Transcription regulation by long non-coding RNAs: mechanisms and disease relevance

Jorge Ferrer; Nadya Dimitrova

doi:10.1038/s41580-023-00694-9

. Author manuscript; available in PMC: 2024 May 1.

Published in final edited form as: Nat Rev Mol Cell Biol. 2024 Jan 19;25(5):396–415. doi: 10.1038/s41580-023-00694-9

Transcription regulation by long non-coding RNAs: mechanisms and disease relevance

Jorge Ferrer ^1,^2,^3,^✉, Nadya Dimitrova ^4,^✉

PMCID: PMC11045326 NIHMSID: NIHMS1982683 PMID: 38242953

Abstract

Long non-coding RNAs (lncRNAs) outnumber protein-coding transcripts, but their functions remain largely unknown. In this Review, we discuss the emerging roles of lncRNAs in the control of gene transcription. Some of the best characterized lncRNAs have essential transcription cis-regulatory functions that cannot be easily accomplished by DNA-interacting transcription factors, such as XIST, which controls X-chromosome inactivation, or imprinted lncRNAs that direct allele-specific repression. A growing number of lncRNA transcription units, including CHASERR, PVT1 and HASTER (also known as HNF1A-AS1) act as transcription-stabilizing elements that fine-tune the activity of dosage-sensitive genes that encode transcription factors. Genetic experiments have shown that defects in such transcription stabilizers often cause severe phenotypes. Other lncRNAs, such as lincRNA-p21 (also known as Trp53cor1) and Maenli (Gm29348) contribute to local activation of gene transcription, whereas distinct lncRNAs influence gene transcription in trans. We discuss findings of lncRNAs that elicit a function through either activation of their transcription, transcript elongation and processing or the lncRNA molecule itself. We also discuss emerging evidence of lncRNA involvement in human diseases, and their potential as therapeutic targets.

Introduction

In the mid-1980s, transcripts that lacked obvious open reading frames were identified in the Drosophila melanogaster bithorax locus¹. At the time, the authors of the study hypothesized that these transcripts might be by-products of enhancer activity, that their transcription could regulate adjacent genes, that they might regulate splicing or translation in trans, or that they might encode atypical polypeptides. Others speculated that bithorax non-coding RNAs promote or inhibit the activity of nearby enhancers². Decades later, these conjectures have been proven to be remarkably insightful, as careful analyses of individual long non-coding RNAs (lncRNAs) have largely confirmed each of these models.

lncRNAs are defined as transcripts that are longer than 500 nucleotides and do not encode proteins³. In practice, this is a catch-all definition that encompasses different types of transcripts that do not have an obvious protein-coding potential, and some lncRNAs defined in this fashion have turned out to encode micropeptides^4,5. The number of annotated lncRNAs has been growing steadily, and currently includes more than 20,000 lncRNAs⁶, most of which have no known function. A relatively small number of lncRNAs, however, has been linked to a wide range of biological processes through disparate molecular mechanisms³.

Many of the best characterized lncRNAs have been implicated in the regulation of gene transcription^7,8, often through defined molecular interactions. These studies have raised several crucial questions. Importantly, are there lncRNA types that execute distinct regulatory functions? Considering our current models of gene transcription, which are largely shaped by our understanding of how regulatory protein complexes are recruited to cis-acting DNA elements, what is the purpose of lncRNAs that regulate gene transcription? Which functions do lncRNAs carry out that cannot be easily enacted by DNA-interacting proteins, and what are the underlying mechanisms?

In this Review, we discuss the accumulating evidence that points to major modes through which lncRNAs participate in the regulation of gene transcription. For the purpose of this Review, we have considered lncRNA transcription units regardless of whether their regulatory function is conferred by the lncRNA molecules, the process of lncRNA transcription or the lncRNA promoter. Given that experimental perturbations of lncRNAs can theoretically disrupt other established transcription-regulating components, we specifically focused on lncRNA functions that cannot be easily ascribed to conventional enhancers, promoters or regulatory proteins. We emphasize functions and mechanisms that have been shown to operate at more than one lncRNA locus, in particular those that have been supported through orthogonal experimental tools. We discuss lncRNAs that act in cis or in trans to promote gene transcription, and lncRNA loci that act as cis-regulatory elements to stabilize the transcription of genes encoding transcription factors (TFs), or as allele-specific repressors. Finally, we discuss how our understanding of lncRNA function is helping unravel the impact of non-coding genome variation in human diseases, and the potential of lncRNAs as therapeutic targets.

Transcription activation in cis by lncRNAs

Genes with complex expression patterns are controlled by enhancers and other DNA elements located in their surrounding genomic regions, which often extend to hundreds of thousands of base pairs. These regulatory domains adopt 3D configurations known as topologically associating domains (TADs), which enable enhancers to gain spatial proximity to the genes they regulate, while restricting their interactions with genes from nearby TADs. This spatial framework enables regulatory elements to act in cis, that is, to control genes located on the same DNA molecule. Multiple lncRNAs have been shown to function in such cis-regulatory domains, sometimes through poorly understood mechanisms.

Many lncRNAs spatially interact with neighbouring mRNA-expressing genes, and the expression of these lncRNA–coding gene pairs correlates across tissues and individuals^9–11. Genetic experiments have revealed that some of these lncRNAs have a cis-activating function^12–20. Although active enhancers also produce non-coding transcripts called enhancer RNAs (eRNAs)^21,22, cis-activating lncRNAs are often distringuished from eRNAs because the promoters of cis-activating lncRNAs are flanked by nucleosomes with histone modifications typical of promoters, such as high levels of histone H3 trimethylated at lysine 4 (H3K4me3), instead of conventional enhancer modifications such as H3 methylated at lysine 4 (H3K4me1)²³. Furthermore, lncRNAs are preferentially spliced and are stable, as opposed to eRNAs, which are generally shorter than 500 nucleotides, unspliced and rapidly degraded^21,22. Despite these differences, the distinction between putative cis-activating lncRNAs and eRNAs can be blurry, as stable multi-exonic transcripts are also formed at regions carrying classic active-enhancer chromatin signatures.

The null hypothesis for candidate cis-activating lncRNAs is that they are passively produced from active DNA enhancer regions, and that the lncRNA molecules are thus merely ‘transcription noise’. An example is mouse Lockd, a spliced lineage-specific lncRNA which, as its name indicates, is expressed 5 kb downstream of Cdkn1b, the gene that encodes the cell cycle regulator p27 (ref. 24). Whereas deletion of the entire Lockd locus in mouse erythroblasts results in reduced transcription of Cdkn1b, premature termination of Lockd does not affect Cdkn1b levels²⁵. This finding was taken to indicate that Cdkn1b is positively regulated by a cis DNA element, but that the transcription of Lockd or the transcribed Lockd itself is dispensable for Cdkn1b expression²⁶.

Studies at other cis-activating lncRNA loci, however, support the notion that lncRNAs are not simply passive by-products of transcription. In this section, we discuss different mechanisms through which lncRNA transcripts or the process of lncRNA transcription can contribute to cis-activating functions^12–19.

lncRNAs that function as scaffolds

Some lncRNAs, exemplified by the lncRNA HOTTIP, have been proposed to form a local concentration gradient that provides a scaffold for locus-specific recruitment of regulatory complexes, which in turn regulate the transcription of neighbouring genes^27–29 (Fig. 1a). A related proposed mechanism is the RNA-mediated feedback model³⁰. In this model, low levels of nascent RNA initially enhance the formation of transcriptional condensates that promote transcription, whereas transcript elongation elevates local RNA concentration, which dissolves the condensates and reduces transcription (Fig. 1b). The key effector in this process is the charge balance of electrostatic interactions provided by RNA molecules, which is proportional to RNA length and local abundance, whereas the importance of the RNA sequence itself is minor. Notably, the cyclic nature of this process suggests that the RNA concentration is the basis of the burst kinetics of promoters’ activity, which cannot be explained by classic promoter–enhancer interaction and function³¹.

