Review

Enhancer RNAs and regulated

transcriptional programs

1 2 2 1,2

Michael T.Y. Lam , Wenbo Li , Michael G. Rosenfeld , and Christopher K. Glass

1

Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, USA

2

Howard Hughes Medical Institute, Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA,

USA

A large portion of the human genome is transcribed into Recent genomic and epigenomic advances have contrib-

RNAs without known protein-coding functions, far out- uted many insights into the properties, activity, and selec-

numbering coding transcription units. Extensive studies tion of enhancer elements. Interestingly, the selection of a

of long noncoding RNAs (lncRNAs) have clearly demon- large fraction of cis-acting regulatory elements for enhanc-

strated that they can play critical roles in regulating gene er activity appears to depend on relatively simple combi-

expression, development, and diseases, acting both as nations of the lineage-determining transcription factors

transcriptional activators and repressors. More recently, (LDTFs) for cell type identity. Through cooperative binding

enhancers have been found to be broadly transcribed, to closely spaced recognition motifs, LDTFs initiate bind-

resulting in the production of enhancer-derived RNAs, or ing to otherwise closed chromatin and facilitate chromatin

eRNAs. Here, we review emerging evidence suggesting accessibility by recruitment of ATP-dependent nucleosome

that at least some eRNAs contribute to enhancer func- remodeling complexes. Histone modifiers are recruited to

tion. We discuss these findings with respect to potential deposit histone marks that demarcate enhancer regions

1

mechanisms of action of eRNAs and other ncRNAs in (e.g., monomethylation of histone 3 lysine 4, H3K4me ) [6].

regulated gene expression. Although a large fraction of enhancers requires cooperative

binding of TFs [6,7], another group of enhancers is activat-

Enhancers in development and diseases ed by sequential binding of TFs at different development

Functional specialization of cell and tissue types is vital for stages [8]. A group of nucleosome-avid ‘pioneering’ TFs,

all metazoans. This requires cells to respond to develop- exemplified by Forkhead box A (FoxA) and GATA binding

mental and environmental cues by generating specific gene protein (GATA), initiate this sequential binding to con-

expression patterns on the basis of an identical set of densed chromatin to prime a ‘soon-to-be’ enhancer [9].

genetic material. Enhancers are the principle regulatory Binding of either LDTFs or pioneering factors permits

components of the genome that enable such cell-type and the binding of other TFs and cofactors upon signal trans-

cell-state specificities of gene expression. Enhancers were duction events resulting in cell-type specific, signal-depen-

initially defined as DNA elements that act over a distance dent gene expression (Figure 2A). For more information on

to positively regulate expression of protein-encoding target the physiology, clinical implication, and biochemical and

genes [1]. Enhancers contain specific recognition sequences molecular perspectives of enhancers, we refer our readers

required for binding of transcription factors (TFs) that to several excellent reviews [2,10–12].

regulate gene expression in a spatial and temporal fashion Recent findings that enhancers can be transcribed added

(Figure 1A). An estimated 400 000 to >1 million putative a new layer of complexity to gene regulation [13]. In addition

enhancers exist in the human genome [2,3], vastly out- to mRNAs, ncRNAs represent a highly functional class of

numbering protein-coding genes, therefore suggesting a molecules because they can possess enzymatic activity (e.g.,

high complexity of enhancer utilization in gene regulation. spliceosomes, ribozyme), have structural roles (e.g., tRNA,

Indeed, a precise pattern of activation of specific cohorts of ribosomes), and transcriptional functions (e.g., lncRNAs)

enhancers is critical for cell-type development and cell [14,15] (Box 1). Understanding how RNAs contribute to

lineage determination, as well as cellular responses to enhancer function has thus become an area of active inter-

stimuli (Figure 1A,B). By contrast, genetic variance in est. We will discuss some of the historical aspects of enhan-

enhancer sequences can alter TF binding, predisposing cers as transcription units and recent studies suggesting

the organism to ‘improper’ gene expression and, ultimate- functional roles of enhancer-derived RNAs, noting similari-

ly, susceptibility to diseases (Figure 1C) [4,5]. ties and differences with other classes of ncRNAs that can

exert positive effects on gene regulation.

Corresponding authors: Rosenfeld, M.G. ([email protected]); Glass, C.K.

([email protected]).

Keywords: noncoding RNA; enhancer; eRNA; transcription.

Evidence of RNA transcription from enhancers

0968-0004/$ – see front matter

Evidence of pervasive RNA transcription at active enhanc-

ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tibs.2014.02.007

er elements became apparent with recent advances in

sequencing technology. Interestingly, several reports over

the past half a century had already hinted at the existence

of short-lived nuclear RNAs (Table 1). Using pulse-chase

170 Trends in Biochemical Sciences, April 2014, Vol. 39, No. 4

Review Trends in Biochemical Sciences April 2014, Vol. 39, No. 4

(A) (B) (C) Physiology

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TiBS

Figure 1. Roles of enhancers in development, signaling events, and diseases. (A) Differential enhancer binding patterns dictate temporal and spatial gene regulations

during development. Colored nodes with ‘e’ indicate potential enhancer elements, which can be activated during specific developmental windows upon recruitment of

certain transcription factors (TFs) for coordinated tissue or cell-type specific gene expression patterns. Tissue-specific enhancers are color coordinated. Relative expression

levels are denoted by the proportional sizes of the arrowheads. ‘p’ represents the promoter; the double line represents the linear genomic distance between enhancer and

promoter that can range from several kilobase pairs (kbps) to megabase pairs (Mbps). (B) During gene transcriptional activation events in response to differentiation or

environmental cues [e.g., lipopolysaccharide (LPS) stimulation in macrophages], a group of pre-existing enhancers (highlighted green) bearing mono- or di-methylation of

1/2

histone lysine 4 (H3K4me ) modification is readily activated. A small subset of enhancers is generated de novo in response to LPS stimulation resulting in deposition of

1/2

H3K4me enhancer marks [27]. This group of enhancers, highlighted in blue, will be readily activated upon repeated stimulation [71]. The order from top to bottom

indicates the sequential cascade of events (from stimulus activation to restoration of resting state); the distance between two histone methylation marks represents

nucleosome spacing and chromatin accessibility; and brown lines on ‘e’ indicates enhancer-derived RNAs (eRNAs). (C) Dysregulated enhancer functions are involved in

human diseases, as exemplified in breast cancer and coronary artery disease (CAD) [5,72,73]. Genetic variation in an enhancer (denoted as e’) could alter TF binding or

create or destroy functional enhancers, resulting in differential gene regulation. This is also often paralleled by differential chromosomal looping. Some of these scenarios

can be directly linked to disease-associated genetic variants that occur at the DNA level of enhancers (e.g., single nucleotide polymorphism, insertion, deletion, and copy

number variants). The sizes of arrowheads of genes or symbols of TFs are proportional to their expression levels or binding intensities, respectively.

