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# Epromoters are new players in the regulatory landscape
with potential pleiotropic roles

Juliette Malfait, Jing Wan, Salvatore Spicuglia

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## To cite this version:

Juliette Malfait, Jing Wan, Salvatore Spicuglia. Epromoters are new players in the regulatory landscape with potential pleiotropic roles. BioEssays, 2023, 10.1002/bies.202300012. hal-04200857

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HYPOTHESES

Insights & Perspectives

# Epromoters are new players in the regulatory landscape with potential pleiotropic roles

Juliette Malfait<fnref n="1" />,<fnref n="2" /> | Jing Wan<fnref n="1" />,<fnref n="2" /> | Salvatore Spicuglia<fnref n="1" />,<fnref n="2" /> [ID]

**Correspondence**

Salvatore Spicuglia, Aix-Marseille University, Inserm, TAGC, Marseille, UMR1090, France.
Email: salvatore.spicuglia@inserm.fr

**Funding information**

Institut National de la Santé et de la Recherche Médicale (INSERM); Ligue contre le Cancer (Equipe Labellisée Ligue 2023); ANR, Grant/Award Numbers: ANR-18-CE12-0019, ANR-17-CE12-0035; Bettencourt Schueller Foundation (Prix coup d'élan pour la recherche française); French Ministry of Education and Aix-Marseille University; Horizon 2020 research and innovation program, Grant/Award Number: 860002

**Abstract**

Precise spatiotemporal control of gene expression during normal development and cell differentiation is achieved by the combined action of proximal (promoters) and distal (enhancers) cis-regulatory elements. Recent studies have reported that a subset of promoters, termed Epromoters, works also as enhancers to regulate distal genes. This new paradigm opened novel questions regarding the complexity of our genome and raises the possibility that genetic variation within Epromoters has pleiotropic effects on various physiological and pathological traits by differentially impacting multiple proximal and distal genes. Here, we discuss the different observations pointing to an important role of Epromoters in the regulatory landscape and summarize the evidence supporting a pleiotropic impact of these elements in disease. We further hypothesize that Epromoter might represent a major contributor to phenotypic variation and disease.

**KEYWORDS**

diseases, enhancer, epromoter, gene regulation, pleiotropy, promoter, variants

## INTRODUCTION

In higher eukaryotes, gene transcription is regulated through the involvement of regulatory elements that are located near the transcription start site (TSS), called promoters, and those that are located far from TSS, called enhancers. This classical definition implies that enhancers activate gene expression at a distance while promoters induce local gene expression. Although these two elements are distinguishable by their genomics and epigenomics characteristics, a strict dichotomy between cis-regulatory elements is being challenged by the broad mechanistic similarities between promoters and enhancers.[1,2] On one hand, active enhancers are able to initiate transcription and recruit general transcription factors as promoters do. On the other hand, several lines of evidence have shown that a subset of coding-gene promoters is able to function as bona fide enhancers (hereafter named Epromoters), as detailed below.

Given the potential regulation of proximal and distal genes by Epromoters, we hypothesized that genetic variation or mutation at Epromoters might play an important role in physiological and pathological traits. In this review, we first describe the different studies

supporting the physiological relevance of genomic regulation by Epromoters. We then discuss the intrinsic features that might drive the enhancer and promoter activity of Epromoters and whether these are shared or specific properties as compared with typical enhancers and promoters. Finally, we provide current observations supporting the hypothesis of an important and pleiotropic role of Epromoter variation on the ontogeny of different diseases.

## SHORT AND LONG-RANGE GENE REGULATION BY EPROMOTERS

Dissection of cis-regulatory elements is classically based on gene reporter assays where the tested DNA regions are either placed immediately before the reporter gene (to assess for promoter activity) or placed upstream or downstream of a basic promoter (to assess for enhancer activity). Surprisingly, many of the early characterized enhancers in the 80s and 90s overlapped the promoter of inducible genes,[2,3] including the first identified enhancer which corresponded to the promoter of the simian virus 40 (SV40) early gene.[4] Currently,

<footnote n="1">¹Aix-Marseille University, Inserm, TAGC, UMR1090, Marseille, France</footnote>