Fig. 1 | — a, The long non-coding RNA (lncRNA) *Hottip* is expressed from the HoxA locus and serves as a scaffold for the local recruitment of the histone methyltransferase complex comprising MLL1 (also known as KMT2A) and WDR5 to HoxA gene (A1–*A13*) sites of transcription. Consistent with local activity, RNAi-mediated depletion of *Hottip* preferentially affects *Hottip* proximal, compared with distal, HoxA genes (fading colour gradient). b, Local RNA abundance provides feedback on transcription initiation. Left: the Mediator complex, the histone-acetylation reader bromodomain-containing protein 4 (BRD4) and RNA polymerase II (Pol II) are present in low abundance at transcriptionally inactive promoter and enhancer elements. Middle: upon transcription initiation, nascent RNAs produced from promoter and enhancer regions nucleate the formation of a condensate, which increases the local concentration of transcription regulators, thereby causing a burst in transcription. Right: as transcription proceeds, the increase in local RNA abundance beyond a certain threshold generates electrostatic repulsive forces that disperse the transcriptional condensates, thereby ending the transcription burst. c, Many transcription factors (TF) have RNA-binding domains, which potentially interact with nascent transcripts, including of lncRNAs. These lncRNA–TF interactions could contribute to the targeting or the strength of association of the TFs to their genomic target sites by taking advantage of their pre-existing 3D proximity. d, *lincRNA-p21* and its *cis*-activated target, the neighbouring gene *Cdkn1a* (encoding p21), are in 3D proximity, and are co-regulated by the TF p53. Transcription initiation of *lincRNA-p21* is sufficient to enhance the expression of *Cdkn1a* by creating a scaffold for the recruitment of the *Cdkn1a* transcriptional co-activator heterogeneous nuclear ribonucleoprotein K (hnRNPK). e, Transcription elongation of the lncRNA *Maenli* increases local histone H3 trimethylated at lysine 4 (H3K4me3), which is a mark of transcriptionally active chromatin, and promotes the expression of the neighbouring gene *En1*. f, Transcription of a lncRNA in the *Bcl11b* locus named thymocyte differentiation factor (*ThymoD*) promotes the demethylation of CTCF-binding sites and, therefore, CTCF recruitment and chromatin reorganization. This process brings the *ThymoD*-associated enhancer region in proximity with their target, the promoter *of Bcl11b*, resulting in transcription activation. g, Pol II recruitment to enhancers and promoters blocks chromatin-loop extrusion and stabilizes the loops at a configuration that brings active enhancers in proximity of active promoters. Experimental degradation of Pol II leads to the formation of larger loops, extrusion of which is now limited by CTCF.

A broad range of TFs were recently shown to harbour RNA-binding domains³². The resulting RNA–TF interactions, which also have limited RNA sequence specificity, were shown to enhance the binding of TFs to chromatin and to promote their ability to activate transcription of episomal reporters³². Although the effects and biological contexts in vivo of these types of RNA–TF interactions remain to be elucidated, this model suggests a plausible function for some cis-activating lncRNAs (Fig. 1c). The notion that lncRNAs modulate TFs is supported by earlier findings on individual lncRNAs, such as a report that the human lncRNA A-ROD recruits TFs upon its release from chromatin to promote the expression of its neighbouring protein-coding gene DKK1 (ref. 33).

Cis-activation by transcription initiation of lncRNAs

Mouse lincRNA-p21 (also known as Trp53cor1) is a spliced and polyadenylated lncRNA transcribed 17 kb upstream of Cdkn1a (encoding the cell cycle inhibitor p21). lincRNA-p21 illustrates the functional importance of transcription initiation³⁴. In this locus, the promoters of lincRNA-p21 and Cdkn1a engage in constitutive 3D chromatin interactions, and both harbour p53 response elements that confer responsiveness to p53 during cellular stress¹⁵ (Fig. 1d). Deletion of the lincRNA-p21 p53 response element, the lincRNA-p21 promoter or the entire locus in mice led to reduced p21 levels and function, indicating the presence of a Cdkn1a cis-activating element in lincRNA-p21 (refs. 13,15,35). Indeed, interference with lincRNA-p21 transcription initiation significantly reduced Cdkn1a expression, indicating that the earliest steps of production of nascent lincRNA-p21 are required for Cdkn1a cis-activation^15,36 (Fig. 1d). By contrast, premature transcription termination, abrogation of splicing or ribozyme-mediated degradation of lincRNA-p21 had no effect on Cdkn1a, indicating that the mature transcript is dispensable for p21 upregulation¹⁵. A similar mechanism was observed for the activation of the developmental gene Eomesodermin (Eomes) (a TF) by the lncRNA Meteor (mesendoderm transcriptional enhancer organizing region)¹⁶ and for the stimulation of the inflammation regulator Ptgs2 by lincRNA-Cox2 (ref. 18). Collectively, these examples indicate that lncRNA transcription initiation and minimal elongation are cis-activating processes of a class of lncRNAs.

It remains to be determined how transcription activation of some lncRNA promoters evokes them to function as enhancers, although several mechanisms have been proposed, including the possibility that nascent lncRNA transcripts tethered to the locus by RNA polymerase II (Pol II) may enhance the recruitment of additional factors. The recruitment includes factors that can contribute to the formation of transcription condensates (Fig. 1b), TFs (Fig. 1c) or transcriptional cofactors, as exemplified by the recruitment of the Cdkn1a activator heterogeneous nuclear ribonucleoprotein K (hnRNPK) by lincRNA-p21 (refs. 13,34) (Fig. 1d).

Cis-activation by lncRNA-transcript elongation and processing

For some lncRNAs, transcript elongation or processing, rather than initiation, is functionally important. Maenli (also known as Gm29348), a multi-exonic mouse lncRNA transcribed 300 kb upstream of the homeobox TF En1, illustrates a transcription-based activation mechanism with important implications for limb development¹⁷. A series of genetically engineered mouse strains revealed that Maenli elongation is required to establish a permissive chromatin environment for En1 expression in cis in limbs¹⁷ (Fig. 1e). Elongation and splicing of the lncRNA Blustr were also proposed to regulate the neighbouring Polycomb-group gene Sfmbt2 (ref. 37). Blustr length, splicing and rate of transcription — but not specific sequence elements in the mature Blustr transcript — contribute to its cis-activating function³⁷.

Evidence for the role of splicing in promoting cis-activation of transcription comes also from genome-scale observations that the production of multi-exonic non-coding transcripts from strong enhancers is evolutionarily constrained³⁸. Accordingly, splicing motifs in lncRNAs, rather than exonic sequences or expression levels of lncRNAs, are under strong purifying selection^39,40. Importantly, individuals who carry nucleotide variants at splice sites of enhancer lncRNAs exhibit changes in local chromatin epigenetic signatures and altered expression of target mRNAs⁴¹.

Cis-activation through 3D genome reconfiguration

lncRNA transcription has been shown to influence 3D chromatin architecture and enhancer–promoter interactions at several loci, including the lncRNAs PLUT in human or thymocyte differentiation factor (ThymoD) in mouse, as well as the protocadherin-α gene cluster lncRNAs^12,19,42. The transcription of ThymoD lncRNA from an enhancer of the Bcl11b gene was linked to the demethylation of CpG dinucleotides at CTCF-binding sequences in the locus, which increases CTCF occupancy¹⁹ (Fig. 1f). The resulting changes in chromatin topology reposition the enhancer region in greater proximity to its target, the Bcl11b gene promoter¹⁹ (Fig. 1f). A related mechanism was described for the stochastic selection of alternative protocadherin-α promoters⁴². Each protocadherin-α promoter overlaps with an antisense lncRNA, and transcription activation of individual antisense lncRNAs causes demethylation of CTCF-binding sites and increased CTCF binding, which enables the formation of long-range chromatin loops with a distal cluster of enhancers⁴².