labelling of RNA (Table 2), Harris found that the turnover the beta-globin LCR orchestrates temporal and spatial

rates of RNA in the nuclei of macrophages or fibroblasts expression of globin genes during development. It consists

were too high to account for the relatively steady level of of five erythroid-specific DNAse-I hypersensitivity sites

cytoplasmic RNA [16]. This study provided the first evi- (HS) (Table 2), and binding elements for erythroid LDTFs

dence that the majority of the nascent RNA produced in the such as GATA binding protein 1 (GATA1), suggesting

nucleus is rapidly turned over and does not contribute to enhancer-like properties of these regions. Importantly,

mRNAs. The discovery of pervasive enhancer transcription transcriptional initiation sites were found in several of

suggests that eRNAs, in addition to intronic RNA, are these DNAse-I hypersensitivity regions [18–20], and are

quantitatively important contributors to this rapidly de- distinct from alternative start sites of the globin gene itself

graded pool of nuclear RNA [17]. Reports of specific en- [20,21]. The description of enhancer RNA transcripts was

hancer-derived transcription were first documented in the subsequently extended to the LCR of MHC class II [22] and

locus control region (LCR) of the beta-globin gene clusters human growth hormone (hGH) [23] loci.

[18–20]. Located 10–15 kb upstream of the gene cluster, A demonstration of a broad pattern of transcription at

active enhancers was uncovered by deep sequencing

approaches, with total RNA sequencing (Table 2) revealing

Box 1. Conceptual distinctions between eRNAs, 1D-eRNAs, enhancer transcription in neurons and T cells [13,24].

2D-eRNAs, lncRNAs, and ncRNA-a Similarly, genome-wide detection of nascent RNA tran-

scripts using Global Run-On sequencing (GRO-seq; Table

eRNAs are noncoding RNA transcripts produced from genomic

1 3 2) demonstrated robust expression and regulation of eRNA

regions that are marked by high H3K4me and low H3K4me histone

modifications that are presumably enhancer DNA elements. eRNAs in macrophages, breast, or prostate cancer cells [25–28].

can be either polyadenylated or non-polyadenylated and appear as

RNA polymerase II (RNA PolII) complexes were noted to

unidirectional (1D-eRNA) or bidirectional transcripts (2D-eRNA) in

be enriched at enhancer elements [24] and to respond

RNA-seq or GRO-seq profiles [13,24–26,32,38]. lncRNAs are noncod-

dynamically to signal transduction events [13,29]. Thus,

ing RNA transcripts generally longer than 200 nucleotides (nt) [74].

ncRNA-a (noncoding RNAs activating) were transcripts annotated in enhancer transcription is a widespread phenomenon ob-

the GENCODE database with an average size of 800 nt, with served across multiple cell types in different species.

subsequent studies showing enhancer-like properties by activating

neighboring genes [43]. Although many ncRNA-a transcripts might be

Properties of eRNAs compared to mRNAs and other

more suitably classified as lncRNAs because they are often spliced

3 3 ncRNAs

and frequently bear H3K4me and H3K36me , histone marks for gene

promoters and transcription elongation, respectively [43], some RNA-seq, chromatin immunoprecipitation (ChIP)-seq, and

ncRNA-a transcripts are very similar to eRNAs, especially unidirec- chromatin-conformation-capture studies (Table 2) have

tional 1D-eRNAs. Functional distinctions between these groups are

collectively defined the following characteristics of eRNA

not fully clarified (see Figure 3 in main text for more information).

transcripts (Figure 3). (i) eRNAs are transcribed from

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Review Trends in Biochemical Sciences April 2014, Vol. 39, No. 4

(A) (1) Closed chroman Pioneer factors and LDTFs (3) Histone modificaons and recruitment of transcripon machinery

RNA Pol II

(2) Collaborave DNA binding and nucleosome remodeling

eRNA Key: Coacvator Pioneer/lineage-determining TFs Histone tail acetylaon complex Histone tail methylaon Signal-dependent TFs Looping complexes Coacvator complexes RNA polymerase II Histone methyltransferases (4) Signal transducon and eRNA elongaon

(B) Enhancers (C) Signal-induced enhancer formaon and eRNA elongaon

Cohesin pTEFb eRNA Elongaon-dependent histone methylaon RNA PoIII Mediator Looping MLL loading complex complex

TFIID Intervening DNA

Target gene mRNA promoters Chroman looping de novo enhancer formaon

TiBS

Figure 2. Proposed functional mechanisms of enhancer transcription and transcripts. (A) Collaborative binding of pioneer or lineage-determining transcription factors (TFs)

leads to nucleosome remodeling, increased chromatin accessibility, histone modifications (mono- and di-methylation on H3K4, blue circles on histone tails), and assembly

of basal transcription machinery. Upon stimulation, signal-dependent transcription factors (SDTFs) bind to recognition sequences at enhancers and recruit coactivator

complexes. This leads to further epigenetic modifications (e.g., histone acetylation) and transcriptional activation of the enhancer. (B–C) The enhancer transcription and

transcripts, under different circumstances, may have independent functional roles. (B) (A proposed model) Enhancer-derived RNA (eRNA) functionally contributes to

enhancer-mediated coding gene expression. Signal-dependent activation of enhancers leads to increased production of eRNAs, which interact with looping factors (e.g.,

cohesin complex) and facilitate/stabilize chromosomal looping between the enhancer and promoter(s) of cognate target gene(s) [28]. eRNA mediates the loading of RNA

PolII, and likely the transcription initiation complex (denoted by TFIID) at the promoter of the target gene [34]. Whether chromosomal looping facilitates RNA PolII loading is

2

unknown. (C) Transcription elongation is required for the deposition of di-methylation of histone lysine 4, or H3K4me , also a histone mark for an enhancer, during signal-

dependent activation of de novo enhancers. The coactivator complexes generate acetylation of histones, which recruits the positive transcription elongation factor (pTEFb,

orange) to promote elongation of RNA PolII. Subsequently, the elongating PolII recruits histone methyl transferases, mixed lineage leukemia complexes (MLLs), for di-

methylation deposition on H3K4 at enhancers [27]. The key in panel (A) also refers to panels (B) and (C).

