Molecular Cell
Perspective Enhancer Malfunction in Cancer Hans-Martin Herz,1 Deqing Hu,1 and Ali Shilatifard1,* 1Stowers Institute for Medical Research, Kansas City, MO 64110, USA *Correspondence:
[email protected] http://dx.doi.org/10.1016/j.molcel.2014.02.033
Why certain point mutations in a general transcription factor are associated with specific forms of cancer has been a major question in cancer biology. Enhancers are DNA regulatory elements that are key regulators of tissue-specific gene expression. Recent studies suggest that enhancer malfunction through point mutations in either regulatory elements or factors modulating enhancer-promoter communication could be the cause of tissue-specific cancer development. In this Perspective, we will discuss recent findings in the identification of cancer-related enhancer mutations and the role of Drosophila Trr and its human homologs, the MLL3 and MLL4/COMPASS-like complexes, as enhancer histone H3 lysine 4 (H3K4) monomethyltransferases functioning in enhancer-promoter communication. Recent genome-wide studies in the cataloging of somatic mutations in cancer have identified mutations in intergenic sequences encoding regulatory elements—and in MLL3 and MLL4 in both hematological malignancies and solid tumors. We propose that cancer-associated mutations in MLL3 and MLL4 exert their properties through the malfunction of Trr/MLL3/MLL4-dependent enhancers. Introduction It has been more than 30 years since Schaffner, Chambon, and colleagues demonstrated that a DNA element from Simian virus 40 (SV40) could drive expression of the T-antigen or a b-globin reporter gene in mammalian cells (Banerji et al., 1981; Moreau et al., 1981). The ability of the SV40 DNA element to activate expression of the b-globin gene was demonstrated to be independent of its distance to the transcription start site, and the element was demonstrated to be functional both up- or downstream of the transcription start site in either orientation (Banerji et al., 1981). Subsequently, DNA elements with similar properties were also discovered in other animal viruses, some of which displayed tissue- and host-specific preferences (de Villiers et al., 1982; Hansen and Sharp, 1983; Schirm et al., 1985; Spandidos and Wilkie, 1983). Based on their ability to enhance the transcription of their target genes, such DNA elements were named enhancers by Schaffner and colleagues (Banerji et al., 1981). Further mechanistic insight into the function of these viral enhancers was derived shortly afterward by the finding that certain transcription factors bind to specific sites within enhancers and regulate their activity (Figure 1A) (Lee et al., 1987). These basic characteristics of viral enhancers are also conserved in metazoans where the coordinated regulation of gene expression is of utmost importance for proper differentiation, morphogenesis, and development. Metazoan enhancers can work over great distances (up to a megabase) and generally range from a few hundred base pairs to several kilobases. Like viral enhancers, they contain binding sites for activating or repressing transcription factors (Figure 1A), which in their wake recruit chromatin-modifying coactivators or corepressors to achieve tissue-specific gene activation or repression, respectively (Figure 1B). The currently prevailing model for enhancer function suggests a looping mechanism by which enhancers are brought into close proximity to their cognate promoters (Dorsett, 1999; Dorsett and Merkenschlager, 2013). Originally
discovered in Drosophila as effectors of enhancer-promoter communication (Dorsett et al., 2005; Rollins et al., 2004; Rollins et al., 1999), members of the cohesin complex and their loading factors such as Nipped-B are involved in stabilizing these longrange interactions and, via the Mediator complex, support the interaction of enhancer-bound transcription factors with the basal transcription machinery on promoters (Figure 1C) (summarized in Dorsett and Merkenschlager, 2013; Levine, 2010; Ong and Corces, 2011; Spitz and Furlong, 2012). Enhancer Signatures The identification of DNA elements encoding enhancers within metazoan genomes has been a major challenge. More recently, genome-wide studies have uncovered various enhancer ‘‘signatures’’ that serve as hallmarks to define cis-regulatory elements that are active