<footnote n="2">²Equipe Labélisée Ligue Contre le Cancer, LIGUE, Marseille, France</footnote>
<figure><img src="image_5.png" /><figcaption>**FIGURE 1** Proximal and distal gene regulation by Epromoters. (A) Example of high-throughput reporter assays to assess enhancer activity of genomic regions. A genomic library is cloned downstream of a basic promoter and a GFP reporter gene. With this construct, enhancers will activate their own transcription. The transcripts are then quantified to assess the enhancer activity. In particular, this type of strategy allows identifying of promoter elements with intrinsic enhancer activity (i.e., Epromoters). pA: Poly-adenylation. (B) In wild-type (WT) cells, the Epromoter (yellow) regulates a proximal gene (promoter activity; blue arrow) and distal genes (enhancer activity; red arrows). In Epromoter-deleted cells (KO: knock out), both the proximal and distal gene expressions are impaired.</figcaption></figure>

powerful techniques incorporating high-throughput sequencing into reporter assays enable systematic and straightforward quantification of enhancer activity of cis-regulatory elements. Two similar high-throughput reporter assays have been widely used in recent years: Massively Parallel Reporter Assay (MPRA) and Self-Transcribing Active Regulatory Region sequencing (STARR-seq).[5] One striking observation of these episomal reporter assays, when assessing large genomic regions from Drosophila to different mammal cell types, is that many promoters display enhancer activity[6-15] (Figure 1A). In particular, by assessing all human core promoters of coding genes by STARR-seq, we found that ~3% of promoters exhibited enhancer activity in a given cell line.[8]

The presence of enhancer activity in some promoters, when tested in episomal reporter assays, does not necessarily mean that they could regulate other promoters in their endogenous context. Thus, whether gene promoters may function as bona fide enhancers by controlling distal gene expression is a critical issue. Several independent studies using mouse transgenics or CRISPR/Cas9 genome editing have demonstrated that the deletion or mutation of some promoters reduces the expression of a distally located gene,[8,16-19] implying they function as enhancers in their natural context (Figure 1B). So far, around 20 Epromoters have been validated experimentally at their endogenous loci by the different genome editing approaches in mouse or human cell lines (detailed in ref.[2]). High-throughput mapping of regulatory DNA by CRISPR-based screens also found evidence of distal gene regulation by gene promoters.[20-23] In particular, the repression of 30 (~8%) promoters out of 359 tested by CRISPR inactivation screen, resulted in reduced expression of a distal gene,[22] supporting the idea that a

substantial amount of promoters display enhancer function at their endogenous loci. Besides coding-gene promoters, promoters of long non-coding RNAs (lncRNAs) have also been shown to display enhancer activity independently of the transcript itself.[16,24,25] Nevertheless, in such cases, it is challenging to determine whether the tested regulatory element is a distal enhancer associated with a long transcript or rather the promoter of a "functional" lncRNA, which indirectly controls the expression of the neighbor gene.

Further evidence of Epromoter function comes from the analysis of 3D and genetic interactions. In addition to the enhancer-promoter interactions, the analyses of capture Hi-C and similarly derived 3C-based methods have shown that promoter-promoter (P-P) interactions are highly frequent.[26-32] Similarly, studies of expression quantitative trait loci (eQTLs) have revealed enrichment for genetic variants laying within gene promoters and associated with the regulation of distal genes.[8,17,29,33,34] Noticeably, some promoters interacting with other promoters, either at the 3D or genetic levels, indeed displayed enhancer activity.[8,17,23,27,33,35] An important outcome of these observations is that Epromoters often regulate several distal genes (in addition to the proximal one), including clusters of inducible genes,[18,32,36] suggesting that Epromoters might function as regulatory hubs for the coordinated regulation of gene clusters.