putative enhancer regions characterized by high levels of phosphorylated RNA PolII, characteristics of coding gene

1 2 3

H3K4me and H3K4me relative to the H3K4me [30]. (ii) promoter regions. However, in contrast to gene bodies of

These genomic regions are bound by LDTFs and associated protein-coding genes, enhancers are less enriched for ser-

0

transcriptional co-regulators including subunits of Media- ine 2 phosphorylated RNA PolII [24]. (v) eRNAs exhibit a 5

tor, histone acetyltransferase p300 and cAMP response cap [17,31] but are generally not spliced or polyadenylated.

element-binding protein (CREB) binding protein (CBP). Polyadenylated eRNAs are generally unidirectionally

(iii) Expression of eRNAs is positively correlated with an transcribed from enhancers (1D-eRNA) [24]; however,

enrichment of histone marks of activated enhancer, par- enhancers with bidirectional transcription and non-poly-

ticularly H3K27ac, and the lack of the repressive adenylated transcripts (2D-eRNA) are more common

3

H3K27me mark [17,27]. (iv) eRNA-expressing enhancers [24,32]. (vi) eRNAs generally exhibit shorter half-lives

are also enriched with the transcriptional initiation com- compared to mRNAs and lncRNAs, but the frequency of

plex [e.g., TBP (TATA-binding protein), TFIIs] and serine 5 transcription initiation of eRNAs appears comparable to

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Table 1. Timeline of enhancer and eRNA discovery and functional characterization

Year, Refs Brief description of discovery Significance

1958, Pardee et al. [75] Using genetics models of Escherichia coli, the PaJaMa This landmark study laid the groundwork for the concept

studies proposed a general mechanism for transcriptional of regulatory elements and the heritability of gene

regulation via a repressor system to control protein regulation.

production of beta-galactosidase. This led to the finding of

lac operator, or ‘lac operon’ regulatory element [76].

1959, Harris [16] Using pulse-chase of RNA with labelled nucleotide, a large Harris deduced that most nuclear RNAs are likely to be

fraction of nascent RNA was shown to retain in the non-protein-coding. In a recent Nature correspondence,

nucleus. In addition, the half-lives of nuclear RNA were Harris connected his 1959 finding with the recent studies

significantly shorter compared to cytoplasmic RNA. of ncRNA [88]. Given the high number of transcribing

enhancer units, the comparable frequency of

transcription initiation at enhancers, and short eRNA half-

lives, this 1959 study is consistent with the observation of

pervasive transcription at enhancer elements.

1981, Banerji et al. [1] Banerji et al. showed that a 72-repeat sequence motif from Discovery of cis-acting enhancer elements.

a Simian virus 40 (SV40) early gene is sufficient to increase

expression of ectopic beta-globin gene by 200 fold. This

DNA element is functional over long distance in an

orientation-independent fashion relative to the beta-

globin gene.

1990, Collis et al. [18] Using nuclear run-off assays, Collis et al. identified One of the first studies demonstrating transcription from

transcription at the LCR of the beta-globin region, both in enhancer regions.

the endogenous locus in MEL (mouse erythroleukaemia)

cells, as well as a mini-gene construct containing the LCR

and beta-globin gene.

1992, Tuan et al. [19] Using RNA protection assays in transfected recombinant This study suggested that enhancer ncRNA is only

plasmids, the authors identified that a noncoding RNA is generated from active enhancer, and enhancer-derived

transcribed from the HS2 enhancer. Only the active transcription is needed for proper enhancer function.

enhancer can direct synthesis of enhancer-derived RNA;

the same group later showed that disruption of HS2-

derived RNA by insertion of a transcription terminator

reduced HS2 enhancer activity and expression of the

target gene [39].

1997, Ashe et al. [20] Using nuclear run-on assays and in situ hybridization The intergenic ncRNA transcript displayed cell line

analysis, the authors found a novel intergenic specificity.

transcription from the human beta-globin locus in K562 A ‘trans’ effect is implicated given that exogenous

but not in HeLa cells. Exogenous expression of the beta- plasmid-driven transcription of the globin genes can

globin gene in HeLa cells was sufficient to induce the induce intergenic ncRNA transcription at the endogenous

expression of intergenic ncRNAs from the otherwise silent locus. This mechanism, however, is not well understood.

endogenous chromatin.

2000, Gribnau et al. [77] RNA fluorescence in situ hybridization (FISH) and DNase-I Intergenic ncRNA transcription could play a role in

sensitivity assays led to the finding that transcription maintaining open and active chromatin.

activity of the human beta-globin locus correlates to its

sensitivity to DNase-I.

2006, Feng et al. [46] The ultraconserved region between DLX5/6 was identified Because ultraconserved regions are frequently enhancers

to transcribe into Evf-2 ncRNA. This Evf-2 then interacts [89], this study implicated that enhancer-derived RNAs

with DLX2 in vivo to activate the transcription of DLX5 and can be functionally regulatory.

DLX6 genes.

‘Genomic’ era

2010, Kim et al. [13]; Enhancer transcription and transcripts are genome-wide Pervasive genome-wide enhancer transcription. The

2010, De Santa et al. [29]; phenomena. Enhancer-templated noncoding RNAs cognate promoter near the enhancer may be required for

2011, Koch et al. [24] (eRNAs) were usually non-polyadenylated and of lower proper eRNA transcription.

abundance compared to coding genes. Expression levels

of eRNAs positively correlated to expression of nearby

protein-coding genes.

2011, Wang et al. [26]; Using GRO-seq data, eRNA transcription was found to be eRNA transcription serves as a marker of active

2011, Hah et al. [25]; 2011, induced by stimuli and widely distributed in the genome; enhancers.

Melgar et al. [78]; 2013, their transcription correlated well with the activity of active Pharmacological inhibition of eRNA transcription does

Hah et al. [38] enhancers. not inhibit enhancer–promoter looping with 3C.

