Is Enhancer Function Driven by Protein–Protein Interactions? From Bacteria to Leukemia

Nicholas T. Crump 1 Thomas A. Milne 2

1 Hugh and Josseline Langmuir Centre for Myeloma Research, Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, UK 2 MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK

Correspondence: Nicholas T. Crump (n.crump@imperial.ac.uk) Thomas A. Milne (thomas.milne@imm.ox.ac.uk)

Received: 7 July 2024 Revised: 23 March 2025 Accepted: 25 March 2025

Funding: This research was funded in whole, or in part, by the UKRI [MC_UU_00029/6] and a Kay Kendall Leukaemia Fund Intermediate Fellowship [KKL1443].

ABSTRACT

The precise regulation of the transcription of genes is essential for normal development and for the maintenance of life. Aberrant gene expression changes drive many human diseases. Despite this, we still do not completely understand how precise gene regulation is controlled in living systems. Enhancers are key regulatory elements that enable cells to specifically activate genes in response to environmental cues, or in a stage or tissue-specific manner. Any model of enhancer activity needs to answer two main questions: (1) how enhancers are able to identify and act on specific genes and (2) how enhancers influence transcription. To address these points, we first outline some of the basic principles that can be established from simpler prokaryotic systems, then discuss recent work on aberrant enhancer activity in leukemia. We argue that highly specific protein–protein interactions are a key driver of enhancer-promoter proximity, allowing enhancer-bound factors to directly act on RNA polymerase and activate transcription.

1 Introduction

Transcription of DNA is a fundamental process of life. To respond appropriately to environmental cues and drive tissue specificity, organisms need to be able to turn genes on and off, and increase and decrease transcription appropriately. Transcription initiation requires the stable assembly of an RNA polymerase (RNAP) complex at a specific initiation site termed the promoter [1]. The length of the transcript is determined by elongation efficiency, along with termination signals, and the ultimate fate of the RNA product depends on its stability and nuclear export. What happens to RNA after it is transcribed is not the focus of this review; instead we consider what we know about mechanisms that allow for the precise control of gene transcription.

2 Transcription Is Regulated by Enhancers

Enhancers were first identified in SV40 viral DNA, demonstrating an ability to increase transcription in cultured mammalian cells when paired with their cognate promoters [2]. Since then, our knowledge has expanded to recognize enhancers as a universal feature of gene regulation, essential for driving transcription in a highly developmental, tissue, and environment specific manner [3–5]. It is therefore not surprising that aberrant enhancer activity is increasingly being acknowledged as a driver of human disease [6–12]. Although enhancers are generally considered to be a eukary-otic innovation, enhancer-like elements also provide a crucial mechanism for gene regulation in prokaryotes [13]. Despite this ubiquity, the true mechanistic activity of enhancers is still not fully understood. In this review, we discuss what is known about enhancer function, and relate it to our recent work on oncogenic enhancers in leukemia [10, 14]. From there, we explore what this tells us about enhancer function more generally.

3 The Main Questions a Model for Enhancer Function Needs to Answer

Many different models of enhancer function have been proposed (comprehensively reviewed in [5]), but any model must explain two key questions. Firstly, how do enhancers identify and act on specific promoters? This question is underscored by observations that it is common for enhancers to bypass neighboring genes, with studies finding values ranging from 10% to 60% of enhancers that do not interact with the nearest promoter [15–18]. This argues that specificity is controlled by factors other than simple proximity along the linear sequence. A particularly elegant example of this is displayed by extensive work on the regulation of the alpha-globin locus [4], where the globin-specific enhancers are embedded in the nearby NPRL3 gene, but guide erythrocyte specific expression of the globin genes without activating the NPRL3 promoter [19]. Secondly, when an enhancer acts on a promoter, how does it influence transcription? It is worth considering the role of enhancer-like elements in bacteria to identify universal principles that apply across species.