The impact on distal gene expression caused by the deletion or mutation of a promoter might, in principle, be caused by different mechanisms that are independent of a direct enhancer-like function. For instance, a given promoter might regulate distal genes by trans-effects, involving either the transcript itself or the protein-coding gene. However, re-expression of the FAF2[8] or OAS3[18] genes
associated with Epromoters did not rescue the expression of dis-
tal genes perturbed by the deletion of these Epromoters. Similarly,
genetic dissection combining promoter deletion and the introduction
of polyadenylation signals also provided evidence of direct enhancer-
like functions.[16] Another possibility is related to the notion of tran-
scription factories, whereby transcriptional hubs are known to contain
many genes and their promoters, and are thought to share limited
resources for their expression.[36-38] Here, the deletion of a pro-
moter within the hub could affect the expression of other distal genes
within that hub, without necessarily acting as an enhancer. Although
this type of mechanism might be at play in some instances, we have
demonstrated that in the case of the aforementioned FAF2 and OAS3
Epromoters, the deletion of the promoters associated with the distally
regulated genes did not impact the expression of the Epromoter-
associated genes, supporting the directionality of the regulation by
these Epromoters.[8,18] Alternatively, the predicted Epromoter might
be involved in a complex 3D organization, for example, involving
the sequestering of another distal cis-regulatory element and, when
deleted, it could indirectly affect the expression of a distal gene without
acting as an enhancer for that gene. To date, few examples have unam-
biguously demonstrated the direct enhancer function of Epromoters,
and whether the majority of Epromoters have a direct impact on dis-
tal gene expression or work in combination with the aforementioned
mechanisms, will need further investigation.

All in all, the discovery that a subset of promoters also functions as
bona fide enhancers, has important implications for our understanding
of complex gene regulation in normal development and offers a ratio-
nale for the frequent repurposing of promoters and enhancers during
mammalian evolution.[1,39,40] Additionally, they raise the intriguing
possibility that sequence variation found within Epromoters may
have an impact on diseases or physiological traits by directly impact-
ing distal gene expression or changing their relative promoter and
enhancer activities. A concomitant question is to understand which
intrinsic features drive the specific enhancer and promoter function of
Epromoters.

EPROMOTERS SHARE ENHANCER AND PROMOTER PROPERTIES

Although typical enhancers and promoters are logically distinguished
by their relative location with respect to the TSS of genes that they
regulate, their shared architectural properties have suggested a uni-
fying model of gene regulation by cis-regulatory elements.[1,2,41-43]
Alike promoters, enhancers recruit RNA-Polymerase II (Pol II) and
General Transcription Factors (GTF), and transcribe non-coding
RNAs (eRNAs).[41,44-48] Promoters and enhancers are both demar-
cated by divergent transcription initiation, surrounded by a well-
positioned array of nucleosomes, and enriched in core promoter
elements.[41,45,48-51] However, transcripts generated by enhancers
are generally bidirectional and less stable. Being generally depleted
in CpG islands, enhancers recruit master regulators like CpG-poor
promoters,[48] while some developmental enhancers require a prox-

imal CpG island to function.[52] Histone post-translational modi-
fications have been used to discriminate between enhancers and
promoters.[53-55] For instance, gene promoters typically exhibit
trimethylation of the histone H3 Lys4 (H3K4me3), while enhancers
were found to be enriched in monomethylated H4K4 (H3K4me1)
and acetylated H4K27 (H3K27ac). As a consequence, the presence of
H3K27ac along with high levels of H3K4me1 and low H3K4me3 is
commonly used as a proxy for active enhancers.[54] However, the level
of H3K4me3 is positively correlated with the enhancer strength and
eRNA level,[9,41,45,47,48,56,57] and, therefore, the presence of H3K4me3
is fully compatible with the enhancer activity. Thus, the relative enrich-
ment in epigenetic modifications might simply indicate differences in
transcriptional levels between the two types of elements, rather than
reflecting mutually exclusive functions.

A main intrinsic difference between enhancers and promoters relates to the composition of transcription factor binding sites (TFBS). High promoter activity is associated with a high density of overlapping binding sites for different TFs, in particular, ubiquitously expressed ones, while enhancers are less constrained.[1] In addition to binding site complexity, the type of TF that binds a cis-regulatory element might influence the relative enhancer or promoter activity.[58] For instance, the binding of AP1 and NFY is associated with enhancer activity, whereas the binding of CREB, ETS and SP1 is preferentially associated with promoter activity.[13,59-62] This suggests that the nature of bound TFs might directly contribute to the enhancer and promoter properties of cis-regulatory elements.