2010, Orom et al. [43]; From transcripts annotated in GENCODE, the authors This is the first description of lncRNAs having enhancer-

2013, Lai et al. [49] identified a cohort of lncRNAs that exhibit enhancer-like like properties. Later, the same group illustrated that

properties. Unlike eRNAs, this group of ncRNAs, termed ncRNA-a mediated gene regulation via modulation of the

enhancer-like lncRNAs or later as ncRNA-activating Mediator complex and chromatin looping [49]. Recent

(ncRNA-a) are spliced, polyadenylated transcripts studies of other lncRNAs demonstrate novel mechanisms

3

expressed from promoter-like regions (i.e., high H3K4me , of gene activation, including physical interaction of

1

low H3K4me ). They activate gene(s) in their vicinity. lncRNA HOTTIP with methyltransferase to drive H3K4

trimethylation, and gene expression in the HoxA cluster

[35].

2013, Melo et al. [33]; 2013, Using siRNA, antisense oligonucleotides and reporter The eRNA transcript per se plays a functional role in

Lam et al. [31]; 2013, assays, with characterization using qRT-PCR, GRO-seq, regulating gene transcription. A stimulus-induced eRNA

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Table 1 (Continued )

Year, Refs Brief description of discovery Significance

Li et al. [28]; 2013, and 3D-DSL, these studies found that eRNA transcripts transcript is needed for proper enhancer–promoter

Mousavi et al. [34] have functional roles in regulating the transcription of looping formation and gene activation.

neighboring coding genes.

2013, Kaikkonen et al. [27] Using genomic tools, the authors found that RNA PolII elongation has a role independent of the eRNA

pharmacological inhibition of eRNA transcriptional transcript in modulating chromatin structure and

elongation impaired mono- and di-methylation of histone deposition of enhancer histone marks.

H3K4 on signal-induced de novo enhancers.

that of protein-coding genes [31]. (vii) eRNAs are dynami- the authors found RNA PolII at the gene promoter. Sur-

cally regulated upon signal transduction events orches- prisingly, RNA PolII was also found at the HS2 enhancer,

trated by signal-dependent TFs such as nuclear factor consistent with the production of enhancer-derived RNA

kappa-light-chain-enhancer of activated B cells (NFkB), transcript therein. To study the role of RNA transcription

p53, or nuclear receptors [13,25–29,31,33,34]. (viii) Of in RNA PolII recruitment, cells were treated with RNA

particular interest, signal-dependent changes in eRNA PolII elongation inhibitor 5,6-dichlorobenzimidazole

expression are highly correlated with corresponding sig- (DRB). This resulted in decreased recruitment of RNA

nal-dependent transcriptional changes in promoters of PolII to the beta-globin promoter, but not at the HS2

nearby genes [13,27,28,31]. (ix) In addition, enhancer tran- enhancer [40]. This implies that enhancer recruitment of

scripts are preferentially enriched at enhancers engaged in RNA PolII preceded RNA PolII loading at target gene

chromatin looping with promoters of protein-coding genes promoter. This also raised the possibilities that enhancer

and other enhancers, which is a feature correlated with transcription is functionally significant for regulating RNA

enhancer activity [36,37]. Collectively, eRNA transcription PolII ‘loading’ to the target gene promoter. This experi-

is highly correlated to other parameters of active enhancer ment, however, could not differentiate whether the RNA

elements [26,27,38]. PolII loading was mediated by the act of enhancer tran-

scription (i.e., RNA PolII elongation) or by the eRNA

Functional roles of enhancer transcription and transcript itself, because both processes were inhibited

transcripts by DRB.

Whether an enhancer transcript is merely a correlation or Recently, several reports have taken new approaches to

a functional component of enhancer activity generated an test functions of enhancer transcripts. Targeted degrada-

active area of research. Three possibilities have been tion of eRNA using either RNA interference (small inter-

considered with respect to the physiological roles of en- fering RNA, siRNA) or DNA–RNA hybrid-induced

hancer transcription. The first possibility considers degradation via RNase-H (i.e., antisense oligonucleotide

enhancer transcription as ‘noise’ from the spurious en- or locked nucleic acids) proved sufficient to reduce expres-

gagement of RNA PolII complexes to the open chromatin sion of nearby protein-coding genes [28,31,33,34]. To fur-

environment of enhancers. The second possibility ther discriminate the effects of RNA PolII transcription at

hypothesizes that it is the process of transcription, not the enhancer versus the eRNA transcript itself, Li et al.

the features of the eRNA transcript itself, that is necessary and Melo et al. used ‘tethering’ strategies [28,33], where

for the activating functions of enhancers. The third possi- eRNA transcripts were fused with RNA tags (i.e., small

bility is that the RNA transcripts per se functionally RNA hairpins MS2 or BoxB) to generate chimera RNAs

contribute to enhancer activity [32]. These possibilities that can be bound by a recombinant adaptor protein that

are not mutually exclusive. could simultaneously bind to specific sequences on a re-

The early investigations of enhancer transcripts from porter plasmid. This strategy enables experimental teth-

the beta-globin LCR implicated functional significance of ering of eRNA to either promoter [33] or enhancer [28] of a

enhancer transcription. HS2, a hypersensitivity site within reporter plasmid, which was sufficient to increase tran-

the beta-globin LCR, was sufficient for erythroid-specific scriptional activity of the reporter gene. An alternative

enhancer activity when cloned into minigene constructs experimental design also supported the function of eRNA

[19]. The transcription start site for an enhancer transcript to enhancer activity. By cloning different sizes of genomic

was found within HS2 of both the endogenous genomic fragments from an endogenous enhancer locus, Lam et al.

locus and plasmid constructs [18,19]. Interestingly, termi- showed that whereas the ‘core’ enhancer fragments con-

nation of HS2-mediated transcription by inserting a lac taining LDTF binding sites were sufficient for enhancer

operator/R repressor complex downstream of the enhancer activity, the enhancer construct containing eRNA-coding

led to decreased promoter activity in a reporter construct sequence had higher transcriptional activity. Importantly,

[39]. This suggested that the transcription from HS2 is the ‘added’ effect from the eRNA was abolished when the

important for its neighboring promoter activity. A similar orientation of its coding sequence was reversed relative to

result was observed in the hGH locus when a transcription the ‘core’ enhancer [31]. Because this inversion completely

termination sequence TerF was inserted between the LCR changes the sequence of the eRNA product but retains any

and the promoter of hGH. Transgenic mice with this TerF putative TF binding sites, these results implied that the

insertion showed decreased expression of hGH [23]. In sequence of the eRNA is important for its function. Collec-

another line of investigation, analysis of RNA PolII locali- tively, these reports suggest that, at least for some enhan-

zation was performed in the beta-globin locus. Expectedly, cers, sequence-specific eRNA transcripts can contribute to

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Table 2. Brief descriptions of methods to characterize RNAs, TF binding, and chromatin states

Method Description

Nascent RNA analysis Pulse-chase A method to examine properties of cellular processes over time.