in a tissue-specific manner. Generally, enhancers contain specific DNA elements that are recognized by tissuespecific transcription factors. These factors often cooperate in their binding to enhancers and frequently synergize to achieve optimal activation of target genes (Ong and Corces, 2011; Spitz and Furlong, 2012). Transcription factor binding on enhancers correlates very well with increased nucleosome depletion or, alternatively, enhanced nucleosome turnover (Mito et al., 2007; Song et al., 2011). Indeed, highly chromatin-accessible sites might be a better predictor for transcription factor recruitment than the presence of conserved DNA binding motifs (Li et al., 2011). HOT (highly occupied target) regions are a special group of genomic elements that initially were characterized in Drosophila and Cenorhabditis elegans by low nucleosome occupancy and the clustering of many types of transcription factors, many more than usually found on ‘‘regular’’ enhancers (Gerstein et al., 2010; modENCODE et al., 2010; Moorman et al., 2006; Ne`gre et al., 2011). Despite their general scarcity in transcription factor motifs, HOT regions in Drosophila show significant Molecular Cell 53, March 20, 2014 ª2014 Elsevier Inc. 859
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Figure 1. A Model for Transcriptional Activation by cis-Regulatory Elements (A) Metazoan cis-regulatory elements also called ‘‘enhancers’’ can activate gene expression over more than hundreds of kilobases but can also function over very short distances. Enhancers contain binding sites for activating or repressing transcription factors (TFs) that are often recruited in response to environmental or developmental signals. (B) TFs often recruit chromatin-modifying coactivators or corepressors. Coactivators such as CBP/p300 and MLL3/MLL4 have been demonstrated to acetylate histone H3 on lysine 27 (H3K27ac, highlighted in pink) or monomethylate histone 3 on lysine 4 (H3K4me1, highlighted in green) around enhancers, respectively. (legend continued on next page)
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Perspective enrichment for motifs of the early developmentally acting transcription factors, Zelda and GAGA, and act as developmental enhancers with specific spatiotemporal gene expression patterns in vivo (Kvon et al., 2012). It is possible that Zelda and GAGA, which are known for the generation and maintenance of nucleosome-free regions (Nakayama et al., 2007), might provide a recruitment platform for transcription factors independent of their DNA-binding motifs. Similar features to the HOT regions of Drosophila are displayed by the so called ‘‘super-enhancers’’ in mammals where the master transcription factors Oct4, Sox2, and Nanog in embryonic stem cells—and more tissue-specific transcription factors in other cell types—can occur in large domains (averaging approximately 9 kb in length). These enhancers display a high density of many other transcription factors including components of the cohesin complex, mediator complex, and various coactivators and corepressors (Hnisz et al., 2013; Whyte et al., 2013). Among the coactivators and corepressors, many contain the ability to chemically modify histones. So not surprisingly, various histone modifications are ascribed as being particularly enriched around regulatory elements. For example, histone H3 lysine 4 (H3K4) monomethylation is very prominent on active enhancers but can also be found on inactive/‘‘poised’’ enhancers (Heintzman et al., 2007). Subsequently, histone H3 lysine 27 acetylation (H3K27ac) and histone H3 lysine 27 trimethylation (H3K27me3) were shown to be differentially enriched between these active and inactive enhancer states, respectively (Creyghton et al., 2010; Heintzman et al., 2009; Rada-Iglesias et al., 2011; Zentner et al., 2011). Trr/MLL3/MLL4 COMPASS-like Complexes in Enhancer-Associated H3K4 Monomethylation and H3K27 Demethylation Yeast Set1 was identified as the first histone H3K4 methylase to exist in a macromolecular complex named COMPASS (complex of proteins Associated with Set1) (Miller et al., 2001; Shilatifard 2012). All metazoan homologs of Set1 also exist in COMPASSlike complexes, all of which share core subunits but also contain complex-specific components (Shilatifard 2012). While in yeast, all H3K4 methylation states (H3K4 