In addition to these studies of individual loci, recent work suggests that Pol II recruitment to lncRNAs could have a widespread influence on 3D genome organization. Transcript elongation of lncRNAs was shown to dissociate long-range chromatin contacts (loops) by displacing CTCF and cohesin, without necessarily modifying DNA methylation at the CTCF-binding sites⁴³. By contrast, recent genome-scale studies have illustrated how Pol II recruitment and productive elongation from enhancers and promoters can block the extrusion of chromatin loops, thereby increasing contacts between enhancers and promoters^44,45 (Fig. 1g). Although the impact of transcription on 3D genome conformation remains unsettled, these findings raise the possibility that the cis-activation function of some transcribed lncRNAs is mediated by the effects of transcription initiation or processing on spatial genome organization.

eRNA contribution to enhancer function

Early studies proposed that eRNAs mediate essential enhancer functions, such as the recruitment of Pol II and loading of the Mediator complex to protein-coding target genes, or the regulation of CTCF-mediated chromatin remodelling to enable enhancer–promoter interactions^46–49. However, these functions were primarily determined using RNAi and antisense oligonucleotides to deplete eRNAs, which in addition to RNA inhibition have been shown to induce epigenetic changes in the chromatin of target loci^50–52.

More recent work, based on varied perturbation tools, has supported the notion that eRNAs could elicit similar functions to those described for cis-activating lncRNAs. For example, eRNAs, similar to other lncRNAs, have been reported to contribute to the formation of local condensates, which lead to enhanced gene expression through increased formation and stabilization of transcription co-activator complexes^53,54. Furthermore, the suggested regulatory impact of RNA–TF interactions theoretically applies to both eRNAs and lncRNAs³². One study has specifically focused on eRNAs containing Alu repetitive sequences and found that they can promote enhancer–promoter pairing by forming RNA duplexes with promoter-associated RNAs⁵⁵.

The spatial effects of Pol II recruitment could be relevant to the cis-activating function of enhancers and cis-activating lncRNA loci, by blocking loop extrusion and promoting proximity to target genes^44,45 (Fig. 1g). Pol II recruitment to enhancers has also been proposed to increase enhancer–promoter contacts by tethering both elements at transcriptionally active hubs or condensates⁵⁶. These findings suggest that enhancer transcription is important for normal enhancer–promoter interactions.

Although the analysis of cis-activating transcription units has largely focused on lncRNA promoters and eRNAs, active promoters of protein-coding genes can also have long-range enhancer activity⁵⁷. The conditions in which transcription regulation functions are enacted by eRNA, lncRNA or mRNA promoters are poorly understood.

lncRNAs as local rheostats of transcription-factor genes

In contrast to lncRNAs that contribute to the activation of transcription by enhancers, other lncRNAs carry out specialized rheostat-like functions in cis.

Many lncRNA genes are located in the vicinity of TF genes^9,58,59, and in numerous studies the knockout of a lncRNA has led to moderately increased expression of a TF gene in the same TAD (Table 1). Further analyses of some of these individual lncRNAs indicate that they are fundamentally different from transcription silencers, which repress their target genes^60,61, or from enhancers that confer cell type-specific gene activation. Instead, these lncRNAs regulate already-active genes and tune their transcription to ensure appropriate gene product concentrations (Fig. 2a). We refer to such lncRNAs as gene expression stabilizers, in analogy to voltage stabilizers, which are devices used to protect electronic equipment from excessively high voltage levels.

Table 1.

Long non-coding RNAs whose loss results in increased expression of a neighbouring transcription-factor gene

lncRNA	TF gene	Effect in cis or in trans	lncRNA transcription required	Modulation of enhancer– promoter contacts	TF–lncRNA feedback	Response signalling	Mouse phenotype of lncRNA loss	Dosage-sensitive phenotype of the human TF gene	Refs.^a

Halr1	Hoxa1 Hoxa2	Cis	Yes	Yes	Negative feedback	Retinoic acid	-	Haploinsufficiency: ear defects	62–64
HASTER	HNF1A	Cis	No	Yes	Negative feedback	Unknown	Diabetes	Haploinsufficiency: diabetes	70
CHASERR	CHD2	Cis	Yes	Yes	Negative feedback	Unknown	Lethal	Haploinsufficiency: neurodevelopmental	71
Hand2os1	Hand2	Unknown	Unknown	Unknown	Unknown	Unknown	Cardiac abnormalities	Haploinsufficiency: cardiac	65,66
Hdnr	Hand2	Unknown	Yes	Unknown	Possible^b	Unknown	Cardiac abnormalities	Haploinsufficiency: cardiac	67
Flicr	Foxp3	Cis	Yes	Unknown	Unknown	IL-2	Reduced type 1 diabetes	No (X-linked autoimmune disease)	69
PVT1	MYC	Cis	Yes	Yes	Possible^b	Stress, p53	Loss of function: tumour suppression	Gain of function: oncogenic	14,74
METEOR	EOMES	Unknown	No	Yes	Unknown	Unknown	-	-	16
Evf2	Dlx6	Cis	No	Unknown	Unknown	Unknown	Neurodevelopmental	Haploinsufficiency: split-hand and foot malformation	72
Playrr	Pitx2	Unknown	Yes	Unknown	Negative cross-regulation	Unknown	Cardiac arrhythmias	Haploinsufficiency: Rieger syndrome	75
NXTAR	AR	Unknown	Yes	Unknown	Negative cross-regulation	Androgen	-	Gain of function : oncogenic	78
FENDRR	FOXF1	Possibly trans	Yes	Unknown	Negative feedback	Unknown	Vascular malformations (AVCD-MPV)	Haploinsufficiency: vascular malformations (AVCD-MPV)	68

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AVCD-MPV, alveolar capillary dysplasia and misalignment of pulmonary veins.;

References of long non-coding RNA (lncRNA) mutation studies showing increased expression of a proximal transcription regulator gene.

Evidence for regulation of a lncRNA gene by the transcription factor (TF), but no direct evidence for a feedback mechanism.

One of the earliest examples of such a stabilizer is the mouse lncRNA Halr1 (also known as Haunt or linc-HOXA1), which is located ~40 kb from the homeobox TF gene Hoxa1. Three studies showed that Halr1 depletion or mutations in its promoter led to increased expression of Hoxa1 and other genes of the Hoxa cluster in pluripotent stem cells exposed to retinoic acid^62–64 (Fig. 2b), indicating that Halr1 guards against inappropriately elevated Hoxa gene expression.

Similarly, two mouse lncRNA transcription units curtail the expression of Hand2, which encodes a cardiac basic helix–loop–helix TF (Fig. 2c). One is the lncRNA Hand2os1 (also known as Upperhand, lncHand2 or HAND2-AS1), which is transcribed divergently of Hand2 (refs. 65,66). Deletion of two Hand2os1 distal exons or of a much broader Hand2os1 sequence in mice causes increased cardiac expression of Hand2 mRNA, which leads to augmented expression of HAND2-dependent genes in specific cardiac cell subpopulations⁶⁶. The other lncRNA, Hdnr (also known as Handsdown), is located downstream of Hand2. Insertion of a polyadenylation signal to prematurely terminate Hdnr transcription also increases Hand2 levels, whereas its CRISPR-based induction reduces Hand2 expression⁶⁷.

The lncRNA FENDRR and the TF gene FOXF1 share an ~1.4 kb promoter region in the mouse and human genomes (Fig. 2d). The replacement of mouse Fendrr exon 1 with a polyadenylation cassette that interrupts Fendrr transcription resulted in increased Foxf1 mRNA levels in the caudal end of embryonic day 8.5 and 9.5 mouse embryos, and ectopic expression of Foxf1 mRNA in the heart of embryonic day 12.5 embryos⁶⁸. Mutations in several other lncRNA genes similarly elicit moderately increased expression of adjacent transcription regulator genes, including Flicr⁶⁹, HASTER (also known as HNF1A-AS1)⁷⁰, CHASERR⁷¹, Evf2 (also known as Dlx6os1)⁷², Hotair⁷³, Pvt1^14,74 and Playrr⁷⁵ (Table 1 and Fig. 2e–g).