The cells are first shortly exposed to a labeled compound (pulse, e.g., radioactivity),

and then to the same compound in an unlabeled form (chase). The amount of the

labeled compound will change over time to reflect the effects from the interrogated

cellular processes. In the context of this review, Harris measured the half-lives of

nuclear and cytoplasmic RNA [16].

Global Run-On sequencing A method to identify and quantify the genome-wide transcription of nascent RNA.

(GRO-seq) [79] At a given time in cells, nascent RNAs are synthesized from actively engaged RNA

polymerases. GRO-seq directly measures the frequency of transcription initiation and

transcriptional rate and is largely independent of the effect of RNA stability.

Precision Global Run-On This method is an adapted version of GRO-seq.

sequencing (PRO-seq) [56] Biotinylated nucleotides are used during in vitro transcription, enabling the mapping

of the positions of the engaged RNA polymerases more precisely by sequencing.

0

RNA 5 cap site analysis; These methods introduced modifications based on GRO-seq and PRO-seq protocols.

0

5 GRO-seq [31]; PRO-cap [56] They allow mapping of the transcription initiation site and assessment of the

0

transcription frequency by measuring 5 capped nascent RNAs at a given time in cells.

RNA sequencing Total RNA-seq This method measures the overall amounts of cellular RNA species by high-

throughput sequencing.

Different categories of RNAs are represented proportionally to their expression levels

in the final sequencing results. Because cellular RNA consists of a large amount of

ribosomal RNA, rRNA removal steps are often incorporated to increase signals for

mRNAs and other ncRNAs of interest. RNA-seq can also provide other information

about splicing, exon inclusion, and exon exclusion, etc.

Poly-A RNA-seq A modified version of total RNA-seq.

This is based on using oligo-dT primer during cDNA generation and library

preparation to enrich RNA species with poly A tails (e.g., most mRNAs and many

lncRNAs).

Chromosomal DNAse-I hypersensitivity A method to assess open chromatin status.

accessibility assay [80] This method measures the susceptibility to DNase-I cleavage locally (e.g., beta-globin

locus control region) or globally when coupled with high-throughput sequencing.

Genomic localization Chromatin A method to study genomic localization of TFs, co-regulatory complexes, post-

immunoprecipitation translational modifications, and variants of histone marks by sequencing DNA

coupled with high- fragments associated with these factors.

throughput sequencing The utility of this method depends on the availability of high-quality antibodies for the

(ChIP-seq) [81,82] proteins of interest. Alternatively, a high affinity tag (i.e., a flag tag) could be fused to

the protein of interest to facilitate affinity purification of the protein–chromatin

complexes.

Chromosomal Chromosome conformation A method to analyze chromosomal interactions to understand the spatial

interaction capture (3C) [83] organization of the chromosomes.

Because interacting genomic regions are in close 3D proximity, the likelihood of DNA

ligation between two interacting regions after chemical fixation and fragmentation

(e.g., restriction enzyme digestion) will be significantly higher than that between any

non-interacting regions of similar linear distance. Interactions could be quantified

using PCR or qPCR. Control experiments are crucial to minimize misinterpretation due

to random ligations.

Chromosome interaction A method to identify a subgroup of chromosomal interactions mediated by a specific

analysis with pair end Taq TF of interest.

sequencing (ChIA-PET) [41] Analogous to 3C, ChIA-PET detects regions of chromosomal interactions that are

bound by a factor of interest (e.g., RNA PolII) by ChIP followed by 3C. The selected

interactions are processed for high-throughput sequencing, which enables an

unbiased, genome-wide approach for detecting long-range chromosomal

interactions mediated by this factor.

3D DNA selection and A method to identify chromosomal interactions at pre-selected genomic regions of

ligation (3D-DSL) [5] interest.

On the basis of 3C, the digested and ligated chromatins are hybridized with a pool of

oligonucleotides pre-designed to detect chromatin interactions at selected genomic

regions. The limitation of resolution is dependent on the distribution of the restriction

sites used for 3C and positions of the pre-designed oligonucleotides. Other methods

for chromatin interactions include 4C, 5C, and Hi-C, but are not discussed here

Fluorescence in situ A cytogenetic technique to detect and localize specific DNA sequences on

hybridization (FISH) [84] chromosomes.

A fluorescent probe (e.g., oligonucleotides or long stretches of DNAs) was pre-

designed to specifically hybridize to DNA/chromatin regions of interest based on

sequence complementarity. Using multiple probes conjugated to multicolor

fluorophores, interactions of two or more genomic regions could be approximate by

tabulating the frequency of these probes being in close physical proximity by

fluorescence microscopy. The resolution of this method is limited by the wavelength

5 6

of the lower light spectrum (200–250 nm), which could be in the range of 10 to 10

base pairs depending on the chromatin state.

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Table 2 (Continued )

Method Description

RNA–chromatin interaction Chromatin isolation by RNA A method to identify chromatin sites bound by an RNA of interest.

purification coupled with The target RNA of interest is hybridized and retrieved by biotin-tagged

sequencing (ChIRP-seq) [85] oligonucleotides by sequence complementarity. The RNA-interacting chromatin

regions can be recovered by streptavidin affinity purification and determined by high-

throughput sequencing. ChIRP-seq usually uses a tiling array of oligonucleotides to

enrich one RNA target and requires glutaraldehyde to irreversibly crosslink RNA

targets and chromatin.

Capture hybridization Another method to capture the chromatin binding sites of an RNA of interest.

analysis of RNA targets Similar to ChIRP, CHART uses a similar hybridization-based strategy to enrich the RNA

coupled with sequencing target and its associated chromatin DNA and proteins, but uses only one cDNA

(CHART-seq) [86] oligonucleotide, which is pre-designed based on analysis of the target RNA structure.

Unlike ChIRP-seq, CHART allows the use of a mild and reversible crosslinking method.