mono-, di-, and trimethylation) are implemented by Set1/COMPASS (Schneider et al., 2005), these activities are divided among the metazoan COMPASS family (Shilatifard 2012). The Drosophila genome contains three Set1-related proteins: Set1, Trithorax (Trx), and Trithorax-related (Trr); and the mammalian genome possesses six yeast Set1 homologs (Shilatifard, 2012; Mohan et al., 2011): SET1A/SET1B (homologous to Drosophila Set1), MLL1/MLL2 (homologous to Trx), and MLL3/MLL4 (homologous to Trr). Drosophila Set1 and mammalian SET1A/SET1B in a redundant fashion are known to be major H3K4 di- and trimethyltransferases (Ardehali et al., 2011; Hallson et al., 2012; Mohan et al., 2011; Wu et al., 2008), whereas Drosophila Trr and MLL3/ MLL4 together in mammals constitute major H3K4 monomethyl-
transferases and also function through Utx (a subunit of the complex) as an enhancer-specific H3K27 demethylase (Herz et al., 2012; Hu et al., 2013; Kanda et al., 2013).The Drosophila Trr/ COMPASS-like complex also contains a protein, LPT, with multiple PHD domains and an HMG box, which is vitally required for proper H3K4 monomethylation and is homologous to the N-terminal portion of mammalian MLL3/MLL4, while Trr containing the catalytic SET domain represents the C-terminal part of MLL3/MLL4 (Herz et al., 2012; Mohan et al., 2011). Thus, in Drosophila, two proteins, LPT and Trr, constitute the functional equivalent of mammalian MLL3/MLL4. Genome-wide studies demonstrated that Trr and MLL3/MLL4 are localized to promoter-distal elements but can also be found on transcription start sites (Herz et al., 2012; Hu et al., 2013) (data not shown). Upon impairment of Trr or MLL3/MLL4 function, specific genome-wide changes in H3K4 monomethylation and H3K27 methylation/acetylation can mainly be observed at enhancer regions, suggesting a direct role for Trr/MLL3/MLL4 in enhancermediated processes (Herz et al., 2012; Hu et al., 2013). Indeed, Trr/MLL3/MLL4 loss results in decreased H3K4 monomethylation on a subset of enhancers (more than two-thirds of enhancers marked by H3K4 monomethylation in human HCT116 cells) referred to as Trr/MLL3/MLL4-dependent enhancers, (Figure 2) (Hu et al., 2013). Additionally, other regulatory elements and enhancers also exist whose H3K4 monomethylation is Trr/MLL3/ MLL4 independent. It is not clear at this time which enzymes are involved in the regulation of H3K4 monomethylation on these Trr/MLL3/MLL4-independent loci (Figure 2). MLL3/MLL4/ COMPASS-like complexes have also been identified to function as histone H3K4 monomethylase on facultative heterochromatin functioning in the assembly and partitioning of chromatin interacting factors (Cheng et al., 2014 in this issue). Enhancer Malfunction and Disease Considering the importance of enhancers in the maintenance of the tissue-specific expression pattern of developmental genes, it is easily conceivable how changes in enhancer activity—either by mutations in enhancer DNA sequences or mutations in transcription factors that either directly interact with enhancers or regulate enhancers (such as MLL3 and MLL4)—could also contribute to the misregulation of disease-relevant genes. Indeed, genome-wide and other studies have shown that single-nucleotide polymorphisms (SNPs), insertions, or deletions at disease-associated enhancers can alter the gene expression of respective target genes by positively or negatively affecting the recruitment of transcription factors and cofactors, thus changing the epigenetic enhancer landscape. Also, specific enhancer mutations have been correlated with various diseaselinked genes for X-linked deafness, Hirschsprung’s disease, Crohn’s disease, multiple sclerosis, systemic lupus, and others (Corradin et al., 2013; de Kok et al., 1996; Emison et al., 2005; Kasowski et al., 2013; Kilpinen et al., 2013; Maurano et al., 2012; McVicker et al., 2013; Noonan and McCallion, 2010). As
(C) A looping mechanism mediated by factors such as cohesin and the Mediator complex can bring enhancers into close proximity to promoters. Longrange interactions between enhancers and promoters have been shown to be stabilized by members of the cohesin complex, and the Mediator complex supports the interaction of enhancer-bound transcription factors and coactivators such as MLL3/MLL4 and CBP/p300 with the basal transcription machinery on promoters.
