Architectural and Functional Commonalities between Enhancers and Promoters

With the explosion of genome-wide studies of regulated transcription, it has become clear that traditional definitions of enhancers and promoters need to be revisited. These control elements can now be characterized in terms of their local and regional architecture, their regulatory components, including histone modifications and associated binding factors, and their functional contribution to transcription. This Review discusses unifying themes between promoters and enhancers in transcriptional regulatory mechanisms. With the explosion of genome-wide studies of regulated transcription, it has become clear that traditional definitions of enhancers and promoters need to be revisited. These control elements can now be characterized in terms of their local and regional architecture, their regulatory components, including histone modifications and associated binding factors, and their functional contribution to transcription. This Review discusses unifying themes between promoters and enhancers in transcriptional regulatory mechanisms. Recent genome-wide studies have significantly advanced our understanding of the genomic architecture that underlies gene expression in higher eukaryotes. Integrative analyses of the transcriptome, transcription factor (TF) binding profiles, and epigenomes reveal complex organization of individual transcription units scattered throughout the genome and causal relationships among the regulatory DNA sequences, chromatin state, and transcriptional activity. In particular, a considerable amount of data have established that enhancers are not merely a collection of TF binding sites, but also have the capacity to drive transcription independent of their target promoters. This feature of enhancers suggests that they serve more regulatory functions than previously appreciated. Transcription of a gene in eukaryotes is a highly complex process that requires precise coordination in the assembly of trans-acting factors through the recognition of various types of regulatory DNA sequences. The promoter and the enhancer represent DNA regulatory regions responsible for ensuring proper spatiotemporal expression patterns of eukaryotic genes. The promoter generally refers to a DNA region that allows accurate transcription initiation of a gene (Smale and Kadonaga, 2003Smale S.T. Kadonaga J.T. The RNA polymerase II core promoter.Annu. Rev. Biochem. 2003; 72: 449-479Crossref PubMed Scopus (609) Google Scholar). The core promoter is a minimal stretch of DNA sequences (e.g., the TATA box, initiator, and downstream core promoter element) surrounding the transcription start site that directly interacts with the components of basal transcription machinery, including RNA polymerase II (RNAPII). Although the DNA sequences or motifs comprising the core promoter region for individual genes can be structurally and functionally diverse, its universal role is thought to drive accurate transcription initiation (Smale and Kadonaga, 2003Smale S.T. Kadonaga J.T. The RNA polymerase II core promoter.Annu. Rev. Biochem. 2003; 72: 449-479Crossref PubMed Scopus (609) Google Scholar). Transcription factors that bind ∼100–200 bp upstream of the core promoter can increase the rate of transcription by facilitating the recruitment or assembly of the basal transcription machinery onto the core promoter or by mediating the recruitment of specific distal regulatory DNA sequences to the core promoter (Akbari et al., 2008Akbari O.S. Bae E. Johnsen H. Villaluz A. Wong D. Drewell R.A. A novel promoter-tethering element regulates enhancer-driven gene expression at the bithorax complex in the Drosophila embryo.Development. 2008; 135: 123-131Crossref PubMed Scopus (0) Google Scholar). These distal sequences, known as enhancers, activate or increase the rate of transcription from the target gene promoter independent of their position and orientation with respect to target genes (Maniatis et al., 1987Maniatis T. Goodbourn S. Fischer J.A. Regulation of inducible and tissue-specific gene expression.Science. 