Because RNA can interact with chromatin by direct DNA binding or through an indirect

protein intermediate, the sensitivity of CHART-seq or ChIRP-seq to these different

interactions needs to be assessed. Importantly, whether the different modalities in

RNA:chromatin interactions correlate with biological significance is not known.

enhancer-mediated transcriptional activation of neighbor- RNA PolII elongation), or it may reflect different mecha-

ing coding genes. nisms at specific gene loci or enhancers. In another recent

study, Mousavi et al. knocked down eRNA transcripts

Mechanisms of eRNA actions produced in each MyoD-bound enhancer across the MyoD

A question immediately emerges as to how eRNAs might locus, showing that only the eRNA from the core enhancer

contribute to enhancer function. Chromatin interaction (CE) was critical for MyoD expression [34]. Furthermore,

CE

studies demonstrated that enhancers engaged in looping knockdown of eRNA decreased RNA PolII recruitment at

with promoters of protein-coding genes possess higher the promoter and gene body of MyoD, but not at the core

expression of eRNAs [36,37]. These studies suggested a enhancer itself. The consequences of eRNA knockdown at

potential role of eRNAs in the process of proper formation the MyoD locus, as well as RNA synthesis inhibition at HS2

of chromosomal looping between enhancers and transcrip- of the beta-globin LCR mentioned earlier [40], suggest that

tion start sites (TSSs). Indeed, in nuclear receptor-regu- eRNA transcripts facilitate RNA PolII recruitment to the

lated gene activation events, ligand treatment induces promoter of the target gene (Figure 2B). It seems that

formation of chromatin looping between the enhancer targeted downregulation of eRNA by knockdown or inhibi-

and the cognate TSS, which could be measured by chromo- tion of RNA PolII elongation did not affect the pre-estab-

some interaction analysis with pair end Taq sequencing lished ‘enhancer assembly’, in terms of binding of TFs,

ChIA-PET [41] and 3D DNA selection and ligation (3D- recruitment of RNA PolII or enrichment in histone marks

1

DSL) (Table 2) [28]. More importantly, knockdown of eRNA (e.g., H3K4me ), suggesting that production of eRNA prob-

from the estrogen receptor alpha (ERa)-bound enhancers ably represents one of the final steps during activation of

at NR1P1 or GREB1 loci reduced enhancer–promoter pre-established enhancers (Figure 2A).

interaction and concomitantly decreased coding gene acti- However, inhibition of enhancer transcription (e.g., by

vation [28]. The potential role of estrogen-induced eRNA in pharmacological inhibitor of RNA PolII elongation)

the modulation of chromosomal looping was suggested by impairs signal-induced de novo activation of enhancers,

the observation that eRNAs could interact with the SMC3 indicated by decreased acetylation of H3K9 [29] or attenu-

(structural maintenance of chromosomes 3) and RAD21 ated mono- and di-methylation of H3K4 [27]. In the latter

(double-strand-break repair protein rad21 homolog) sub- case, deposition of H3K4 mono- and di-methylation to

units of the cohesin complex, which has been shown to enhancers is dependent on histone methyltransferases

control enhancer–promoter looping in stem cells [42]. Tar- [i.e., mixed lineage leukemia-1 (MLL1), MLL2/4, and

geted degradation of eRNAs attenuated estrogen-induced MLL3] coupled to RNA PolII elongation, but independent

cohesin increment to several ERa-bound enhancers, and of the eRNA transcript [27], suggesting a key functional

knockdown of cohesin almost completely abolished both role of the process of enhancer transcription, at least for

the observed induced looping and gene activation events some enhancer cohorts (Figure 2C). Collectively, these

[28] (Figure 2B). These data suggested that eRNA may findings suggest that both enhancer transcription and

play a role in the initiation or stabilization of enhancer– the resultant enhancer RNAs can contribute to enhancer

promoter looping, but its quantitative effect on cohesin function.

recruitment to the enhancers remains unclear. However,

Hah et al. found that reducing the amount of eRNA and Mechanism of other ncRNAs that positively regulate

coding gene transcripts through chemical inhibition of gene expression

RNA PolII elongation (i.e., flavopiridol) had no significant In addition to eRNAs, a plethora of lncRNAs are already

effect on estrodiol-induced enhancer–promoter looping at reported to play activating roles in gene transcription

the P2RY2 or the GREB1 loci, as measured by chromosom- regulation [35,43]. These lncRNAs are generally tran-

3

al conformation capture (3C) (Table 2) [38]. This difference scribed from regions exhibiting high H3K4me at the

3

may be explained by the different experimental techniques promoter and H3K36me at gene body (a histone mark

used (eRNA knockdown vs pharmacological inhibition of of elongation), resembling the chromatin properties of

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Protein-coding geneLong noncoding Unidireconal Bidireconal RNA enhancer RNA enhancer RNA 3 H3K4me 1 H3K4me

Histone modificaons ? H3K27ac

LDTF1 LDTF2 LDTF3 LDTFs factors Transcripon p300/CBP Tol-Pol II

RNA Pol II ? PoI lI-Ser2P

+strand ? (GRO-Seq)

Nascent RNA –strand +strand RNA

Gene

body

–strand PolyA RNA

– TSS + – TSS + – TSS + – TSS +

CpG 70% ∼20% ? <5% splicing site Yes Yes ?No

TiBS

Figure 3. Molecular characteristics of protein-coding RNAs, long noncoding RNAs (lncRNAs), and enhancer RNAs. Schematic diagrams of chromatin immunoprecipitation

coupled with high-throughput sequencing (ChIP-seq) or RNA-seq analysis centered on the transcription start sites (TSS) to depict the differences and similarities between

3 1

these four categories of transcription units. Briefly, protein-coding genes have a higher level of H3K4me , lower level of H3K4me , relatively lower enrichment of lineage-

determining transcription factors (LDTFs), and higher CpG islands at TSSs; transcription orientation is predominantly unidirectional, transcripts are polyadenylated, and

3 hi low

gene bodies are demarcated with the elongating histone mark H3K36me . Transcripts of lncRNAs resemble protein-coding genes: H3K4me3 /H3K4me1 promoters with

1

polyadenylated and predominately unidirectional transcripts [43]. Compared to protein-coding genes, lncRNAs possess higher H3K4me , higher LDTF enrichment, and

high low

lower CpG density [24]. For enhancer-derived noncoding RNAs (eRNAs), TSSs are demarcated with H3K4me1 /H3K4me3 modifications and low CpG density [24].

eRNAs, in a majority, are non-polyadenylated bidirectional transcripts (2D-eRNAs), but also comprise a less common group of unidirectional transcripts (1D-eRNAs). Both