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Perspective homolog NIPBL can frequently be found in patients with Cornelia de Lange Syndrome (Dorsett and Krantz, 2009). Therefore, the genetic defects observed in Cornelia de Lange Syndrome might be the direct result of changes in enhancer-mediated processes. More recently, some studies have also linked changes in the enhancer ‘‘landscape’’ with an increased risk to develop cancer, providing evidence that enhancer inactivation of tumor suppressor genes or enhancer activation of oncogenes might contribute to tumorigenesis (Akhtar-Zaidi et al., 2012; Aran and Hellman, 2013; Aran et al., 2013; Jia et al., 2009; Kurdistani, 2012; Love´n et al., 2013; Sur et al., 2012). MLL3 and MLL4 Branches of the COMPASS Family Are Frequently Mutated in Various Cancers The MLL3 and MLL4 genes have been reported to be frequently mutated in many different forms of cancer, some of which include bladder cancer, breast cancer, colon cancer, gastric cancer, liver cancer, medulloblastoma, non-Hodgkin’s lymphoma, and others (Ashktorab et al., 2010; Ellis et al., 2012; Fujimoto et al., 2012; Gui et al., 2011; Jones et al., 2012; Kandoth et al., 2013; Morin et al., 2011; Parsons et al., 2011; Watanabe et al., 2011; Zang et al., 2012). According to the ‘‘Catalogue Of Somatic Mutations In Cancer’’ (COSMIC), MLL3 and MLL4 nonsense and missense mutations are distributed across the whole length of the protein, respectively. However, a particular enrichment for mutations can be observed over more highly conserved regions including the two N-terminally located PHD repeats and the C termini of MLL3/MLL4, which comprise many domains including a PHD finger, FYRN, FYRC, and the catalytic SET domain. Furthermore, in vivo evidence suggests that at least under certain conditions, the catalytic activity of MLL3/ MLL4 might be required to suppress tumorigenesis, as mice containing a SET domain deletion of MLL3 develop ureter epithelial tumors (Lee et al., 2009). Similarly, studies on the Drosophila homologs of the human histone 3 lysine 27 demethylase, UTX (a complex-specific subunit of the MLL3/MLL4 COMPASS-like complexes), and MLL3/MLL4 -Utx, and Trrsupport a role for these proteins in growth control (Herz et al., 2010; Kanda et al., 2013).
Figure 2. Trr/MLL3/MLL4 COMPASS-like Complex-Dependent Enhancers Coverage profiles arranged for enrichment of H3K4 monomethylation (H3K4me1) at promoter-distal sites (>1 kb) in the human colorectal carcinoma cell line, HCT116 (MLL3Dset, column 1), or HCT116 cells with a MLL4 deletion (MLL3Dset/4Dset, column 2). Sites are arranged in a vertical gradient with a higher enrichment of H3K4 monomethylation at the top and lower levels of H3K4me1 toward the bottom. A large percentage (70%) of putative enhancers exhibits a significant loss in H3K4 monomethylation (p < 1e 3) in the absence of both MLL3 and MLL4 (compare column 2 with column 1 in first panel), while a minority of putative enhancers (30%) is independent of MLL3/ MLL4 for their H3K4me1 status (we call these enhancers Trr/MLL3/MLL4independent enhancers; compare column 2 with column 1 in second panel). Data for this figure were adopted from Hu et al. (2013) with permission from the American Society for Microbiology.
mentioned above, Nipped-B is a loading factor of the cohesin complex that was initially identified in genetic screens in Drosophila as a regulator of enhancer-promoter communication. Interestingly, dominant loss-of-function mutations in the human 862 Molecular Cell 53, March 20, 2014 ª2014 Elsevier Inc.
A Model for Enhancer Malfunction in Cancer Pathogenesis Based on recent findings that the Trr/MLL3/MLL4 complexes constitute enhancer-associated H3K4 monomethyltransferases (Herz et al., 2012; Hu et al., 2013) and our knowledge of the frequency of MLL3/MLL4 mutations in various cancers, we propose a model that might explain how the misregulation of MLL3/MLL4-dependent enhancer function of tumor suppressor genes or oncogenes could drive cancer (Figure 3). The different modes of action described here exemplary for mutations of MLL3/MLL4 would also be applicable to any other specific mutations in enhancers, mutations in enhancer-binding transcription factors, or mutations in cofactors with disease relevance. In the first scenario, a somatic mutation would result in a loss of function of MLL3/MLL4 (Figure 3A). The effect of this mutation would either lead to a destabilization of MLL3/MLL4 within COMPASS or the reduced association of MLL3/MLL4 with recruiting transcription factors on the specific enhancers
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Figure 3. Misregulation of MLL3/MLL4-Dependent Enhancer Function in Cancer Pathogenesis Two models proposing how MLL3/MLL4 mutations can either result in inappropriate enhancer-mediated inactivation of tumor suppressor genes or activation of oncogenes are described here. In (A), MLL3/MLL4 loss-of-function scenario on the enhancer of a tumor suppressor gene is shown. MLL3/MLL4 mutations could either result in a destabilization of MLL3/MLL4, reduced association of MLL3/MLL4 with recruiting TFs on enhancers, or catalytic inactivation of MLL3/MLL4. In any case, the outcome would be diminished or eliminated enhancer activation of tumor suppressor genes. In (B), MLL3/MLL4 gain-of-function scenario on the enhancer of oncogenes is shown. MLL3/MLL4 mutations could either result in increased MLL3/MLL4 stability, increased affinity of MLL3/MLL4 toward MLL3/ MLL4-recruiting TFs, or catalytic hyperactivation of MLL3/MLL4 on enhancers regulating the expression of oncogenes.