1987; 236: 1237-1245Crossref PubMed Google Scholar). In multicellular organisms, enhancers are primarily responsible for the precise control of spatiotemporal patterns of gene expression. Enhancer elements were initially discovered in the early 1980s in studies that characterized eukaryotic gene promoters. Functional tests of sea urchin histone gene expression in the Xenopus oocyte identified DNA sequences located upstream of the TATA box motif that positively influence H2A gene transcription, originally termed transcriptional “modulators” (Grosschedl and Birnstiel, 1980Grosschedl R. Birnstiel M.L. Spacer DNA sequences upstream of the T-A-T-A-A-A-T-A sequence are essential for promotion of H2A histone gene transcription in vivo.Proc. Natl. Acad. Sci. USA. 1980; 77: 7102-7106Crossref PubMed Google Scholar). Deletion of the modulator resulted in 15- to 20-fold decrease in H2A gene expression. Interestingly, the modulator activity was retained even when its DNA sequence was inverted. Similarly, the tandem 72 bp DNA repeats located upstream of viral SV40 early gene were found to be indispensable for SV40 early gene expression (Benoist and Chambon, 1981Benoist C. Chambon P. In vivo sequence requirements of the SV40 early promotor region.Nature. 1981; 290: 304-310Crossref PubMed Google Scholar). Shortly after those initial observations, a series of studies on the SV40 enhancer established the conceptual framework for defining enhancers as follows (Atchison, 1988Atchison M.L. Enhancers: mechanisms of action and cell specificity.Annu. Rev. Cell Biol. 1988; 4: 127-153Crossref PubMed Google Scholar, Banerji et al., 1981Banerji J. Rusconi S. Schaffner W. Expression of a β-globin gene is enhanced by remote SV40 DNA sequences.Cell. 1981; 27: 299-308Abstract Full Text PDF PubMed Scopus (0) Google Scholar, Fromm and Berg, 1982Fromm M. Berg P. Deletion mapping of DNA regions required for SV40 early region promoter function in vivo.J. Mol. Appl. Genet. 1982; 1: 457-481PubMed Google Scholar, Fromm and Berg, 1983Fromm M. Berg P. Simian virus 40 early- and late-region promoter functions are enhanced by the 72-base-pair repeat inserted at distant locations and inverted orientations.Mol. Cell. Biol. 1983; 3: 991-999Crossref PubMed Google Scholar, Khoury and Gruss, 1983Khoury G. Gruss P. Enhancer elements.Cell. 1983; 33: 313-314Abstract Full Text PDF PubMed Google Scholar, Moreau et al., 1981Moreau P. Hen R. Wasylyk B. Everett R. Gaub M.P. Chambon P. The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants.Nucleic Acids Res. 1981; 9: 6047-6068Crossref PubMed Google Scholar): (1) Enhancers increase transcription of a linked gene from its correct initiation site specified by the core promoter, (2) enhancer activity is independent of orientation relative to its target gene, (3) enhancers can function independent of their position relative to the target genes, and also over long distances, (4) enhancers can function with a heterologous promoter, (5) enhancers exhibit DNase I hypersensitivity (HS), which reflects a less compacted chromatin state as a result of the binding of various transcription factors. Although these properties were defined more than three decades ago, they are still widely used to classify enhancers. Subsequent studies identified the first mammalian cellular enhancer, which is required for efficient expression of the immunoglobulin (Ig) heavy-chain gene (Banerji et al., 1983Banerji J. Olson L. Schaffner W. A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes.Cell. 1983; 33: 729-740Abstract Full Text PDF PubMed Scopus (552) Google Scholar, Gillies et al., 1983Gillies S.D. Morrison S.L. Oi V.T. Tonegawa S. A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene.Cell. 1983; 33: 717-728Abstract Full Text PDF PubMed Scopus (0) Google Scholar, Neuberger, 1983Neuberger M.S. Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells.EMBO J. 1983; 2: 1373-1378Crossref PubMed Scopus (259) Google Scholar). Importantly, the Ig enhancer studies provided the first evidence demonstrating that enhancer activity exhibits tissue or cell-type specificity. When various cell lines were tested, Ig enhancer activity was observed only in lymphocyte-derived cell lines (Banerji et al., 1983Banerji J. Olson L. Schaffner W. A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes.Cell. 1983; 33: 729-740Abstract Full Text PDF PubMed Scopus (552) Google Scholar, Gillies et al., 1983Gillies S.D. Morrison S.L. Oi V.T. Tonegawa S. A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene.Cell. 1983; 33: 717-728Abstract Full Text PDF PubMed Scopus (0) Google Scholar). Since then, a variety of cell-type- or developmental stage-specific enhancers have been determined to regulate the expression of genes in higher organisms (Müller et al., 1988Müller M.M. Gerster T. Schaffner W. Enhancer sequences and the regulation of gene transcription.Eur. J. Biochem. 1988; 176: 485-495Crossref PubMed Google Scholar). Transcriptional activation of yeast genes was also shown to be mediated by enhancer-like sequences, known as upstream activation sequences (UASs), although their distances from the core promoters are much shorter (within a few hundred base pairs) than the typical distances between enhancers and promoters in mammals (Guarente, 1988Guarente L. UASs and enhancers: common mechanism of transcriptional activation in yeast and mammals.Cell. 1988; 52: 303-305Abstract Full Text PDF PubMed Google Scholar). These results led to the realization that enhancer activity is the primary mechanism for determining the spatiotemporal gene expression pattern in eukaryotes. The ability to recruit RNAPII and initiate transcription has generally been considered the most unique property of promoters. However, even before the genomics era, several studies found that RNAPII can be directly recruited to enhancers upon transcriptional induction, potentially serving as a regulatory checkpoint for RNAPII delivery to the target promoter. Interestingly, an early study of the SV40 enhancer found that, in the absence of any known promoter sequence, the 72 bp DNA repeats can also “promote” gene expression, although this was deemed to be inefficient (Benoist and Chambon, 1981Benoist C. Chambon P. In vivo sequence requirements of the SV40 early promotor region.Nature. 1981; 290: 304-310Crossref PubMed Google Scholar, Moreau et al., 1981Moreau P. Hen R. Wasylyk B. Everett R. Gaub M.P. Chambon P. The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants.Nucleic Acids Res. 1981; 9: 6047-6068Crossref PubMed Google Scholar). This finding suggested the possibility that the 72 bp sequence might serve as a general entry site for a component of the transcription machinery, such as RNAPII, which could then track along the DNA to a transcription initiation site (Moreau et al., 1981Moreau P. Hen R. Wasylyk B. Everett R. Gaub M.P. Chambon P. The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants.Nucleic Acids Res. 1981; 9: 6047-6068Crossref PubMed Google Scholar). Another proposed mechanism that may not be mutually exclusive with the RNAPII tracking model is the chromatin remodeling effect. As various chromatin modifying enzymes such as histone acetyltransferases and methyltransferases can be part of the RNAPII transcription complex (Cho et al., 1998Cho H. Orphanides G. Sun X. Yang X.J. Ogryzko V. Lees E. Nakatani Y. Reinberg D. A human RNA polymerase II complex containing factors that modify chromatin structure.Mol. Cell. Biol. 1998; 18: 5355-5363Crossref PubMed Google Scholar, Gerber and Shilatifard, 2003Gerber M. Shilatifard A. Transcriptional elongation by RNA polymerase II and histone methylation.J. Biol. Chem. 2003; 278: 26303-26306Crossref PubMed Scopus (0) Google Scholar), transcription initiated from the enhancer proceeding across the intervening regions between the enhancer and the target promoter might be responsible for establishment and/or maintenance of an active chromatin conformation required for efficient gene transcription. Initial studies of enhancer identification and characterization were carried out by transient transfection experiments, which means that enhancer activity may be subject to position-effect variegation, depending on the chromatin configuration at the genomic site of integration. However, a study of a transgene containing the human β-globin locus discovered that five DNase-I-hypersensitive sites scattered in a ∼70 kb region surrounding the β-globin gene were sufficient to overcome the positional effect (Grosveld et al., 1987Grosveld F. van Assendelft G.B. Greaves D.R. Kollias G. Position-independent, high-level expression of the human beta-globin gene in transgenic mice.Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1197) Google Scholar). These cis-regulatory regions (e.g., enhancers) conferring activation of a linked gene in a tissue-specific, copy-number-dependent manner, independent of its position of integration, were collectively termed a locus control region (LCR) (Orkin, 1990Orkin S.H. Globin gene regulation and switching: circa 1990.Cell. 1990; 63: 665-672Abstract Full Text PDF PubMed Scopus (0) Google Scholar). Notably, transcription activity was detected at enhancers located within the β-globin LCR region and throughout the intervening DNA into the globin genes (Ashe et al., 1997Ashe H.L. Monks J. Wijgerde M. Fraser P. Proudfoot N.J. Intergenic transcription and transinduction of the human beta-globin locus.Genes Dev. 1997; 11: 2494-2509Crossref PubMed Google Scholar, Routledge and Proudfoot, 2002Routledge S.J. Proudfoot N.J. Definition of transcriptional promoters in the human beta globin locus control region.J. Mol. Biol. 2002; 323: 601-611Crossref PubMed Scopus (0) Google Scholar, Tuan et al., 1992Tuan D. Kong S. Hu K. Transcription of the hypersensitive site HS2 enhancer in erythroid cells.Proc. Natl. Acad. Sci. USA. 1992; 89: 11219-11223Crossref PubMed Scopus (0) Google Scholar). These LCR-driven intergenic transcripts are relatively short (< 3 kb), remain in discrete foci in the nucleus, and do not encode proteins (Ling et al., 2005Ling J. Baibakov B. Pi W. Emerson B.M. Tuan D. The HS2 enhancer of the beta-globin locus control region initiates synthesis of non-coding, polyadenylated RNAs independent of a cis-linked globin promoter.J. Mol. Biol. 2005; 350: 883-896Crossref PubMed Scopus (0) Google Scholar). Transcription predominantly occurred toward the downstream globin genes but was independent of the orientation, position, and distance of the enhancers with respect to the gene (Kong et al., 1997Kong S. Bohl D. Li C. Tuan D. Transcription of the HS2 enhancer toward a cis-linked gene is independent of the orientation, position, and distance of the enhancer relative to the gene.Mol. Cell. Biol. 1997; 17: 3955-3965Crossref PubMed Google Scholar, Routledge and Proudfoot, 2002Routledge S.J. Proudfoot N.J. Definition of transcriptional promoters in the human beta globin locus control region.J. Mol. Biol. 2002; 323: 601-611Crossref PubMed Scopus (0) Google Scholar). RNAPII recruitment and transcription activity have also been observed in other LCRs, including those that control expression of major histocompatibility complex (MHC) class II in antigen-presenting immune cells and pituitary-specific expression of the human growth hormone (hGH) gene (Ho et al., 2006Ho Y. Elefant F. Liebhaber S.A. Cooke N.E. Locus control region transcription plays an active role in long-range gene activation.Mol. Cell. 2006; 23: 365-375Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, Masternak et al., 2003Masternak K. Peyraud N. Krawczyk M. Barras E. Reith W. Chromatin remodeling and extragenic transcription at the MHC class II locus control region.Nat. Immunol. 2003; 4: 132-137Crossref PubMed Scopus (0) Google Scholar). Interestingly, insertion of an exogenous RNAPII termination sequence within the hGH-LCR blocked hGH regulation, suggesting that transcription through the LCR domain is a functionally important event. In both the human and murine β-globin gene loci, RNAPII interacts with the LCR, but not directly with the β-globin gene prior to erythroid differentiation, whereas it is associated with both in differentiated erythroid cells (Levings et al., 2006Levings P.P. Zhou Z. Vieira K.F. Crusselle-Davis V.J. Bungert J. Recruitment of transcription complexes to the beta-globin locus control region and transcription of hypersensitive site 3 prior to erythroid differentiation of murine embryonic stem cells.FEBS J. 2006; 273: 746-755Crossref PubMed Scopus (29) Google Scholar, Vieira et al., 2004Vieira K.F. Levings P.P. Hill M.A. Crusselle V.J. Kang S.