Department of Pediatrics, Division of Pediatric Hematology and Oncology, Department of Developmental Biology, School of Medicine, Washington University in St. Louis, 660 South Euclid Avenue, St. Louis, MO 63110, USA; moc.liamg@tbardneliahs or ude.ltsuw@ardneliahs.ayruam
Received 2021 Jul 28; Accepted 2021 Sep 28. Copyright © 2021 by the author.Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Enhancers are cis-regulatory elements containing short DNA sequences that serve as binding sites for pioneer/regulatory transcription factors, thus orchestrating the regulation of genes critical for lineage determination. The activity of enhancer elements is believed to be determined by transcription factor binding, thus determining the cell state identity during development. Precise spatio-temporal control of the transcriptome during lineage specification requires the coordinated binding of lineage-specific transcription factors to enhancers. Thus, enhancers are the primary determinants of cell identity. Numerous studies have explored the role and mechanism of enhancers during development and disease, and various basic questions related to the functions and mechanisms of enhancers have not yet been fully answered. In this review, we discuss the recently published literature regarding the roles of enhancers, which are critical for various biological processes governing development. Furthermore, we also highlight that altered enhancer landscapes provide an essential context to understand the etiologies and mechanisms behind numerous complex human diseases, providing new avenues for effective enhancer-based therapeutic interventions.
Keywords: enhancer, poised, cis-regulatory, lineage, spatio-temporalThe term “enhancer” was first coined based on studies of Simian virus 40 (SV40) when, in 1981, Banerji et al. observed for the first time that a viral DNA element from SV40 had the ability to enhance activity towards a T-antigen or β-globin reporter in mammalian cells [1]. Further research has explored endogenous sequences with similar functions in the immunoglobulin heavy chain locus. These preliminary studies have established that enhancers function as short DNA elements that trigger a gene’s transcription from a long distance in an orientation-independent manner. Preliminary studies have determined the exact mechanism of how distal regulatory elements regulate gene transcription through distal enhancers [2]. Enhancers are cis-regulatory elements that carry epigenetic information in DNA sequences through specific histone modifications [3]. Studies assessing the characteristics of enhancers have reported that they can function independently of the orientation and distance to their cognate target genes, at distances sometimes of several hundred kilobases or megabases [2,4,5]. Enhancers can be identified and characterized by various factors, including histone modifications, their transcription into non-coding RNAs, and their epigenetic features [6]. The prominent feature of enhancers is their ability to serve as a docking platform for transcription factor binding, where developmental signaling (intrinsic or extrinsic) cues are interpreted in a highly context-specific manner [7]. The signatures of these enhancers are highly cell type-specific; such cell type-specific use of the epigenetic information has demonstrated the combinatorial function of transcription factors in maintaining cell identity and lineage determination [8]. The cell type-specific enhancer pattern provides a unique cis-regulatory platform, in which transcription factors are activated by developmental cues and modify the transcriptome [9,10,11]. This model revealed that every developmental stage has a cell type-specific transcription factor, which functions by cell type-specific enhancer signatures [12]. Enhancers are actuated in a stage-specific manner, correlated with cell type-specific histone modifications. This combinatorial mechanism of transcription factor binding on cell type-specific enhancers results in the so-called enhancer signature, which serves as a readout to define enhancers in a cell type-specific manner at a global scale [13]. Precise spatial and temporal control of gene expression and the correct interpretation of the enhancer signature are crucial in the development process. Any alterations in the enhancer signature can modify the gene expression pattern and ethe capacity of the enhancer to respond to developmental signals, narrowing cell differentiation and affecting correct lineage formation. Heinz et al. (2010) have shown that lineage-determining transcription factors bind to a genomic region in a cell-specific manner. They showed the genome-wide locations of PU.1 binding patterns in macrophages, B-cells, and diffuse B-cell progenitors [14]. Similarly, Xu et al. (2012) analyzed chromatin state maps, transcription factor occupancy rates, and gene expression profiles during the development of human erythroid cells at the fetal and adult stages, and carried out a comparative analysis to determine the specific procedures of the development stage [15]. Here, it is also important to discuss the study of Choukrallah et al. (2015), who found that the enhancer landscape is dynamically reshaped in each differentiation step. Interesting changes include creating new enhancers and the closing and re-opening of the pre-existing enhancer landscape [16]. The authors also reported that the regulatory signatures of two related types of myeloid leukemia expression fusion proteins (RUNX1-ETO and RUNX1-EV1) display a distinct binding pattern and interact with different transcription factors to impact the epigenome [17]. A study has also reported that MLL-Af9 and MLL-AF4 oncofusion proteins showed distinct binding patterns in the enhancer region and targeted the RUNX1 program in 11q23 acute myeloid leukemia [18]. Enhancers are essential for normal functioning, and the loss of enhancer elements can cause abnormalities; for example, Groschel et al. (2014) showed that the removal of the distal enhancer essential for the GATA2 gene resulted in insufficient Functional GATA2 haploids, which only reduced the expression of the remaining normal alleles [19]. This review focuses on a concise and brief overview of the roles of enhancers in development and disease. We attempt to discuss how enhancers are activated and coordinated with transcription factors, as well as the roles of enhancers in mammalian development. This may comprise the first attempt to compile recently published research on development and disease, focusing on enhancers.