of tumor suppressor genes resulting in a lower expression of the tumor suppressor (Figure 3A). If the catalytic activity of MLL3/MLL4 was required to convey enhancer activation, inactivation of MLL3/MLL4 (by a point mutation in the SET domain) would pose another alternative. In any of these cases,
the outcome would be the same and result in diminished or eliminated enhancer activation for tumor suppressor genes (Figure 3A). It might also be anticipated that MLL3/MLL4destabilizing or -inactivating mutations are not as gene-specific but have broader effects on all or most MLL3/MLL4-associated Molecular Cell 53, March 20, 2014 ª2014 Elsevier Inc. 863
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Perspective enhancers, which would result in lower enhancer activity in general. An MLL3/MLL4 gain-of-function scenario on the enhancers of oncogenes would provide a second explanation of how MLL3/ MLL4 mutations might contribute to the development of cancer (Figure 3B). MLL3/MLL4 mutations could either increase the affinity toward specific MLL3/MLL4-recruiting transcription factors or increase cellular MLL3/MLL4 levels by affecting MLL3/MLL4 protein stability. Hyperactivating mutations in the catalytic SET domain would have a similar effect if enhancer activation required the catalytic activity of MLL3/MLL4. Alternatively, MLL3/MLL4-stabilizing or -hyperactivating mutations could also result in a general increase of enhancer activity on MLL3/ MLL4-associated enhancers. Changes in enhancer activity brought about by both context- and gene-specific MLL3/MLL4 loss-of-function (Figure 3A) or gain-of-function (Figure 3B) scenarios, as described above, are also supported by the COSMIC database. Missense mutations constitute more than 60% of reported MLL3 mutations (630 total) and more than 50% of reported MLL4 mutations (481 total), which allows for the possibility of transcription factor-specific, and thus in certain cases, tissue-specific exclusion (e.g., on tumor suppressor genes) or inclusion (e.g., oncogenes) of MLL3/MLL4 mutants in enhancer recruitment. The higher frequency of MLL3/MLL4 mutations in certain cancers might be explained by recruitment of MLL3/ MLL4 via tissue-specific transcription factors to the enhancers of tumor suppressor genes under wild-type conditions. Thus, MLL3/MLL4 would generally function as coactivators on the enhancers of tumor suppressor genes, but upon mutation, could either occupy the role of a transcriptional silencer of tumor suppressor genes or of an activator of oncogenes as discussed above. Alternatively, MLL3/MLL4 could also occupy the role of a corepressor on cis-regulatory ‘‘silencer’’ elements. Under wild-type conditions, MLL3/MLL4 recruitment to ‘‘silencers’’ by transcriptional repressors would keep oncogenes in a deactivated state. MLL3/MLL4 mutations that affect protein stability, recruitment, or catalytic activity as described for Figure 3A might in this case enhance the expression of oncogenes in a tissuespecific manner but differ in nature from the MLL3/MLL4 mutations outlined in Figure 3B. Future Directions for ‘‘Enhancer Therapy’’ in Cancer Treatment Given the potential roles for mutations in enhancer elements and in enhancer regulators such as MLL3/MLL4 in cancer pathogenesis, increasing our mechanistic understanding of how enhancer-mediated processes function in cellular growth should lie at the forefront of future research efforts. Despite the fact that MLL3/MLL4 regulate H3K4 monomethylation on many enhancers genome-wide, we currently do not know whether the context of MLL3/MLL4 and/or their H3K4 monomethyltransferase activities are instructive for the regulation of enhancer activities in general or only apply to particular cases. New techniques that allow the genome-wide identification of all active enhancers in a given cell type in combination with high-throughput sequencing approaches (Arnold et al., 2013) for MLL3/MLL4 would permit the direct and genome-wide determination of MLL3/MLL4-dependent enhancers under MLL3/MLL4 mutant 864 Molecular Cell 53, March 20, 2014 ª2014 Elsevier Inc.