-H.L. Engel J.D. Bungert J. Recruitment of transcription complexes to the beta-globin gene locus in vivo and in vitro.J. Biol. Chem. 2004; 279: 50350-50357Crossref PubMed Scopus (0) Google Scholar). In an in vitro assay using nuclear extracts from MEL cells, RNAPII and other basal transcription factors associated with immobilized LCR templates could be transferred to a β-globin gene template, which was facilitated by the erythroid transcription factor NF-E2 (Vieira et al., 2004Vieira K.F. Levings P.P. Hill M.A. Crusselle V.J. Kang S.-H.L. Engel J.D. Bungert J. Recruitment of transcription complexes to the beta-globin gene locus in vivo and in vitro.J. Biol. Chem. 2004; 279: 50350-50357Crossref PubMed Scopus (0) Google Scholar). Although performed in vitro, these results suggest a model in which the β-globin LCR functions to assemble and hold the RNAPII transcription complex for timely delivery to the β-globin gene to ensure the developmentally stage-specific expression. Furthermore, blocking transcription elongation between the LCR and the promoter by inserting a transcription terminator sequence significantly decreased the β-globin mRNA level, suggesting that the β-globin LCR facilitates a tracking and transcription mechanism (Ling et al., 2004Ling J. Ainol L. Zhang L. Yu X. Pi W. Tuan D. HS2 enhancer function is blocked by a transcriptional terminator inserted between the enhancer and the promoter.J. Biol. Chem. 2004; 279: 51704-51713Crossref PubMed Scopus (0) Google Scholar). A similar mechanism has been proposed for other LCRs and enhancers (Ho et al., 2006Ho Y. Elefant F. Liebhaber S.A. Cooke N.E. Locus control region transcription plays an active role in long-range gene activation.Mol. Cell. 2006; 23: 365-375Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, Wang et al., 2005Wang Q. Carroll J.S. Brown M. Spatial and temporal recruitment of androgen receptor and its coactivators involves chromosomal looping and polymerase tracking.Mol. Cell. 2005; 19: 631-642Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). In a contrasting model, transfer of the RNAPII machinery from the α-globin LCR to the promoter appears to be mediated by formation of a DNA loop between the LCR and the promoter, as no RNAPII signal is detected in the intervening DNA between the LCR and the promoter (Vernimmen et al., 2007Vernimmen D. De Gobbi M. Sloane-Stanley J.A. Wood W.G. Higgs D.R. Long-range chromosomal interactions regulate the timing of the transition between poised and active gene expression.EMBO J. 2007; 26: 2041-2051Crossref PubMed Scopus (144) Google Scholar). These initial insights into the complex roles of enhancers and LCRs set the stage for thinking about regulatory elements in a more global manner. Early genome-wide studies identified RNAPII binding at intergenic loci, which suggested the existence of enhancer-like sequences across the genome; however, there were questions regarding the functional relevance of such RNAPII occupancy (Barrera et al., 2008Barrera L.O. Li Z. Smith A.D. Arden K.C. Cavenee W.K. Zhang M.Q. Green R.D. Ren B. Genome-wide mapping and analysis of active promoters in mouse embryonic stem cells and adult organs.Genome Res. 2008; 18: 46-59Crossref PubMed Scopus (0) Google Scholar, Brodsky et al., 2005Brodsky A.S. Meyer C.A. Swinburne I.A. Hall G. Keenan B.J. Liu X.S. Fox E.A. Silver P.A. Genomic mapping of RNA polymerase II reveals sites of co-transcriptional regulation in human cells.Genome Biol. 2005; 6: R64Crossref PubMed Google Scholar, Carroll et al., 2006Carroll J.S. Meyer C.A. Song J. Li W. Geistlinger T.R. Eeckhoute J. Brodsky A.S. Keeton E.K. Fertuck K.C. Hall G.F. et al.Genome-wide analysis of estrogen receptor binding sites.Nat. Genet. 2006; 38: 1289-1297Crossref PubMed Scopus (831) Google Scholar, Heintzman et al., 2007Heintzman N.D. Stuart R.K. Hon G. Fu Y. Ching C.W. Hawkins R.D. Barrera L.O. Van Calcar S. Qu C. Ching K.A. et al.Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome.Nat. Genet. 