1D- and 2D-eRNAs often bear shorter half-lives (low signal from total RNA sequencing), but comparable initiation rates of transcription compared to protein-coding genes

and lncRNAs, as measured by total RNA polymerase II (RNA PolII) enrichment or nascent RNA transcripts (GRO-seq). The elongating form of RNA PolII with serine 2

phosphorylation (PolII-Ser2p) mainly enriches at protein-coding genes. Chromosomal interaction studies indicated higher frequency of looping events, either enhancer–

promoter, enhancer–enhancer, or promoter–promoter, at actively transcribed enhancers and protein-coding genes [36,87].

protein-coding genes [43]. They are also often spliced, specific [17]. Given this overlap in genomic characteristics

polyadenylated, and unidirectionally transcribed. Accord- of these two species of noncoding nuclear RNAs, there may

ing to these criteria, most eRNAs and activating lncRNAs also be overlap in mechanisms by which they influence

are very distinct (Box 1). However, as noted above, some gene expression.

transcripts derived from enhancer-like regions of the ge- Although each lncRNA activator studied to date appears

nome are polyadenylated and transcribed unidirectionally to function through distinct mechanisms, three general

[24,38], and both eRNAs and lncRNAs tend to be cell-type themes have emerged: (i) lncRNAs recruit activating

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(A) Acvang ncRNAs AAAAAAAA RNA PolII GTF Promoter ncRNAs

Intervening DNA (kbps– AAAAAAAA Mbps) Acvator RNA PolII GTF complex Promoter Target genes Model 1: ncRNAs collaborate with transcriponal acvators

(B) RNA PolII Promoter ncRNAa GTF AAAA ncRNAa Intervening DNA (kbps– Mbps) AAAA GTF RNA PolII Promoter Targen genes

Model 2: ncRNAs modulate chroman loops

(C) Repressors RNA PolII

Promoter Target genes

Repressors

RNA PolII ncRNA Promoter Target genes

Model 3: ncRNAs evict transcriponal repressors

Key: Histone tail methylaon Signal dependent TFs Looping complexes

Coacvator complexes GTF General TFs RNA polymerase II

Transcriponal repressors

TiBS

Figure 4. Three mechanistic models underlying functions of activating noncoding RNAs (ncRNAs): (A) by directly recruiting a transcriptional activator or activator complex;

(B) via mediating chromatin looping; and (C) through evicting transcriptional repressors. (A) Several ncRNAs {e.g., HOTTIP (HOXA transcript at the distal tip) [35], Mira [47],

steroid receptor RNA activator (SRA) [44], and Evf-2 [46]} physically interact with and recruit transcriptional activators [e.g., the WD repeat-containing protein 5 (WDR5)

subunit of the mixed lineage leukemia-1 (MLL) complex] to promote target gene activation. This type of activation is achieved, so far, only by polyadenylated ncRNAs, and

can function either in cis (e.g., HOTTIP) or in trans (e.g., SRA). (B) Another group of ncRNAs (e.g., noncoding RNAs activating, ncRNA-a) can modulate chromatin looping

between enhancers and promoters through interacting with and recruiting or stabilizing the looping factors (e.g., Mediator and cohesin complexes). This is similar to the

mechanism of some estrogen-induced enhancer-derived RNAs (eRNAs; Figure 2B) for gene activation. (C) Under some circumstances, some ncRNAs (Braveheart [53], Jpx

[51,52]) can abolish the effects of transcriptional repressors by titrating them away from the promoters of target genes.

proteins and protein complexes; (ii) they mediate chromatin as a transcriptional coactivator; it functions in the tran-

interactions; and (iii) they have roles in eviction of repressive scriptional activator complex steroid receptor coactivator-1

machineries (Figure 4). (SRC1) to enhance transcription mediated by steroid hor-

The first category constitutes the most common mecha- mone receptors [44]. Further, Evf-2, a ncRNA generated

nism of activating ncRNAs (Figure 4A). Steroid receptor from an ultraconserved intergenic region, was discovered

RNA activator (SRA) was the first ncRNA discovered to act to recruit TF distal-less homeobox 2 (DLX2) and methyl

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Review Trends in Biochemical Sciences April 2014, Vol. 39, No. 4

CpG binding protein 2 (MECP2) to regulate expression of A corollary issue is whether eRNAs play general roles in

the neighboring Dlx5/6 genes [45,46]. Another recent enhancer function. For those enhancers in which it exerts a

example is HOTTIP (HOXA transcript at the distal tip), functional role, we also need a deeper understanding of

which drives the transcriptional activation of the homeo- their mechanism of action. In view of the multiple mecha-

box A (HOXA) gene cluster by directly interacting with the nisms of action described thus far for lncRNAs, similar

WD repeat-containing protein 5 (WDR5) subunit of the complexity may be expected for the action mechanisms of

MLL methyltransferase complex, leading to deposition of eRNAs. Given the initial evidence for roles of eRNAs in

3

active promoter histone mark, H3K4me , at target genes enhancer/promoter interactions, it will be of particular

[35]. Consistent with this, the MLL complex can also be interest to systematically examine the consequences of

recruited by Mistral (Mira) ncRNA to activate homeobox A 6 loss of eRNA function on short- and long-range interactions

(HOXa6) and HOXa7 during stem cell differentiation [47] of enhancers with coding gene promoters and other enhan-

and by NeST (Nettoie Salmonella pas Theiler’s) ncRNA to cers [57].

activate the interferon-gamma locus [48]. Although the evidence suggests that eRNA transcripts

The second category of activating ncRNAs is exemplified per se and eRNA transcription can both play certain func-

by ncRNA-a (noncoding RNAs activating; Figure 4B) [43]. tional roles, it is currently not clear whether these func-

This group of ncRNAs regulates gene activation through a tions are primarily performed in trans or in cis. Current

distance by recruiting the Mediator complex to, and con- findings regarding eRNAs – their low copy numbers per

trolling the phosphorylation of histone H3S10 at its target cell [13,25], the predominant absence of eRNA localization

gene promoter [49]. Intriguingly, ncRNAs might, in specific at other genomic regions, and the minimal effect of eRNA

circumstances, indirectly recruit complexes that exert knockdown on expression of genes on different chromo-

functions in enhancer–promoter looping [50]. somes [28] – are most consistent with function in cis