Given the aforementioned duality of cis-regulatory elements, cur-
rent models do not propose that promoter and enhancer activities
are mutually exclusive, rather different regulatory elements might
accommodate different proportions of these activities[1] (Figure 2).
In such scenarios, Epromoters represent remarkable examples of cis-
regulatory elements that share functional and architectural properties
with both types of cis-regulatory elements.[1,2]

High-throughput reporter assays have allowed a systematic com-
parison of cis-regulatory elements independently of their relative
proximity with TSS.[5] These studies have revealed specific proper-
ties of Epromoters that distinguish them from typical enhancers and
promoters. When compared to distal enhancers, Epromoters mainly
differ by the type of associated TFBS. While distal enhancers are pref-
erentially associated with TFBS for developmental and tissue-specific
transcription factors (TFs), Epromoters appear to be associated with
TFBS for ubiquitous or inducible TFs.[7,10,12,15,18] When compared to
typical promoters, Epromoters differ by the higher level of unstable
bidirectional transcripts,[8,45,50,51,63] association with co-activators,
such as p300[8] and more frequent P-P interactions.[8,29] Moreover,
Epromoters appear to be associated with a higher density of TF bind-
ing and a higher quality of binding sites.[8,18] The above results are
consistent with the fact that Epromoters are preferentially associated
with housekeeping and stress response genes,[7,8,10,12,15] including
interferon-response genes.[8,12,18,64] For instance, we found that Epro-
moters are significantly associated with the induction of gene clusters
during the inflammatory response and that induced Epromoters are
characterized by a higher density and quality of Interferon-Stimulated
<figure><img src="image_6.png" /><figcaption>**FIGURE 2** A general model defining cis-regulatory function. Regulatory elements are composed of different ranges of binding sites for transcription factors associated with either enhancer (red) or promoter (blue) properties. Enhancers are mostly composed of binding sites associated with enhancer activity and initiate bidirectional transcription of short and unstable transcripts. Promoters are principally composed of binding sites associated with promoter activity and initiate strong unidirectional transcription towards the coding gene. Epromoters share structural features of both promoters and enhancers.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**FIGURE 3** Epromoters might function as regulatory hubs for the coordinated induction of gene clusters. During the interferon response, clusters of induced genes are frequently associated with Epromoters. In this context, the Epromoter recruits the key interferon-response TFs STAT1, STAT2 and IRF9, and simultaneously regulates co-induced genes within the same cluster. Legend is as in Figure 1.</figcaption></figure>

Response Elements (ISRE), as compared with typically induced pro-
moters, which, in turn, results in the Epromoter-specific recruitment
of STAT1/2 and IRF TFs[18] (Figure 3). Moreover, inhibition of inter-
feron signaling in HeLa cells drastically reduced the number of active
Epromoters without affecting distal enhancers.[12,18,64] These results
suggest that at least a subset of Epromoters plays an essential role
in the coordination of rapid gene induction upon cellular response to

intra- and extra-cellular signals which might require a high efficiency of
TF recruitment.

We speculate that Epromoters represent a combination of the two
types of cis-regulatory elements, thus combining features that are
associated with enhancer and promoter activities within an enhancer-
promoter continuum of cis-regulatory elements (Figure 2). This inter-
mediated position implies that Epromoters might display a higher
density and complexity of TFBS because it has to accommodate the
binding of TFs for both enhancer and promoter functions. In this
scenario, typical promoters are enriched in binding sites for TF con-
ferring promoter activity and enhancers enriched in binding sites for
TF conferring enhancer activity, while Epromoters will be enriched
for both types of binding sites leading to a higher density of TFBS.
An alternative model will imply that Epromoters are associated with
a unique combination of TFBS providing specific enhancer-promoter
features. Future works should apply systematic assess the contribution
of TFBS and associated transcription factors to enhancer and promoter
activity in combination with machine learning models to understand
the molecular features that determine the intrinsic promoter and
enhancer potentials of cis-regulatory elements, and in particular of
Epromoters.[1,10,65] This, in turn, might help to better predict the
impact of mutations or natural variants of Epromoters that might affect
either proximal or distal gene regulation.