conditions. These studies would have the potential to reveal MLL3/MLL4-dependent enhancers of tumor suppressor genes and/or oncogenes based on the nature of MLL3/MLL4 mutations as discussed above (Figure 3). Select representative MLL3/ MLL4-dependent enhancers could be further investigated for their dependency on the catalytic activity of MLL3/MLL4 and should be followed up by genome-wide RNAi screens to identify potential recruiters of MLL3/MLL4 COMPASS-like complexes to these enhancers. This will ultimately provide a network of general enhancer regulators and more context-dependent enhancerspecific factors and, thus, significantly improve our understanding of enhancer-mediated processes in cancer pathogenesis. Pharmacological or even gene therapeutic approaches that target transcription factors and cofactors that are recruited to the enhancers of tumor suppressor genes or oncogenes, such as MLL3/MLL4, which we refer to as ‘‘Enhancer Therapy,’’ should in the long run yield fruitful results in the fight against enhancer-mediated cancers. REFERENCES Akhtar-Zaidi, B., Cowper-Sal-lari, R., Corradin, O., Saiakhova, A., Bartels, C.F., Balasubramanian, D., Myeroff, L., Lutterbaugh, J., Jarrar, A., Kalady, M.F., et al. (2012). Epigenomic enhancer profiling defines a signature of colon cancer. Science 336, 736–739. Aran, D., and Hellman, A. (2013). DNA methylation of transcriptional enhancers and cancer predisposition. Cell 154, 11–13. Aran, D., Sabato, S., and Hellman, A. (2013). DNA methylation of distal regulatory sites characterizes dysregulation of cancer genes. Genome Biol. 14, R21. Ardehali, M.B., Mei, A., Zobeck, K.L., Caron, M., Lis, J.T., and Kusch, T. (2011). Drosophila Set1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription. EMBO J. 30, 2817–2828. , L.M., Rath, M., and Stark, A. Arnold, C.D., Gerlach, D., Stelzer, C., Boryn (2013). Genome-wide quantitative enhancer activity maps identified by STARR-seq. Science 339, 1074–1077. Ashktorab, H., Scha¨ffer, A.A., Daremipouran, M., Smoot, D.T., Lee, E., and Brim, H. (2010). Distinct genetic alterations in colorectal cancer. PLoS ONE 5, e8879. Banerji, J., Rusconi, S., and Schaffner, W. (1981). Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell 27, 299–308. Cheng, J., Blum, R., Bowman, C., Hu, D., Shilatifard, A., Shen, S., and Dynlacht, B.D. (2014). A Role for H3K4 Monomethylation in Gene Repression and Partitioning of Chromatin Readers. Mol. Cell 53this issue, 979–992. Corradin, O., Saiakhova, A., Akhtar-Zaidi, B., Myeroff, L., Willis, J., Cowper-Sal Lari, R., Lupien, M., Markowitz, S., and Scacheri, P.C. (2013). Combinatorial effects of multiple enhancer variants in linkage disequilibrium dictate levels of gene expression to confer susceptibility to common traits. Genome Res. Creyghton, M.P., Cheng, A.W., Welstead, G.G., Kooistra, T., Carey, B.W., Steine, E.J., Hanna, J., Lodato, M.A., Frampton, G.M., Sharp, P.A., et al. (2010). Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA 107, 21931–21936. de Kok, Y.J., Vossenaar, E.R., Cremers, C.W., Dahl, N., Laporte, J., Hu, L.J., Lacombe, D., Fischel-Ghodsian, N., Friedman, R.A., Parnes, L.S., et al. (1996). Identification of a hot spot for microdeletions in patients with X-linked deafness type 3 (DFN3) 900 kb proximal to the DFN3 gene POU3F4. Hum. Mol. Genet. 5, 1229–1235. de Villiers, J., Olson, L., Tyndall, C., and Schaffner, W. (1982). Transcriptional ‘enhancers’ from SV40 and polyoma virus show a cell type preference. Nucleic Acids Res. 10, 7965–7976. Dorsett, D. (1999). Distant liaisons: long-range enhancer-promoter interactions in Drosophila. Curr. Opin. Genet. Dev. 9, 505–514.
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