Finally, the third type of activating mechanism can be (Figure 5A). Similar conclusions were made for activating

summarized as an ‘eviction’ model (Figure 4C), which is lncRNA [35,58]. Although depletions of lncRNAs were

exemplified by X chromosome inactivation (XCI). The sufficient to decrease expression of their target genes,

Xist gene initiates XCI, and is only expressed when more ectopic expressions of lncRNAs had minimal effect on

than one X chromosome is present. At the pre-XCI stage, target gene expression at the endogenous loci [35,58]. This

Xist is silenced by the binding of CCCTC-binding factor points to activity in cis, where lncRNAs are likely to exert

(CTCF) at its promoter. At the onset of XCI, a ncRNA function within a domain consisting of interactions be-

transcript Jpx is upregulated from both X chromosomes, tween neighboring and distal genomic regions (i.e., intra-

which directly binds to CTCF and titrates it away from chromasomal interactions) refined within a close 3D space.

the Xist promoter. This allows expression of Xist to Whether lncRNA and eRNA mediate expression of other

initiate XCI on one of the X chromosomes [51,52]. Simi- genes in trans within the ‘interacting domain’ has not been

larly, activation of lineage-specific genes during cardio- systemically addressed (Figure 5D). Several observations

vascular development requires the removal of polycomb suggest this possibility. ncRNA-a depletion resulted in a

3

repressive complexes (PRC2) and H3K27me marks from greater number of changes in gene expression than would

those gene promoters. This process depends on the be accounted for by its target protein-coding gene alone

ncRNA Braveheart, which binds to the suppressor of [43]. This suggests trans activity, although the results

Zeste 12 (SUZ12) subunit of PRC2 and titrates the whole could be confounded by secondary effects (i.e., changes in

PRC2 complex away [53]. other unforeseen cis-regulated direct target genes)

Together these data suggest a complex network of in- (Figure 5C). The observations that eRNA contributes to

teraction between ncRNA and regulatory protein machin- chromatin looping [28] and RNA PolII loading at target

eries to precisely control gene transcription programs. gene promoters [34] raised an intriguing possibility of

Although the list of proteins that interact with ncRNAs trans function within the ‘interacting domain’ that has

is still limited (e.g., PRC1/2 and MLL), significant growth not been ruled out by current studies.

of this list is very likely in the near future because many Elucidating the cis versus trans mechanism of eRNA

transcriptional regulators may contain RNA binding and lncRNA helps tease out allelic specificity in gene

domains [54]. regulation, which is an important component of under-

standing genetic diseases and therapeutic applicability.

Concluding remarks Further understanding of the 3D organization of the ge-

A detailed understanding of the molecular mechanisms by nome and genomic localization of eRNA or lncRNA by

which enhancers become activated as transcription units identifying RNA–chromatin association will be informa-

remains an important question. For example, what is the tive (Table 2). Given the rapid technological advances in

mechanism underlying biogenesis and regulation of genome editing [59,60], it is now feasible to investigate the

eRNAs? Is the transcription machinery on enhancers iden- in cis and in trans mechanism by examining allelic speci-

tical to that on promoters? The precise initiation site of ficity with carefully designed genetic experiments or ge-

enhancer transcription, and the mechanisms that deter- nome editing experiments coupled with sequencing

mine elongation and termination, can be approached with modalities.

0

contemporary technologies. For example, 5 RNA cap site eRNAs that contribute to enhancer activity presumably

analysis by GRO-seq and PRO-seq [31,55,56] could help do so by interacting with other effector molecules. One

delineate distinctive features of the TSSs of protein-coding approach to investigating the function of a particular

genes, ncRNAs, 1D-eRNAs, and 2D-eRNAs. eRNA will be to identify the factors it interacts with

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(A) (B) ncRNA ncRNA Translocate

RNA PolII RNA PolII

ncRNA

ncRNA Target gene Target gene/genes Classic cis Classic trans

(C) Assumed as trans targets??

ncRNA

RNA PolII

ncRNA Target gene/genes

Intermediate cis target Indirect effects

(D)

RNA PolII Chromosome B

ncRNA Target gene Intervening DNA in cis (kbps–Mbps) looping

Target gene in trans Chroman Interacng Chromosome A Domain

TiBS

Figure 5. In trans versus in cis action of noncoding RNAs (ncRNAs) in gene transcriptional regulation. (A,B) Classic in cis (A) or in trans (B) actions of regulatory ncRNAs in

transcriptional activation. (C) A scenario in which an ncRNA is assumed to regulate a target gene in trans, but the trans effect of the ncRNA is actually mediated indirectly

through its primary effect on a cis-target gene. (D) The postulated dual in cis and in trans role of ncRNA within the domain of interacting chromatin. ncRNAs [e.g.,

noncoding RNAs activating (ncRNA-a) and enhancer-derived RNA (eRNAs)] have been shown to regulate chromatin looping formation, which raised the possibility that

ncRNAs can stay where they are produced (in cis) but exert long-distance regulations on a target gene (i.e., interchromosomal interactions), mimicking a trans effect.

biochemically. At a more general level, efforts have been targets of therapeutic small molecules that modulate spe-

made to delineate the dependence of activating ncRNAs on cific classes of epigenetic regulators. The further explora-

substructure to interact with cofactors. For instance, the tion of enhancer transcription and functional roles of

RNA motifs of the RNA coactivator SRA are critical for its eRNAs will therefore not only be of central importance

coactivation function [61]. Case studies of other RNA for the further understanding of regulation of gene expres-

species indicate that simple secondary structures (e.g., sion in homeostasis and development, but also for advanc-

tandem stem loops) enable protein–RNA complex forma- ing novel therapeutic interventions for human diseases.

tion [62,63]. Additionally, repeat elements or GC-rich

sequences in ncRNAs have been implicated in gene regu- Acknowledgements

lation in trans through RNA–DNA–DNA triplex formation These studies were supported in part by National Institutes of Health

(NIH) Grant CA173903, Department of Defense breast cancer fellowship

with similar elements at promoters of target genes [63–65].

(BC110381 to W.L.), and National Institute of General Medical Science T32

As progress is made in understanding protein–RNA bind-

GM007198-37 and T32 GM008666 (M.T.Y.L.). We apologize to our many

ing specificity and RNA secondary structures [54,66], a

colleagues for not being able to cite all relevant papers due to space

major challenge will be to elucidate hidden sequence or limitations.

structural codes underlying the behaviors of different

types of ncRNAs in interacting with their protein partners. References

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