GENETIC VARIATION AT EPROMOTERS MIGHT HAVE PLEIOTROPIC ROLES

There is growing evidence that a wide range of human diseases is influ-
enced by dysfunctions of cis-regulatory elements caused by genetic,
structural, or epigenetic mechanisms.[66] These processes frequently
underpin the susceptibility to common diseases but can be also directly
involved in cancer or Mendelian diseases. The advent of genome-
wide association studies (GWASs) in the past decade has been one of
the great endeavors in genomic research toward identifying genetic variants associated with candidate genes for common diseases. The majority of these genetic variants are found in non-coding regions, therefore are likely to be involved in regulatory mechanisms controlling gene expression.[67-69] However, a major challenge in interpreting the impact of genetic mutation or variation in disease is to identify the targets that are impacted by the genomic alteration, which are not necessarily the closest genes and might have confounding features.[70] Despite this, most studies select the closest gene to the associated GWAS variant to establish possible causal mechanisms, namely when the variant lies in the vicinity of a TSS or within an intronic region. However, this assumption has been shown to be biased in examples like the FTO[71] and TCF7L2[72] loci, in which obesity and type 2 diabetes GWAS data, respectively, were interpreted to implicate the nearest genes, while 3D epigenomics and functional follow-up showed that the disease variants reside in elements that regulate distant genes. In a similar way, it might be envisioned that GWAS variants lying within Epromoters might regulate the expression of distal disease-causing genes.

The discovery of Epromoters thus opens a new paradigm in the study of regulatory variants as a mutation in a promoter could potentially influence the expression of several genes or change the relative ratio of promoter versus enhancer activity. Thus, resulting in a variety of potential changes in the relative expression of neighboring genes (Figure 4A). In addition, it is plausible that the same cis-regulatory element displays preferential promoter activity in some tissues while displaying increased enhancer activity in other tissues, depending on the expressed combination of TFs and the epigenetic context.[8,33,73] For instance, Leung et al. found frequent examples of dynamic epigenetic switches where active promoters in one tissue displayed a histone modification signature of enhancers in other tissues/cell types.[73] Similarly, Chandra et al. found that a substantial number of promoter-promoter interactions involved transcriptionally inactive genes, suggesting that non-transcribing promoters may function as active enhancers for distal genes.[33]

The complex regulation by Epromoters might therefore have two predicted consequences. On one hand, there might be a general underestimation of the impact of Epromoter variation in disease because the causal gene might not be the closest one and therefore the link between genotype and phenotype might be missed in many case studies. On the other hand, as Epromoters potentially control several genes at the same time and efficiently recruit key TFs, mutations in these regulatory elements are expected to have a stronger pathological impact, as compared to typical promoters. This might result from the regulation of multiple genes either involved in the same (additive or synergistic effects) or different (pleiotropy) pathways (Figure 4B). Here, pleiotropy refers to a single cis-regulatory element affecting more than one trait independently.[74] Pleiotropy could be due to the perturbation of a single gene playing multiple functions in different tissues [75,76] or the regulation of multiple genes in the same or different tissues.[77,78] Several genomic features, such as a higher number of regulated genes and more abundance and diversity of encoded TFBS, are indicative of increasing variant pleiotropy.[78-80] Notably, these two features are

readily associated with both Epromoters and typical enhancers. Thus, it is fair to hypothesize that genetic alterations affecting Epromoters are likely to be involved in disease, as previously suggested,[2,18,27] and that Epromoters might have a stronger impact on the regulation of disease-associated genes, as compared with typical promoters. Although the hypothesis is not fully validated yet, several lines of observations support this assumption, as detailed below.

There are several pieces of genetic evidence indicating that promoter variants affecting distal genes are physiologically relevant. One way to connect GWAS-reported genetic variants with effects on gene function is to associate the genetic polymorphisms with eQTLs.[81] eQTLs with a higher probability to directly impact gene expression variation tend to be found in open chromatin regions, such as promoters and enhancers,[82] supporting the hypothesis of a possible effect through changes on regulatory mechanisms. Studies of natural genetic variation through eQTLs thus provide important insights into the mechanisms of specific diseases and gene control, and can point to the possible gene regulatory function of specific sequences based on their allelic associations with gene expression.[66]