2007; 39: 311-318Crossref PubMed Scopus (1444) Google Scholar). Moreover, it was difficult to classify RNAPII binding sites as possible enhancer or un-annotated promoter of a protein-coding gene by the virtue of RNAPII association alone. It became clear that additional criteria would be needed to identify enhancers. Given their association with transcription factors, computational analysis of TF binding motifs combined with the assessment of evolutionary conservation within the DNA was used as a popular approach in identifying enhancers (reviewed in Aerts, 2012Aerts S. Computational strategies for the genome-wide identification of cis-regulatory elements and transcriptional targets.Curr. Top. Dev. Biol. 2012; 98: 121-145Crossref PubMed Scopus (0) Google Scholar). More recently, chromatin-immunoprecipitation-based analysis of TF binding in vivo (e.g., ChIP-chip and ChIP-seq) has been widely used to experimentally determine actual TF binding sites in vivo. This approach revealed that only a small fraction of TF binding motifs are actually bound by TFs in vivo in a given tissue and/or stage (ENCODE Project Consortium et al., 2007Birney E. Stamatoyannopoulos J.A. Dutta A. Guigo R. Gingeras T.R. Margulies E.H. Weng Z. Snyder M. Dermitzakis E.T. et al.ENCODE Project ConsortiumIdentification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.Nature. 2007; 447: 799-816Crossref PubMed Scopus (0) Google Scholar). TF binding per se does not signal a functional outcome. Functional activation requires recruitment of additional cofactors or mechanisms involving a combinatorial coordination of multiple TFs. Therefore, analysis of evolutionarily conserved TF motifs or TF binding alone has a limited power for identification and prediction of functional enhancers (see also Kellis et al., 2014Kellis M. Wold B. Snyder M.P. Bernstein B.E. Kundaje A. Marinov G.K. Ward L.D. Birney E. Crawford G.E. Dekker J. et al.Defining functional DNA elements in the human genome.Proc. Natl. Acad. Sci. USA. 2014; 111: 6131-6138Crossref PubMed Scopus (153) Google Scholar for review). Transcriptional coactivators p300 and CBP interact with a large number of transcriptional activators and the general transcription machinery, including RNAPII. Moreover, both p300 and CBP display acetyltransferase activity toward the tails of histones localized near cis-regulatory regions, which is thought to create a transcriptionally permissive chromatin structure. Therefore, although not perfect, genome-wide analysis of p300/CBP binding sites has been commonly used as a method for identifying enhancer elements in vivo without having to investigate individual TFs (May et al., 2012May D. Blow M.J. Kaplan T. McCulley D.J. Jensen B.C. Akiyama J.A. Holt A. Plajzer-Frick I. Shoukry M. Wright C. et al.Large-scale discovery of enhancers from human heart tissue.Nat. Genet. 2012; 44: 89-93Crossref Scopus (0) Google Scholar, Visel et al., 2009Visel A. Blow M.J. Li Z. Zhang T. Akiyama J.A. Holt A. Plajzer-Frick I. Shoukry M. Wright C. Chen F. et al.ChIP-seq accurately predicts tissue-specific activity of enhancers.Nature. 2009; 457: 854-858Crossref PubMed Scopus (833) Google Scholar). A complementary approach in identifying enhancers takes advantage of their chromatin accessibility. The assembly of various TF complexes at cis-regulatory regions is considered to compete with stable association of nucleosomes. As a result, active enhancers and promoters have reduced nucleosome density and display hypersensitivity to DNase I digestion. This feature of chromatin accessibility has been utilized in next-generation sequencing-based techniques such DNase-seq, FAIRE-seq, and ATAC-seq (Boyle et al., 2008Boyle A.P. Davis S. Shulha H.P. Meltzer P. Margulies E.H. Weng Z. Furey T.S. Crawford G.E. High-resolution mapping and characterization of open chromatin across the genome.Cell. 2008; 132: 311-322Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar, Buenrostro et al., 2013Buenrostro J.D. Giresi P.G. Zaba L.C. Chang H.Y. Greenleaf W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.Nat. Methods. 