication timing with these other nuclear processes ,146,182 To further clarify the relationship between RT and cancers, the development of cell models that perfectly characterize the onset of the expression of associated oncogenes would be valuable and permit analysis at the moment when RT is affected during its set-up. We could thus identify the first process disturbed—replication, transcription, epigenetic landscape or 3D organization of the genome—during these initial steps and thus understand the critical level of oncogenesis and genetic instability, which could be targeted by future therapeutic approaches.Furthermore, single-cell approaches to studying RT ,184 such