Several studies have observed significant enrichment for eQTLs located within gene promoters which are associated with the regulation of distal genes,[8,17,29,33,83] pointing out to Epromoter-like regulation. A study, integrating predicted allelic variation in TF binding affinity in human lymphoblastic cell lines with their putative target genes inferred from Promoter Capture Hi-C, observed that a large proportion of regulatory variants associated with distal gene expression localized to the promoter regions of other genes, supporting the notion of Epromoters.[29] Interestingly, some of these variants were co-associated with the expression of both proximal and distal genes, while others were uniquely associated with distal genes. Using a set of Epromoters identified by STARR-seq, we observed that eQTLs lying within Epromoters are more likely to be associated with the expression of a distal gene as compared to other promoters and tend to have stronger effects on distal gene expression.[8] By analyzing promoter-centered long-range chromatin interactions in the human genome, Jung et al.[17] and Chandra et al.[33] found that P-P interactions were significantly enriched in eQTLs where a genetic variant in one of the interacting promoters was associated with the expression of the other interacting promoter. In these three studies, CRISPR-mediated gene editing recapitulated the predicted function of the promoter-associated eQTL variants in the regulation of distal gene expression.[8,17,33] A similar study found that the majority of genetic variation affecting TF binding at Epromoters in Lymphoblastoid cell lines was associated with the expression of distal genes alone, independently of whether the proximal gene was transcriptionally active.[29] While it is difficult to ensure that all promoter-distal eQTLs are bona fide distal regulators, we noticed that taking into consideration functional assays for enhancer activity (STARR-seq) allows to significantly enrich for promoter-eQTLs regulating distal genes with a higher probability of perturbing TF binding affinity.[8] Taken together, these findings support the functional significance of long-range transcriptional regulation by Epromoters and imply that regulatory variants within these elements may have both independent
<figure><img src="image_8.png" /><figcaption>**FIGURE 4** Effects of Epromoter variation on gene regulation. (A) Different potential impacts of Epromoter genetic variation on proximal and distal gene expression. In the reference (ref) haplotype, proximal and distal genes transcription are regulated by the Epromoter. In the alternative (alt) haplotypes, the promoter (blue arrows) and enhancer (red arrows) activities could increase (thicker arrows) or decrease (thinner and dashed arrows), resulting in up-or down-regulation of the associated genes. (B) Genetic variants at Epromoters might result in either synergistic/additive (all affected genes are involved in the same disease/trait) or pleiotropic (each affected gene is involved in a different disease/trait) effects.</figcaption></figure>

and shared effects on the expression of their proximal and distal target genes.

Besides the global genomic evidence, several specific examples are pointing toward the relationship between disrupted Epromoters and variants associated with a variety of diseases, including autoimmunity (Crohn's disease, Lupus),[30,33,34,83,84] cardiovascular diseases,[19,85,86] diabetes,[2,35,87] infection diseases,[88,89] and cancer[90-93] (Table 1). However, to our best knowledge only in four cases, have the link between the disease-associated variant and the Epromoter function been experimentally validated[84,88,91,92] (Figure 5). In the first case, the alternative variant of a promoter-overlapping SNP associated with prostate cancer changes the relative affinity for two transcription factors resulting in promoter-enhancer switching and the corresponding increase of the expression of two distal transcripts directly involved in cancer progression (Figure 5A).[91,92] In the second case, the promoter of the BAZ2B gene was identified as Epromoter based on STARR-seq, while CRISPR-mediated deletion resulted in decreased expression of the MARCHF7 gene located 95 kb