Fine-tuning the expression of transcription-factor genes through feedback

The dampening effect of lncRNAs on adjacent TF genes could theoretically represent a steady-state tonic inhibition, but some lncRNAs have been shown to act as robust feedback circuits that maintain stable concentrations of regulatory proteins within a narrow concentration range. An example of such a rheostat-like mechanism is the human lncRNA named HASTER. HASTER transcription starts in the first intron of HNF1A, which encodes a homeodomain TF, and proceeds in antisense orientation to HNF1A (ref. 70) (Fig. 2f). High HNF1A protein concentrations result in the direct activation of the HASTER promoter, and this leads to inhibition of HNF1A transcription⁷⁰. Consequently, deletion of the HASTER promoter in mice or human stem cell-derived hepatocytes boosted HNF1A expression⁷⁰. This indicates that HNF1A and HASTER form a classic negative feedback loop that prevents HNF1A overexpression (Fig. 2f).

This type of feedback likely exists at other loci. The lncRNA Halr1, which limits retinoic acid-induced transcription of Hoxa1, is positively regulated by HOXA1 (ref. 63) (Fig. 2b). Depletion studies have shown that FOXF1 is a positive regulator of the lncRNA FENDRR⁷⁶, which in turn inhibits FOXF1 (ref. 68) (Fig. 2d), and that HAND2 binds at two sites in the Hdnr locus, which could conceivably modulate the inhibitory function of Hdnr⁷⁷.

Other types of feedback appear to operate in some lncRNA–TF gene pairs. A recent study reported in a preprint points to a negative cross-regulatory feedback loop formed by the mouse lncRNA Playrr and its adjacent homeodomain TF gene Pitx2⁷⁵ (Table 1). These two genes are expressed in mutually exclusive heart domains, and loss of a Playrr splice site leads to increased and inappropriate Pitx2 expression patterns, causing cardiac arrythmia⁷⁵. The human lncRNA NXTAR also forms negative cross-regulatory feedback with the AR gene, which in turn inhibits NXTAR⁷⁸.

These examples raise the question of why TF genes require feedback. Negative feedback systems dampen fluctuations and can provide a cell type-specific range for TF concentrations. This is important because TF concentrations are crucial determinants of genomic-site binding choices⁷⁹. Lineage-selective TFs are often expressed at different levels across cell types, and typically bind to different cell type-specific genomic sites^80,81. Tight regulation of TF concentrations may be particularly relevant for pioneer TFs, which bind DNA sequences in inaccessible chromatin. HNF1A, in particular, has typical pioneer factor properties, such as the ability to bind nucleosomal DNA⁸², and a capacity to activate silent genes in fibroblasts⁸³. Accordingly, livers of Haster mutant mice, in which HNF1A concentrations are abnormally high, exhibit widespread genomic binding of HNF1A and chromatin opening at HNF1A recognition sequences that are normally inaccessible in hepatocytes, leading to aberrant, ectopic gene transcription⁷⁰. Feedback from lncRNAs could therefore tune TF concentrations to ensure the specificity of cell type-specific gene programmes.

Studies of mouse and human CHASERR lncRNA have exemplified another remarkable feedback system that controls a chromatin remodeller instead of a DNA-binding TF⁷¹. The lncRNA CHASERR is transcribed upstream on the same strand of the CHD2 gene (Fig. 2b). Heterozygous deletions of mouse Chaserr promoter or gene body, or lncRNA depletion, increased Chd2 expression, indicating that Chaserr inhibits Chd2 (ref. 71). The authors postulated that Chaserr transcription interferes with transcription of the downstream Chd2 gene. They also found that CHD2 forms an autoregulatory feedback loop by binding to Chaserr transcripts — and to the Chaserr gene — which promotes Chaserr interference of Chd2 (ref. 71) (Fig. 2g). Failure of this feedback in Chaserr mutants causes increased CHD2 expression, which in turn decreases the expression of many other genes that are located downstream of CHD2-bound transcription units.

Mechanisms of modulation of transcription-factor genes

Genetic experiments have begun to shed light on how stabilizer lncRNAs tune the expression of TF genes. Although lncRNA–TF gene pairs vary greatly in their relative orientations or genomic distance (Fig. 2b–g), perturbation studies have revealed commonalities in their mode of action.

Several studies have demonstrated that the mechanism by which transcription stabilizer lncRNAs modulate the expression of adjacent TF genes occurs in cis. The demonstration that a lncRNA exerts its effects in cis rules out the possibility that the local regulatory activity is carried out by RNA-encoded polypeptides, as well as other indirect mechanisms. cis effects have been demonstrated using heterozygous mutant models that can unequivocally ascertain whether only the chromosome that carries the mutant allele exhibits altered expression of the TF gene. In practice, this analysis can be carried out using either heterozygous lncRNA mutations bred on hybrid mouse strain backgrounds, thereby allowing to distinguish between the two chromosomes, or with compound heterozygotes in which the lncRNA and TF mutations are on separate chromosomes^70,71. Other studies have pointed to a cis effect by showing that ectopic lncRNA expression does not rescue the lncRNA-null mice⁶⁹. The case of Fendrr differs from that of other stabilizer lncRNAs in that ectopic expression was shown to partially rescue the mouse Fendrr mutant phenotype⁶⁸, which suggests that at least some effects of Fendrr occur in trans. This possibility was supported by another study, which deleted a sequence in Fendrr forming a putative DNA–DNA–RNA triplex with various potential target sequences, and found that it partially phenocopies other Fendrr mutants⁸⁴. However, these experiments have not fully addressed whether the rheostat-like function of Fendrr on the Foxf1 gene, with which it shares a promoter region and a closely related developmental phenotype, also occurs through this type of trans mechanism⁶⁸.

Cis-regulatory effects can be mediated through RNA-dependent or DNA-dependent mechanisms. A functional requirement of lncRNA transcription for expression stabilization has been demonstrated using transcription termination alleles or CRISPR-based transactivation for some, but not all, stabilizer lncRNAs^66,67,69,71. Likewise, genetic perturbations or RNA degradation experiments have shown that the RNA itself is functionally important for the inhibitory effects of Chaserr⁷¹, Flicr⁶⁹ and Hdnr⁶⁷ on adjacent genes. By contrast, blocking transcription of HASTER using nuclease-deficient Cas9 as a roadblock or by inserting a polyadenylation site, as well as activation of HASTER transcription through a modified CRISPR–Cas9 system did not have an effect on HNF1A mRNA expression⁷⁰. Moreover, overexpression of HNF1A separation-of-function mutants that lacked the ability to transactivate genes, and therefore did not activate HASTER transcription, still resulted in feedback inhibition of the endogenous HNF1A gene, an effect that required an intact HASTER promoter⁷⁰. Thus, HNF1A interactions with the HASTER promoter, but not HASTER transcript elongation or transcripts, appear to be important for the HASTER–HNF1A transcription feedback.

Chromatin conformation capture studies have shown that the promoters of several transcription stabilizer lncRNAs, including HASTER⁷⁰, PVT1⁷⁴, Meteor¹⁶, Halr1^63,64 and Chaserr⁷¹, limit interactions between enhancers and their target genes, which consequently dampens gene transcription (Fig. 2h–j). In the case of HASTER, increased HNF1A concentrations led to enhanced binding to the HASTER promoter, which further limited interactions between HNF1A intronic enhancers and the HNF1A promoter (Fig. 2i). These independent studies suggest that enhancer competition may be a prevalent mode through which lncRNAs control the expression of TF genes (Fig. 2h–j). In summary, to maintain homeostatic expression levels of TFs, cis-acting lncRNAs deploy transcription-dependent and RNA-dependent mechanisms, but also compete with enhancers of their target genes, and several lncRNAs appear to simultaneously use more than one of these molecular mechanisms^14,71,74.