Enhancers have been identified in the form of various regulatory domains, primed enhancers, active enhancers, and poised enhancers. Each type of enhancer signature has specific histone modification patterns and can be easily identified by these signatures [13,20,21]. Primed enhancers can be identified by only histone H3K4 mono-methylation, while active enhancers signatures are identified by H3K4 mono-methylation and H3K27ac. Finally, the signatures of poised enhancers are marked with H3K4Me1 with H3K27me3, but not H3K27ac [13,20,21]. Active enhancers are linked to expressed genes, while poised enhancers are always associated to developmental genes, which are inactive in embryonic stem cells or precursor cells and become expressed during different differentiation stages [22]. During differentiation, the poised enhancer’s signature successfully loses the repressive H3K27me3 histone mark, acquires H3K27ac marks, and becomes active. Enhancers are, thus, subject to dynamic change functions, as an on/off switch to tune the target gene expression and changing the cell state from undifferentiated to differentiated phenotype [23]. Hence, the signatures of Poised Enhancers comprise a small set of regulatory signatures in embryonic stem cells that facilitate their timely and stage-specific function, once the correct differentiation signals become available [20]. One term also uses super-enhancers (SEs), which are described as large clusters of active enhancers with robust enrichment for binding transcriptional coactivators [24,25]. These features have also been called stretch enhancers [26], multiple enhancers [27], and enhancer clusters [28], which are similar but not identical between studies (although many of these features overlap). SEs regulate master regulators of pluripotency, such as OCT4, SOX2, and NANOG [24]. It has been reported that the SE signature is often enriched near the oncogene in tumor cells, while an enrichment GWAS has identified SNPs normally associated with several common diseases [29,30].
Many studies have established the idea that the enhancer signature is intricately orchestrated, in a stage-specific pattern, by several proteins complexes during development [31]. Some enhancer signatures are established early during development in precursor cells, and are modified and activated as cells differentiate in terminal steps along specific lineages [32]. All enhancer marks serve different purposes; for example, poised enhancers serve as signatures for future gene expression [2]. In contrast, active enhancer signatures play a functional role in the current transcriptional state [33]. The chromatin state is mostly invariant across different tissues, whereas histone signature patterns at the enhancer level are highly tissue-specific [34]. Mammals, with about 200 specialized cell types, all have different transcriptional outcomes, reflecting the unique coding pattern and regulatory elements during development [34,35,36]. Indeed, the enhancer repertoire active in a particular lineage, as identified by chromatin marks and transcriptional regulators, represents only a small subset of all genomic regulatory domains [33,37,38]. Studies have suggested that the enhancer repertoire is a pre-established landscape, formed and imposed by the lineage, determining TFs that maintain cell identity. All transcriptional regulation and functional outcomes occur within the differentiated cell under this pre-established enhancer landscape [10]. The study also established that enhancers playing a developmental role are evolutionarily conserved sequences [39,40]. Thus, the pre-established enhancer landscape has a crucial role in lineage determination. Any disturbance in the enhancer landscape affects the lineage, determining the potential of cells. This concept suggests that any external cues that trigger the transitory response cannot functionally change the repertoire of genomic regulatory domains, but act on the pre-established epigenomic landscape. This response is a buffer system that ensures cell identity maintenance, despite the changing environment [10]. A schematic diagram explaining enhancer biology is shown in Figure 1 . We also summarize the related studies in Table 1 , which have investigated the roles of enhancers in development through different model systems, including evidence in support of early enhancer establishment reported in B-cell and macrophage specification [14,16], T-cell development [41], early hematopoiesis [42], and the commitment of multipotent endoderm cells to liver and pancreas cell fates [43]. Wang et al. (2015) have also inferred the role of the poised enhancer landscape in endoderm development, as well as established a functional link between the gain of poised enhancer chromatin state and the temporal acquisition of competence during developmental progression [44]. Dynamic and coordinated epigenetic regulation has also demonstrated chromatin transition during cardiac lineage commitment [45]. Stage-specific enhancers are synergistically activated in a genome-wide manner during cardiac development by cardiac reprogramming factors [46]. The combinatorial action of pioneer factor and super-enhancer dynamics has also been reported in stem cell plasticity and lineage choice [47]. Further, Enhancer priming by histone methyl-transferase has also been demonstrated to control cell fate transition [48]. The master regulator ‘Scl’ has been reported to bind to pre-established primed enhancer signatures in the mesoderm, as well as regulating hematopoietic and cardiac fate divergence at terminal differentiation steps. Scl uses the pre-established epigenetic landscape during the specification of lineage choices [49]. The pioneer factor FOXA2 is required for enhancer priming during HPSC differentiation in pancreatic lineages [50]. It has also been shown that ERK directly regulates enhancer priming in lineage choice [51]. Developmental stage-specific enhancers drive lineages and control gene expression programs during hematopoiesis [15,52]. Rubin et al. (2017) have reported that lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation [53]. Recently, Maurya et al. (2021) [54] have shown that the loss of KMT2C in HPSCs cells significantly reprograms the enhancer landscapes in HPSCs cells, leading to the loss of Hemogenic endothelium during in vitro hematopoietic differentiation. Further, it has also been reported that the deletion of KDM6A in HPSCs cells significantly reprograms the Bivalent chromatin in HPSCs cells, suggesting perturbed development at the terminal developmental steps in particular lineages [55].