away.[8] The BAZ2B promoter overlaps with an SNP in eQTL with MARCHF7[8] and is associated with hypothyroidism (Ref.[94]; unpublished observation). Haplotype replacement by CRISPR-mediated homology recombination resulted in reduced expression of MARCHF7, but not BAZ2B,[8] suggesting it is the distal gene regulation of the identified Epromoter's SNP that is involved in the disease (Figure 5B). In the third case, haplotype-specific chromatin looping implicating genetic variants associated with Systemic Lupus Erythematosus (SLE) revealed that the alternative haplotype laying within the promoter of BLK gene decrease the promoter activity while increasing the long-range interaction with the FAM167A promoter resulting in the up-regulation of FAM167A expression (Figure 5C).[84] In the last case, a haplotype of five genetic variants associated with severe Malaria and laying within the internal promoter of the ATP2B4 gene was found to switch the relative promoter and enhancer activity by luciferase reporter assays resulting in the increased expression of the long ATP2B4 isoform initiated from the upstream promoter (Figure 5D).[88]
<table><caption>TABLE 1 List of diseases associated variants in Epromoters.</caption><thead><tr><td>Disease</td><td>Epromoter-containing variant<sup>a</sup></td><td>Affected gene(s)</td><td>Evidence<sup>b</sup></td><td>Ref.</td></tr></thead><tbody><tr><td>Prostate cancer</td><td>PCAT19-short isoform</td><td>PCAT19-long &amp; short isoforms, CEACAM21</td><td>P-P interaction, enhancer &amp; promoter reporter assays, CRISPR-Cas9 genome editing, CRISPR interference/activation</td><td>[91, 92]</td></tr><tr><td>Hypothyroidism</td><td>BAZ2B</td><td>MARCHF7</td><td>P-P interaction, CRISPR-Cas9 genome editing, eQTL</td><td>[8]</td></tr><tr><td>Systemic lupus erythematosus</td><td>BLK</td><td>BLK, FAM167A</td><td>P-P interaction, CRISPR interference,</td><td>[84]</td></tr><tr><td>Severe malaria</td><td>ATP2B4-short isoform</td><td>ATP2B4-long &amp; short isoforms</td><td>Enhancer &amp; promoter reporter assays, CRISPR/Cas9 genome editing</td><td>[88]</td></tr><tr><td>Cardiovascular diseases</td><td>Nppb</td><td>Nppa</td><td>P-P interaction, Mouse transgenic models</td><td>[19, 86]</td></tr><tr><td>Type 2 diabetes</td><td>ARAP1</td><td>PDE2A</td><td>CapSTARR-seq, promoter reporter assays, eQTL</td><td>[2, 87]</td></tr><tr><td>Rheumatoid arthritis</td><td>CCR6</td><td>RNASET2</td><td>P-P interaction, eQTL</td><td>[33]</td></tr><tr><td>Systemic lupus erythematosus</td><td>TREH</td><td>CXCR5</td><td>P-P interaction, eQTL</td><td>[34]</td></tr><tr><td>Crohn disease</td><td>SMAD3</td><td>SMAD3, AAGAB</td><td>P-P interaction, eQTL</td><td>[30, 99]</td></tr><tr><td>Coronary artery disease</td><td>CDKN2B</td><td>IFNA2</td><td>P-P interaction</td><td>[85]</td></tr><tr><td>Multiple cancers</td><td>TERT</td><td>CLPTM1L</td><td>Somatic mutations, correlation with gene expression</td><td>[90]</td></tr><tr><td>Type 2 diabetes</td><td>INS</td><td>SYT8</td><td>P-P interaction, siRNA</td><td>[35]</td></tr></tbody></table>

In addition to small genetic variants, other types of genomic alterations involving enhancer repositioning (a phenomenon also named "enhancer hijacking") by chromosomal translocations, genomic rearrangements, or insulator disruption, have been described as common molecular mechanisms resulting in disease-related gene deregulation, including overexpression of oncogenes.[66] Epromoter hijacking could likely impact diseases through related mechanisms. For instance, in T-acute lymphoblastic leukemia, a large intergenic deletion replaces the TAL1 locus into the vicinity of the CMPK1 promoter, which displays features of an active enhancer, resulting in TAL1 oncogenic expression.[93] More generally, a study, using randomly integrated reporter constructs, found that chromosomal contacts with endogenous promoters

of housekeeping genes is required for the expression of the reporter gene,[95] supporting the idea whereby genomic repositioning due to structural variations might result in gene expression deregulation by Epromoter hijacking.

## CONCLUDING REMARKS AND FUTURE DIRECTIONS

Current results point to an important role of Epromoters in the regulatory landscape. These findings also open up the possibility that disease-associated variants or developmental traits lying within a

<footnote><sup>a</sup>Name of the proximal gene associated with the Epromoter.</footnote>

<footnote><sup>b</sup>Nature of the evidence suggesting an Epromoter-type of regulation.</footnote>
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

**FIGURE 5** Examples of Epromoter variation associated with diseases. (A) The variant rs11672691 is associated with prostate cancer and locate within the internal PCAT19 promoter. The alternative variant switches the relative promoter and enhancer activity resulting in up-regulation of the most upstream PCAT19 promoter and the distal gene CEACAM21. (B) The variant rs1046496 is associated with hypothyroidism and locate within the BAZ2B promoter. The alternative variant decreases the transcription of the MARCHF7 gene. (C) The variant rs922483 is associated with systemic lupus erythematosus and locate within the BLK promoter. The alternative variant decreases the transcription of the BLK gene while increasing the expression of the FAM167A gene. (D) A haplotype of five variants containing the lead variant rs10900585 is associated with severe malaria and is located within the internal promoter of ATP2B4. The alternative variant switches the relative promoter and enhancer activity resulting in up-regulation of the most upstream ATP2B4 promoter.