Dual positive and negative regulatory functions

Numerous lncRNA loci intertwine positive and negative cis-regulatory functions^{14,16,64,70,74}. For example, a comprehensive genetic dissection of Meteor showed that its transcription is required to activate Eomes in pluripotent cells, whereas the Meteor promoter limits Eomes expression during neuronal differentiation¹⁶. Likewise, a deletion of the Haster promoter in mice led to increased HNF1A expression in all hepatocytes, whereas this same deletion caused variegated HNF1A expression in pancreatic β-cells, with some cells showing marked HNF1A overexpression and others complete HNF1A silencing⁷⁰. The Haster promoter, therefore, has a cell type-specific cis-activating function in addition to its negative feedback.

Some of the observed dual phenotypes might occur because stabilizer lncRNAs are often embedded in enhancer clusters. Different genetic alterations in a locus with a complex interspersion of positive and negative regulatory elements can easily lead to opposite phenotypes. For example, an allele that produces a premature termination of Hand2os1 transcription causes a severe loss of cardiac Hand2 expression⁶⁵, whereas Hand2os1 deletions cause Hand2 upregulation^65,66. Likewise, small deletions in Halr1 and RNA perturbations have resulted in increased expression of HoxA genes, whereas other deletions in the Halr1 locus have uncovered HoxA-activating sequences^62–64.

Signal-induced modulation of transcription-factor genes by lncRNAs

For some cis-regulatory lncRNAs, modulation of the neighbouring gene can be triggered by cellular and environmental perturbations. In this manner, lncRNAs can endow signal responsiveness to a single gene, rather than act on a wide gene expression programme. For example, in humans and mice, PVT1 is a collection of alternatively spliced lncRNAs initiated ~50 kb downstream of the TF oncogene MYC, which accumulate locally and downregulate MYC transcription^14,74,85. A study in human breast cancer cell lines showed that the PVT1 promoter limits long-range interactions between the MYC promoter and PVT1 intragenic enhancers, and therefore reduces MYC expression⁷⁴ (Fig. 2j). Human and mouse PVT1 are part of an additional inhibitory mechanism that involves the expression of a stress-dependent, p53-inducible transcript isoform termed Pvt1b, which is initiated at a downstream transcription start site and whose production inhibits transcription of Myc without insulating Myc from its enhancers¹⁴ (Fig. 2j). The stress-induced Pvt1b isoform reverses transcription activation by p53 into a local inhibitory signal, thereby limiting Myc levels and reducing cell proliferation. Interestingly, Pvt1b production leads to both proliferation arrest by Myc downregulation within hours of stress, and to long-term Myc repression, which is associated with activation of cell senescence, suggesting it has a role in epigenetic reprogramming of the Myc locus^14,86.

Flicr is another signal-responsive transcription stabilizer that dampens the expression of Foxp3, which encodes a forkhead TF that controls regulatory T cells⁶⁹ (Fig. 2e). The disruption of mouse Flicr promoters, a mutation of a Flicr splice donor site or targeted degradation of Flicr all led to increased expression of Foxp3 and its target genes, and to a relative depletion of regulatory T cell subpopulations that express low Foxp3 levels⁶⁹. This process can be modulated by interleukin-2 (IL-2), which inhibits Flicr expression, thus promoting Foxp3 expression and regulatory T cell expansion⁶⁹.

Whereas Pvt1 and Flicr tune the transcription level of active genes, other signal-responsive lncRNAs elicit transcription switches. The mouse and human lncRNA MORRBID, for example, rapidly downregulates the neighbouring pro-apoptotic gene BCL2L11 (also known as BIM), in response to cytokines and viral infections, thus promoting the survival of myeloid and CD8⁺ T cells^87,88. Bcl2l11 downregulation is accompanied by deposition of the gene-repressive H3K27me3-modified chromatin⁸⁸. A plant lncRNA named COOLAIR, which inactivates the expression of the vernalization TF FLOWERING LOCUS C (FLC) during the autumn to winter transition⁸⁹, provides fascinating insights into how a lncRNA carries out a signal-responsive binary switch (Box 1).

Box 1. A plant long non-coding RNA as a paradigm of environmental switch.

The Arabidopsis thaliana long non-coding RNA (lncRNA) COOLAIR exemplifies how a lncRNA can function as a binary switch. COOLAIR is a gene comprising a group of antisense, alternatively spliced lncRNA isoforms that overlap the gene body and promoter of the vernalization transcription factor (TF) FLC gene^255–257. COOLAIR responds to environmental cues such as the first seasonal frost, and switches off FLC expression during the autumn to winter transition⁸⁹. In vivo analysis of structural conformations of individual COOLAIR RNA molecules revealed striking cold-dependent enrichment of specific structural isoforms²⁵⁸. Interestingly, these transcript structural variants preferentially occur in a key region of complementarity between COOLAIR and the transcription start site of FLC. The structural variability might influence the ability of the lncRNA to form an R-loop at the 5′ end of FLC, mediate DNA–DNA–RNA triplex formation between COOLAIR and the FLC transcription start site or promote the recruitment of a protein complex to the FLC transcription start site²⁵⁹. Although the exact mechanism by which COOLAIR suppresses FLC is unclear, this finding reveals a new dimension of RNA-based cis-regulation, namely the capacity to be dynamically altered by adopting alternative structural conformations in response to environmental cues²⁵⁸.

Cis-regulatory lncRNAs as allele-specific repressors

Another notable gene regulatory activity that cannot be explained solely through the general framework of TFs interacting with specific DNA sequences is the selective silencing of one of two homologous chromosomal loci. Some of the best characterized lncRNAs accomplish this type of function, including the lncRNA XIST, which regulates X-chromosome inactivation (XCI), and imprinted lncRNAs that control parent-of-origin allele-specific repression.

X-chromosome inactivation

XCI in mammals ensures X-linked dosage compensation between cells of females and males by inactivating one of the two X chromosomes in female cells^90–92. This process is controlled by the X-inactivation centre (Xic), a genomic region that integrates X-chromosome counting information with random selection of one of the two female X chromosomes for inactivation^93–96.

The lncRNA gene XIST, which has a central role in XCI, is located in the Xic and is selectively transcribed from what will become the silenced X chromosome (Xi)^90,97 (Fig. 3a). XIST transcription is regulated by neighbouring activating and repressive cis-regulatory lncRNAs, which are partitioned into two adjacent TADs⁹⁸. In mice, the lncRNAs Jpx, Ftx and Xert are located in the same TAD as Xist and promote its transcription in cis^99,100 (Fig. 3a). This regulation has been demonstrated also in human cell models, and by a 453-kb deletion in a human female that overlaps JPX and FTX and caused markedly skewed XIST expression from the intact chromosome¹⁰¹. Mechanistically, XIST transcription and accumulation depend on JPX transcription in human cells, or on the accumulation of mature Jpx RNA in mice¹⁰⁰ (Fig. 3b). Furthermore, Jpx can also act in cis and in trans to activate Xist by binding and displacing CTCF from the Xist promoter¹⁰².