4 Bacteria: Basic Principles for Enhancer Function

Many of the principles of transcription were first established with experiments in bacteria [20, 21], including purification and structural analysis of the RNAP complex [22–24] and some of the early single molecule work studying transcription dynamics [25]. Initiation of transcription requires two key processes: stable binding of RNAP to DNA (i.e., the promoter), followed by melting of the DNA. A single strand is introduced into the active site, enabling dNTP binding and formation of the first phosphodiester bond. A subsequent transition to stable transcription is required to achieve productive elongation. At high-affinity promoter ele-ments, RNAP binding can be achieved without additional factors. However, where promoter sequences are suboptimal, additional regulatory elements (e.g., enhancer-like elements) are required to enable stable RNAP binding and promoter melting. Suboptimal promoters are not simply a problem for the cell to overcome but represent an opportunity to introduce layers of modulation to the transcription of a gene. Control of the factors present at enhancers therefore provides a mechanism to regulate gene activation, for example, making transcription responsive to the environment or other specific cues. In bacteria, anchoring RNAP to the promoter is achieved by a set of DNA-binding proteins called sigma ( σ) factors [26]. Canonical σ70 factors direct RNAP binding to −35 and −10 promoter elements, and facilitate DNA melting. In contrast, the σ54 family, which recognizes distinct promoter sequences, lacks this unwinding activity (Figure 1A). Its ability to isomerize and activate transcription is entirely dependent on accessory factors, known as bacterial enhancer-binding proteins (bEBPs) [27, 28]. bEBPs bind to sequences typically 100–150 bp away from the promoter [28] but can function up to 3 kb away [29], and actively promote the formation of loops between the enhancer-like element and RNAP-bound promoter, directly binding to σ54 to induce DNA melting [30] (Figure 1B). In this way, bEBP-binding sites act analogously to eukaryotic enhancers. Looping is promoted by the integration host factor (IHF), helping bend DNA to bring bEBPs into the vicinity of the promoter [27, 31]. These loops disappear when transcription is initiated [30], suggesting they are a temporary occurrence needed only for the early stages of transcription (Figure 1C). The observation that transcription disrupts loop formation fits with the specific role for bEBPs in σ54 regulation. Although σ54 binding stabilizes RNAP binding, it also blocks DNA loading into the cleft of RNAP, keeping the complex in an inhibited state [28]. Interaction with bEBPs drives conformational changes, further DNA melting, loading of the DNA into the cleft, stabilization of the transcription bubble, and ultimately productive elongation [28]. Once productive elongation begins, bEBP contact with the σ54 :RNAP complex is no longer necessary and may even be inhibitory. Thus, these enhancer-promoter loops are transient, a point we will return to. Recent 3C work has revealed higher order DNA looping struc-tures in bacteria [32], similar in appearance to higher order struc-tures in eukaryotic cells (discussed in section 5). Importantly, these bacterial DNA loops are driven by transcription [32], likely due to protein–protein interactions such as bEBP: σ54 :RNAP, but could also be driven by the interaction of transcription complexes themselves.

5 General Features of Eukaryotic Enhancer Function

Many aspects of transcriptional regulation in eukaryotes are simi-lar to bacteria, but the protein complexes are bigger, the genomes are larger and contain many more genes, and the DNA has an additional regulatory layer created by the histone protein–DNA complex termed chromatin [33–35]. In multicellular organisms, more intricate control of gene expression is also crucial, adding complexity to the regulatory mechanisms required. As a key tool for driving higher levels of transcription from the promoter, enhancers are crucial regulatory elements, separated from their target genes by much longer distances than in bacteria [3, 5, 28]. In general, eukaryotic enhancers function as binding sites for sequence-specific DNA binding proteins, known as transcription factors (TFs). Active enhancers are associated with nucleosome-free or “open” regions (likely because they are bound by TFs), flanked by high levels of histone acetylation (especially H3 lysine-27, H3K27ac) as well as an enrichment for H3 lysine-4 monomethylation (H3K4me1) [36–39]. They are actively tran-scribed, typically producing short, unstable RNAs termed eRNAs [1] that are often rapidly degraded [40]. However, some enhancers are associated with the transcription of more stable long noncod-ing RNAs (lncRNAs) [41]. Enhancers can exist in large clusters termed super-enhancers that are often associated with cell identify or disease states [42–44]. Whether the super-enhancer concept is a useful model for enhancer function has been the topic of some debate [45], but they are generally recognized to be regions of dense protein binding and activity, and are often visually identifiable. Physical proximity between an enhancer and its target promoter, sometimes over very long distances, is a common feature of active enhancers. Although there may be examples where prox-imity does not appear to be important [46, 47], it is generally accepted to be a prerequisite for gene activation [3, 15, 18, 48– 50] (Figure 1D–F). In eukaryotes, large looping domains of chromatin, bounded by CTCF, are thought to be created through the loop extrusion activity of cohesin [51, 52], termed topologically associated domains (TADs) [53]. However, recent work in yeast has found that loop extrusion-deficient cohesin mutants retain the ability to entrap chromatin and form loops, especially TADs, arguing that extrinsic processes such as transcription may instead drive looping [54]. Within TADs, enhancers and promoters can interact, sometimes as a hub or cluster [55], through as yet unresolved mechanisms. One consequence of TADs is the insulation of enhancers from interacting with promoters outside the TAD [4, 5, 11, 56], providing a mechanism to restrict potential targets for gene activation. Loop formation is likely to be the sum of a complex series of interactions; in this review, we focus specifically on enhancer–promoter interactions and do not address the formation of larger domains such as TADs.

6 Controversies

The universality of histone acetylation at both active promoters and enhancers has been recognized [38, 39],