subset of promoters directly impact distal gene expression. Indeed, the recent observations support the hypothesis whereby Epromoters have a pleiotropic effect on diseases by perturbing the expression of several genes at the same time. Future works, including additional experimental settings where the Epromoter function is assessed in their endogenous loci, should tell us how commonly promoters are used as distal enhancers and better describe the physiological contexts where they are at play. Equally important will be to assess the physiological relevance of genes regulated by Epromoters. Are both proximal and distal gene deregulation directly involved in diseases? In particular, given the preponderant role of Epromoters in the regulation of interferon-response genes, it is expected that they might have an important impact on the etiology of inflammatory diseases.

Although several works have provided clear examples of natural genetic variation within promoters affecting the expression of a distal gene or an isoform regulated by a distal promoter, more

systematic studies are required to ascertain whether Epromoters indeed play a pleiotropic role in disease. While sequence-based models perform well in predicting how the impact of genetic variants in promoters affects local gene expression, they still perform very low to predict distal gene effects.[96] Therefore, we suggest that promoter-associated variants should systematically be tested for their proximal and distal effects. One possibility might be to simultaneously test the same DNA fragment in high-throughput reporter assays designed to assess the enhancer and the promoter activity in parallel.[13,50] This type of approach will help to elucidate whether enhancer and promoter activity of Epromoters are generally correlated amongst different cell types/tissues or whether they exhibit tissue-specific context. Similarly, it will help to assess whether genetic variants generally influence the global regulatory activity of Epromoters or rather affect their relative function to primarily work as a promoter or as an enhancer.
A central goal of biology is to decipher the cis-regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state.[97] Two major advancements provide a paradigm shift in our capacity to integrate mechanistically informed, quantitative models of transcriptional regulation toward cracking the cis-regulatory code. On one side, the development of high-throughput reporter assays allows systematic and quantitative measurements of cis-regulatory activity. On the other side, the ability of recent deep learning models to learn the most relevant features from genomic data and to interpret and extract the features (e.g., DNA sequence) that underlie the predictions. These interpretable rules can then be used to decode the regulatory "syntax" or "grammar," providing detailed information about the arrangement of TFBS, including their number, order, orientation and spacing. Several examples, disentangling cis-regulatory functions by combining high-throughput reporter assays with deep learning methods, have provided remarkable results.[62,65,98] Similar approaches aiming to dissect the intrinsic DNA features required for promoter and enhancer activities should be applied to the study of Epromoters. As Epromoters share features of both enhancer and promoter functions, a clear disentangling of both activities should lead to a better definition of the molecular bases governing gene regulation. In turn, understanding the genetic features driving proximal and distal activities will allow a better prediction of the impact of genetic variants with a pleiotropic effect on disease.

## ACKNOWLEDGMENTS

Work in the laboratory of Salvatore Spicuglia was supported by recurrent funding from Institut National de la Santé et de la Recherche Médicale (INSERM) and Aix-Marseille University, and by specific grants from Ligue contre le Cancer (Equipe Labellisée Ligue 2023), ANR (ANR-18-CE12-0019 and ANR-17-CE12-0035) and Bettencourt Schueller Foundation (Prix coup d'élan pour la recherche française). Juliette Malfait is supported by a fellowship from the French Ministry of Education and Aix-Marseille University. Jing Wan is a fellow of the EU-funded Innovative Training Network "Molecular Basis of Human Enhanceropathies" and received funding from the Horizon 2020 research and innovation program under Marie Sklodowska-Curie grant agreement no. 860002.

## CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

## DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

## ORCID

Salvatore Spicuglia https://orcid.org/0000-0002-8101-7108

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**How to cite this article:** Malfait, J., Wan, J., & Spicuglia, S. (2023). Epromoters are new players in the regulatory landscape with potential pleiotropic roles. *BioEssays*, e2300012. https://doi.org/10.1002/bies.202300012