Fig. 3 | — a, Transcription activation of mouse *Xist*. The X-inactivation centre (Xic) shows two topologically associating domains (TADs) in mouse cells. In one TAD (blue background), the long non-coding RNAs (lncRNAs) *Linx* and *Tsix* — antisense transcript of *Xist* — and the *Tsix* enhancers, termed *Xite*, are located. On the active X chromosome (Xa), *Tsix* transcription suppresses *Xist* transcription, whereas the *Linx* promoter acts across the TAD boundary to limit *Xist* expression in *cis*. In the other TAD (red background), the lncRNAs *Xist*, *Jpx*, *Ftx* and *Xert* are located. *Xert* enhancers, termed *XertE*, promote both *Xert* and *Xist* transcription on the inactive X chromosome (Xi). Following X-chromosome inactivation (XCI), *Jpx* and *Ftx* maintain *Xist* expression and accumulation at Xi. b, Similarities and differences between *XIST* regulation by *JPX* and *FTX* in human and mouse. In human, whereas *FTX* is not essential for *XIST* regulation, *JPX* transcription, but not the mature RNA, contributes to polymerase II (Pol II) loading and *XIST* transcription and accumulation. In mouse, *Ftx* transcription promotes *Xist* transcription, whereas mature *Jpx* transcript is responsible for *Xist* transcriptional activation and accumulation. c, *XIST* mediates transcriptional gene silencing at the X chromosome. *XIST* RNA highlighting its repeat regions A–F and showing the role of A-repeats in promoting the initial steps of gene silencing through SPEN-mediated and histone deacetylase (HDAC)-mediated histone deacetylation and RNA Pol II eviction; the role of B-repeats and C-repeats in heterochromatinization through recruitment of Polycomb repressive complex 1 (PRC1) and PRC2 downstream of heterogeneous nuclear ribonucleoprotein K (hnRNPK); and the role E-repeats in the CIP1-interacting zinc finger protein 1 (CIZ1)-dependent maintenance of *XIST* localization at Xi and in recruiting RNA-binding proteins to mediate the nuclear compartmentalization of Xi. H2AK119ub, histone H2A ubiquitylated at lysine 119; H3K27me3, histone H3 trimethylated at lysine 27; MATR3, matrin 3; PTBP1, polypyrimidine tract-binding protein 1; TDP-43, TAR DNA-binding protein 43.

By contrast, the lncRNA Tsix, which inhibits Xist transcription in cis, is located in an adjacent TAD, along with the Xite enhancer elements, which promote Tsix activation^103,104 (Fig. 3a). The lncRNA Linx also maps to this TAD and acts as a distant cis-inhibitor of Xist¹⁰⁵. This effect is exerted by the active Linx promoter, and is independent of Linx transcript elongation or effects on Tsix¹⁰⁵. Heterozygous inactivation of Tsix, Xite or Linx prior to the onset of XCI shows that they are essential cis-regulators of Xist expression in mice^104–106.

Elegant studies have shown that once the Xi is selected, Xi-specific expression and accumulation of Xist is both necessary and sufficient for chromosome-wide gene repression in cis. Early experiments established that an inducible Xist transgene can silence autosomes in cis in embryonic stem cells^107,108. Molecular and genetic deletion studies have since revealed that repetitive sequences and structural elements within the Xist RNA are central to its ability to recruit regulatory proteins to Xi^109–113 (Fig. 3c). The A-repeats of Xist adopt structural features that are recognized by SPEN (also known as MSX2-interacting protein), a transcription co-repressor that mediates the recruitment of chromatin modifying complexes that promote histone deacetylation, evict Pol II and contribute to the early steps of X-linked gene silencing^{111,114–117}. B-repeats and C-repeats have been implicated in the scaffolding of hnRNPK, which mediates the recruitment of Polycomb repressive complex 1 (PRC1) and the subsequent activity of PRC2 at Xi^118–122, although a recent study also describes independent binding of PRC2 to A-repeats during initiation of XCI¹²³. The E-repeats mediate the assembly of RNA-binding proteins (RBPs) such as CIP1-interacting zinc finger protein (CIZ1), polypyrimidine tract-binding protein 1 (PTBP1), matrin 3 (MATR3), TAR DNA-binding protein 43 (TDP-43) and CELF1, which promotes chromatin compaction and maintenance of late-stage Xi in a phase-separated compartment^124,125.

Ultimately, the accumulation of Xist-scaffolded protein complexes at Xi begins a succession of events that initiate, spread and maintain transcriptional gene silencing^97,126 through the formation of a repressive chromatin state^127–129 and reconfiguration of the chromosomal architecture^112,130,131.

Parent-of-origin allelic repression

Imprinted loci provide another mechanism of lncRNA-dependent cis-regulation. There are at least 29 imprinted domains in the mouse and human genomes, harbouring more than 150 imprinted genes that are organized in clusters¹³². Imprinting of such loci is critically dependent on differentially methylated regions that span ~1–3 kb and acquire parent-of-origin specified epigenetic states during gametogenesis¹³³. A seminal discovery in genomic imprinting was the identification of lncRNAs that are transcribed from the unmethylated allele in a differentially methylated region, and contribute to the repression of imprinted genes from the same locus^134–140.

Although imprinted lncRNAs exhibit considerable sequence and gene-structure diversity, they also share key similarities. Imprinted lncRNAs accumulate in the chromatin at the loci from which they are expressed¹⁴¹, frequently exerting bidirectional, long-range silencing of multiple genes in cis¹⁴². Furthermore, transcription-based and RNA-based mechanisms have been proposed to cooperatively contribute to allele-specific transcription repression by these lncRNAs¹⁴².

Imprinted lncRNAs cause allele-specific silencing of coding genes with which they overlap^143,144. Genetic experiments in mice have shown that promoter deletions or premature transcription termination of imprinted lncRNAs disrupts their silencing functions^145–148. For example, prevention of Airn or Kcnq1ot1 transcription read-through into the coding genes Igf2r and Kcnq1, respectively, causes reactivation of the paternal alleles of these genes^146–150. At the well-studied Airn–Igf2r locus, transcription interference has been ascribed either to promoter occlusion, where Airn transcription of Igf2r antisense prevents the recruitment of the transcription initiation machinery at the Igf2r promoter, or to a mechanism in which the Airn transcript actively removes the transcription machinery from the nascent Igf2r transcript¹⁴⁷ (Fig. 4). A related proposed mechanism is the collision of converging elongating Pol II complexes, exemplified by transcription of the lncRNA Ube3a-ATS, which leads to premature Ube3a termination at the paternal allele¹⁵¹. Support for this model comes from the observation that although both maternal and paternal Ube3a promoters are actively transcribed¹⁵², either premature termination or antisense oligonucleotide-mediated degradation of paternal Ube3a-ATS prior to the overlap with the paternal Ube3a transcripts de-repress paternal Ube3a expression^152,153. Despite the unequivocal evidence that antisense transcription is important for silencing by imprinted lncRNAs, it remains to be established why only some antisense transcripts evoke this effect, given that mammalian genomes harbour a myriad of convergent sense–antisense transcripts that are co-expressed in the same cells¹⁵⁴.

Fig. 4 | — The mouse imprinted *Igf2r* locus, which includes the long non-coding RNA (lncRNA) *Airn*. Maternally expressed (blue), paternally expressed (red), and biallelic-expressed (grey) genes are shown. The circle underneath *Airn* denotes a differentially methylated region, with a black filling indicating methylation of the maternal allele. Bottom right: occlusion and dislodgement models for transcription-mediated repression of *Igf2r* by *Airn*. Bottom left: *Airn* RNA-dependent recruitment of the gene-repressing chromatin modifier complexes Polycomb repressive complex 1 (PRC1), PRC2 and G9a, which repress the *Airn*-distal genes *Slc22a2* and *Slc22a3*. *Airn* RNA is drawn as an unstructured molecule owing to lack of structural information. Pol II, polymerase II.

Imprinted loci, however, harbour multiple genes that do not overlap with imprinted lncRNAs yet show allele-specific silencing. In these cases, lncRNAs serve as scaffolds for other regulatory complexes. Repressed alleles at several imprinted loci are heavily enriched in H3K27me3, and several studies describe cell type-specific direct interactions of the lncRNAs Airn, Kcnq1ot1 and Meg3 with PRC1, PRC2 and the H3K9 methyltransferase G9a (also known as EHMT2)^{139,155–162} (Fig. 4). Two recent studies have highlighted the role of Airn in PRC spreading over a 15 Mb domain in mouse trophoblast stem cells, showing a strong correlation between Airn expression, PRC occupancy at CpG islands and local changes in the chromatin architecture^137,163. This finding is broadly consistent with some studies showing that RNA interactions are essential for genomic occupancy of PRC2 (ref. 164). Such mechanisms therefore explain how local RNA-dependent functions contribute to silencing of non-overlapping genes. One open question is whether the interaction of imprinted lncRNAs with protein-binding factors is mediated by specific lncRNA sequences and/or structures, as proposed for XIST.