2013; 10: 1213-1218Crossref PubMed Scopus (313) Google Scholar, Giresi et al., 2007Giresi P.G. Kim J. McDaniell R.M. Iyer V.R. Lieb J.D. FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin.Genome Res. 2007; 17: 877-885Crossref PubMed Scopus (380) Google Scholar) to identify enhancers without any prior knowledge of TF binding motifs or TF binding. Although not sufficient to pinpoint cell-type-specific enhancers due to its indiscriminate nature, this method can be very useful for enhancer characterization when combined with other mapping techniques. An increasing number of epigenomic studies have illustrated that the chromatin of metazoan genomes is organized into modular domains that represent unique chromatin states formed by a combination of multiple post-translational modifications on histones within the nucleosomes (ENCODE Project Consortium, 2012ENCODE Project ConsortiumAn integrated encyclopedia of DNA elements in the human genome.Nature. 2012; 489: 57-74Crossref PubMed Scopus (3789) Google Scholar, Ernst et al., 2011Ernst J. Kheradpour P. Mikkelsen T.S. Shoresh N. Ward L.D. Epstein C.B. Zhang X. Wang L. Issner R. Coyne M. et al.Mapping and analysis of chromatin state dynamics in nine human cell types.Nature. 2011; 473: 43-49Crossref PubMed Scopus (1153) Google Scholar). For example, nucleosomes within enhancer regions contain histone variants H3.3 and H2A.Z (Goldberg et al., 2010Goldberg A.D. Banaszynski L.A. Noh K.M. Lewis P.W. Elsaesser S.J. Stadler S. Dewell S. Law M. Guo X. Li X. et al.Distinct factors control histone variant H3.3 localization at specific genomic regions.Cell. 2010; 140: 678-691Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, Henikoff et al., 2009Henikoff S. Henikoff J.G. Sakai A. Loeb G.B. Ahmad K. Genome-wide profiling of salt fractions maps physical properties of chromatin.Genome Res. 2009; 19: 460-469Crossref PubMed Scopus (0) Google Scholar, Jin et al., 2009Jin C. Zang C. Wei G. Cui K. Peng W. Zhao K. Felsenfeld G. H3.3/H2A.Z double variant-containing nucleosomes mark ‘nucleosome-free regions’ of active promoters and other regulatory regions.Nat. Genet. 2009; 41: 941-945Crossref PubMed Scopus (398) Google Scholar). These nucleosome variants are deposited into enhancer regions in a replication-independent manner and are more sensitive to high salt than canonical nucleosomes. In contrast, nucleosomes flanking TF-bound sites are stable and undergo various histone modifications that are distinctive to each functional domain and across cell types and also correlate with transcriptional outputs (ENCODE Project Consortium, 2012ENCODE Project ConsortiumAn integrated encyclopedia of DNA elements in the human genome.Nature. 2012; 489: 57-74Crossref PubMed Scopus (3789) Google Scholar, Heintzman et al., 2007Heintzman N.D. Stuart R.K. Hon G. Fu Y. Ching C.W. Hawkins R.D. Barrera L.O. Van Calcar S. Qu C. Ching K.A. et al.Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome.Nat. Genet. 2007; 39: 311-318Crossref PubMed Scopus (1444) Google Scholar, Heintzman et al., 2009Heintzman N.D. Hon G.C. Hawkins R.D. Kheradpour P. Stark A. Harp L.F. Ye Z. Lee L.K. Stuart R.K. Ching C.W. et al.Histone modifications at human enhancers reflect global cell-type-specific gene expression.Nature. 2009; 459: 108-112Crossref PubMed Scopus (1079) Google Scholar, Hon et al., 2009Hon G. Wang W. Ren B. Discovery and annotation of functional chromatin signatures in the human genome.PLoS Comput. Biol. 2009; 5: e1000566Crossref PubMed Scopus (0) Google Scholar, Visel et al., 2009Visel A. Blow M.J. Li Z. Zhang T. Akiyama J.A. Holt A. Plajzer-Frick I. Shoukry M. Wright C. Chen F. et al.ChIP-seq accurately predicts tissue-specific activity of enhancers.Nature. 2009; 457: 854-858Crossref PubMed Scopus (833) Google Scholar). Importantly, such chromatin modifications combined with other measures (chromatin accessibility and TF binding) have proven themselves a useful barometer for active enhancers. Enhancers of active genes generally display a high level of mono- or di-methylation on H3 lysine 4 (H3K4me1/2) but are low or devoid of H3K4me3, whereas promoter sequences show the opposite pattern. In addition to H3K4me1/2, mutually exclusive modifications on H3K27 residues co-segregate with active or inactive/poised enhancers (Creyghton et al., 2010Creyghton M.P. Cheng A.W. Welstead G.G