Imprinted loci also undergo profound monoallelic changes in CTCF binding and local 3D genome organization, which can insulate genes from enhancers¹⁶⁵. There is evidence that RNA–protein interactions are important for CTCF binding^166,167, and imprinted lncRNAs have been proposed to contribute to local allele-specific 3D genome changes, although more evidence is needed to define the precise role of imprinted lncRNAs in 3D genome organization^168,169.

The analysis of imprinted lncRNAs, therefore, has offered unique insights into how transcription-based and RNA-based mechanisms cooperate to enact gene repression in cis.

Transcription regulation by lncRNAs in trans

Single-molecule imaging studies have shown that some lncRNA molecules are exclusively localized at their transcribed locus, whereas other lncRNAs disperse throughout the nucleus and could thus function in trans. In early studies, global changes in gene expression observed following lncRNA inhibition implied that the lncRNAs have such a trans-regulatory function, but in several cases the changes were later attributed to secondary events or to off-target effects of the perturbation tools. The validation of trans-regulatory functions of lncRNAs requires considerations such as the physiological stoichiometry of the lncRNA and its targets²⁹, evidence for direct engagement of the lncRNA with putative target regions and the ability to rescue loss-of-function phenotypes with exogenously expressed lncRNAs¹⁷⁰.

The archetypal trans-acting lncRNA is HOTAIR, which is expressed from the mouse and human HOXC locus and was proposed to regulate the expression of genes in the distant HOXD locus through PRC2 targeting¹⁷¹. A large Hotair deletion in mice confirmed a role in transcription repression of HoxD genes and several imprinted loci, leading to developmental defects¹⁷². However, the contribution of Hotair to homeotic transformation and the specificity of its interaction with PRC2 were challenged by subsequent studies, one of which used more selective mutations to show that Hotair RNA primarily acts as a negative regulator in cis of adjacent HoxC TF genes^73,173–175. Hotair highlights the need for using complementary experimental tools to understand the function of trans-regulatory lncRNAs.

Global transcription control through nuclear assemblies

An emerging concept in transcription control is the role of nuclear compartmentalization of regulatory factors, mediated by interactions between lncRNAs and RBPs with intrinsically disordered regions¹⁷⁶. Two well-characterized examples of lncRNA-containing nuclear assemblies are nuclear speckles and paraspeckles, which compartmentalize the highly abundant lncRNAs MALAT1 and NEAT1, respectively, and have a role in the global regulation of transcription and RNA processing^177,178. Recent studies have expanded the list of lncRNA-scaffolded nuclear assemblies (Fig. 5a). The intron-retaining, nuclear isoform of the lncRNA Charme specifically recruits MATR3–PTBP1 into nuclear aggregates that regulate chromatin at myogenic loci^179–181. Another study directly visualized the definitive endoderm-specific lncRNA DIGIT (also known as GSC-DT) in condensates that contained the acetylated H3K18 reader, bromodomain-containing protein 3 (BRD3)¹⁸². Deletion of the retained intron of Charme or of DIGIT disrupted condensate formation and perturbed their respective downstream developmental programmes¹⁸². Analogously, the breast cancer-associated lncRNA mammary tumour-associated RNA 25 (MaTAR25) was found to interact with the complex purine-rich element-binding protein A (PURA)–PURB and was proposed to guide their association with the promoter of tensin-1, a key mediator of cell–matrix adhesion and metastatic migration¹⁸³. How lncRNAs are targeted to one or many distant genomic sites remains an open question. In the context of nuclear assemblies, it is possible that locus specificity may be determined by either the lncRNA or the RBP.

Fig. 5 | — a, Association of the long non-coding RNAs (lncRNAs) *Charme*, *DIGIT* and mammary tumour-associated RNA 25 (*MaTAR25*) with polypyrimidine tract-binding protein 1 (PTBP1)–matrin 3 (MATR3), bromodomain-containing protein 3 (BRD3) and purine-rich element-binding protein A (PURA)–PURB, respectively, drives condensate formation (blue background) and localization at target genes. This localization promotes the activation of broad developmental or disease-associated transcription programmes. b, The lncRNA *Firre* promotes inter-chromosomal contacts, which facilitates the co-regulation of a genes with shared functions in energy metabolism. c, Sequence complementarity between lncRNAs and one or many genomic regions enables targeting of the lncRNA to specific loci in *trans* through the formation of a DNA–DNA–(lnc)RNA triplex that involves DNA major groove Hoogstein base pairing. Various lncRNAs, including *Fendrr*, *HOTAIR*, *HIF1A-AS1*, *Sarrah* and others, have been shown to engage in triplex formation and exert positive or negative effects on target-gene expression. Pol II, polymerase II.

Recent studies have also demonstrated a more general role for abundant nascent transcripts in maintaining regional chromatin compaction¹⁸⁴. Analysis of chromatin-associated pre-mRNAs, lncRNAs and non-coding RNAs produced from repetitive regions has identified an RNA–protein scaffold that serves to counteract chromatin compaction and maintain active chromosome territories¹⁸⁴. lncRNAs can also promote chromosomal reorganization by bringing genomic locations from different chromosomes in spatial proximity within the nucleus¹⁸⁵. The X-linked lncRNA functional intergenic repeating RNA element (Firre) has been proposed to promote the formation of such an inter-chromosomal nuclear compartment, which contains co-regulated genes with a shared function in energy metabolism^186,187 (Fig. 5b).

Engagement of targets through triplex formation

Some lncRNAs were proposed to specifically target genomic locations through the formation of hybrid DNA–DNA–RNA triplex structures. Initially, this model was put forth to explain cis-regulation by overlapping antisense lncRNAs, such as KHPS1 (refs. 188,189) and PARTICLE¹⁹⁰. This model has been expanded to address genome-wide triplex formation based on computational identification of regions of lncRNA–DNA complementarity^191–193 or experimental identification of RNAse H-resistant lncRNA–DNA heteroduplexes pulled down using an RNA–DNA-specific antibody^193,194. Examples of lncRNAs proposed to engage this mechanism to repress or activate networks of genes in trans include Fendrr in mid-gestational embyos¹⁹¹, HOTAIR in mesenchymal stem cells¹⁹², HIF1A-AS1 in endothelial cells¹⁹³ and Sarrah (also known as Oxct1as) in cardiomyocytes¹⁹⁴ (Fig. 5c). A recent study proposed an additional role for hybrid triplexes, showing that KCNQ1OT1 forms triplexes to target gene-repressing complexes to transposable elements¹⁹⁵. More work, however, is needed to define the extent to which DNA–DNA–RNA triplex structures are formed by lncRNAs for site-specific regulation at distant genomic sites.

Dual cis and trans regulation

Some lncRNAs have been shown to mediate regulatory activities both in cis and in trans. Notable examples include lncRNAs such as MEG3, which controls imprinting in cis but also mediates the p53 stress response¹⁹⁶ or engages in triplex formation¹⁹⁷; the auxin-inducible lncRNA Apolo in forming R-loops in cis and in trans as a regulator of auxin-responsive genes in plants^198,199; and the cis-activating lincRNA-Cox2, which controls the expression of the neighbouring gene Ptgs2, but also modulates the expression of a wide range of immune genes through an unknown mechanism¹⁸. In particular, the trans activity of lincRNA-Cox2 was demonstrated in a mouse model by rescuing a lincRNA-Cox2 deletion with a lncRNA-expressing transgene¹⁸.

Roles of transcription-regulating lncRNAs in disease

As we begin to grasp the biological purpose of different types of regulatory lncRNAs, it becomes possible to explore their involvement in human Mendelian and polygenic diseases and oncogenesis, and their potential role as therapeutic targets.

Mendelian diseases

Identifying lncRNA gene mutations that cause Mendelian diseases poses major challenges because, unlike protein-coding sequences, there are no rules to predict the functionality of lncRNA sequence variants. Even in cases of lncRNA deletions, it is challenging to ascertain that phenotypes are not due to disruption of other functional elements, such as enhancers, located in the deleted region. Making this distinction usually requires complex genetic engineering approaches, such as combining deletions, transcription termination signals and insertion of RNA ribozymes.

Several lncRNA genes are located within genetically mapped loci that harbour Mendelian or monogenic mutations. For example, deletions encompassing the FENDRR locus lead to alveolar capillary dysplasia and misalignment of pulmonary veins (AVCD-MPV), although those deletions also disrupt elements that regulate the nearby gene FOXF1, which also harbours causal AVCD-MPV mutations^76,200 (Table 1). Nonetheless, mice with disrupted Fendrr transcription recapitulate features of AVCD-MPV^68,201. Likewise, variants in the lncRNA HELLPAR co-segregate with haemolysis, elevated liver enzymes, low platelets (HELLP) syndrome, although more conclusive evidence is needed to prove the causality of distinct variants²⁰². These examples, together with knowledge that lncRNA genes often contain or are adjacent to enhancers, highlight some of the serious challenges facing efforts to demonstrate the pathogenicity of lncRNA defects in Mendelian diseases.

The analysis of Maenli, discussed above, has illustrated how a Mendelian phenotype can be followed up with careful mouse genetic studies to specifically assess the role of a lncRNA in the disease. Two children with a limb malformation were found to harbour the same homozygous 27 kb non-coding deletion in the EN1 locus, whereas another individual with the same phenotype had a larger deletion in the same region in one allele, and an insertion in the other allele¹⁷ (Fig. 6a). They examined the syntenic mouse sequence which contains Maenli lncRNA, and either deleted the Maenli promoter or blocked its transcription by inserting a polyadenylation sequence, both of which phenocopied the human disease¹⁷. These cases suggested that the severe human developmental phenotype was caused by germ-line deletions of a cis-activating lncRNA, raising the question of how many rare or common genetic variants in lncRNAs might influence human health.

Fig. 6 | — a, The human *EN1* locus, which encodes a homeobox transcription factor (TF). *EN1* harbours recessive coding mutations in an individual with limb and brain malformations, whereas far-upstream biallelic non-coding deletions (or a compound heterozygous deletion and insertion not shown here) cause dorsal-limb malformations. *Maenli* is a mouse long non-coding RNA (lncRNA) mapped to the orthologous minimal deleted region in humans. Deletion of the *Maenli* promoter, or insertion of polyadenylation signals of transcription termination, recapitulate limb malformations and lead to reduced *En1* expression, whereas an inverted termination signal has no effect. b, Transcription stabilizers control dosage-sensitive TF genes. Small deviations in the expression levels of certain TFs can be caused by heterozygous loss-of-function mutations or duplications of TF genes, or by biallelic loss of function of the stabilizer lncRNA, causing abnormal cellular transcription with organismal phenotypes. In several examples, the defects in stabilizer lncRNAs and the dosage alterations of their target genes have the same phenotype (Table 1). c, An expression quantitative trait locus (eQTL), in which a single nucleotide variant (SNV) influences expression of a lncRNA (left). rs10000X is depicted as the identifier of a fictitious regulatory SNV that is causal for this eQTL. The two graphs on the right depict statistically significant P values of a group of adjacent SNVs for association with the presence of the autoimmune disease systemic lupus erythematosus (SLE) (top) or with a lncRNA eQTL detected in T cell lymphocytes, a cell type that is relevant to SLE (bottom). The co-localization of both sets of association P values means that the lncRNA is a plausible mediator of the disease association. Several hundred instances such as this have been identified, indicating that variation in lncRNA expression contributes to the susceptibility of common diseases. GWAS, genome-wide association studies.

Phenotypic relevance of transcription-stabilizing lncRNAs

So far, FENDRR and PVT1 are the only lncRNAs that restrain the transcription of adjacent TF genes for which there is genetic evidence of a role in human disease^76,200. It is reasonable to expect that large-scale functional screens will uncover many more cis-regulatory lncRNAs, and whole genome sequencing has the potential to discover genetic defects in lncRNAs. However, not all functional genetic elements are vulnerable to disruptive mutations. For example, enhancers are thought to be relatively robust to loss of function owing to functional redundancy^203–205.

Several considerations nevertheless indicate that genetic defects in stabilizer lncRNAs can result in strong phenotypes. Many of the TFs that are controlled by known stabilizer lncRNAs are very sensitive to gene dosage (Table 1). Thus, haploinsufficient germ-line mutations in HAND2, CHD2, FOXF1, HNF1A and PITX2 cause Mendelian diseases, and in some of these cases an increased dosage in mice or humans also has phenotypic consequences^206–213 (Fig. 6b). Interestingly, somatic gain-of-function mutations or increased expression of MYC, the target of the lncRNA PVT1 (Table 1), constitute an established oncogenic mechanism²¹⁴.

It is thus not surprising that mutations in lncRNAs that control the expression levels of these TFs frequently have phenotypes in mice (Fig. 6b). Different Hand2os1 deletions cause either abnormal heart function or heart malformations and embryonic lethality in mice⁶⁶. Likewise, Hdnr disruption leads to increased Hand2 and severe cardiac malformations^67,215, and deletion of Flicr decreases susceptibility for autoimmune diabetes in non-obese diabetic mice⁶⁹. Pancreatic or germ-line Haster mutations cause diabetes⁷⁰, and Chaserr null mutations cause embryonic lethality or severe growth retardation⁷¹ (Table 1).

Importantly, these in vivo lncRNA phenotypes are not simply associated with silencing of their target TF genes, as would be expected if there was inadvertent disruption of an enhancer, but are instead linked to increased expression of the adjacent TF genes. Interestingly, most stabilizer lncRNA mutants with organismal phenotypes exhibit only moderate changes in the expression of their target TF genes, which underscores the importance of maintaining TF concentrations within a narrow range. The strong mutant phenotypes also suggest that, unlike enhancers, cis-inhibitory lncRNAs have limited built-in redundancy. Thus, despite the challenges in annotating lncRNA mutations that are deleterious in humans, mouse genetics indicate that lncRNA defects can lead to disease.

Polygenic diseases

The most prevalent chronic human diseases, such as Alzheimer disease, coronary artery disease or type 2 diabetes, reflect the interplay of environmental factors with a large number of genetic variants. Although individual variants typically have very small effects on disease risk, the fact that they demonstrably influence human disease processes has the potential to shed light on causal mechanisms. A major fraction of susceptibility variants for common diseases identified in genome-wide association studies are non-coding, but the extent to which they act through lncRNAs is still unknown.

Numerous disease-risk variants have tight genetic co-localization with expression quantitative trait loci (eQTLs) for lncRNAs in disease-relevant cells (Fig. 6c). For example, common DNA variants that influence the expression of the lncRNA named ANRIL (also known as CDKN2B-AS1) co-localize with genetic association signals for coronary heart disease and type 2 diabetes^216,217, and pancreatic islet eQTLs for LINC01512 (also known as HI-LNC77) as well as a splicing quantitative trait locus for LINC00261 co-localize with type 2 diabetes genetic association signals²¹⁶. A recent systematic analysis of expression and splicing quantitative trait loci from a broad panel of tissues revealed that DNA variants influencing the expression of more than 100 lncRNAs co-localize with variants associated with 66 polygenic phenotypes. For more than 50% of these loci, the effect on the lncRNA eQTL appears to be exclusive, or stronger than effects on any protein-coding gene eQTL²¹⁸. These data warrant in-depth studies to examine how specific lncRNAs can act as molecular effectors of genetic susceptibility for common diseases.

lncRNA defects in cancer

Recurrent somatic copy number variants have been identified in several lncRNA loci. Examples include PVT1 structural mutations in multiple cancer types^74,219,220, amplification of FAL1 in approximately 10% of liver cancer²²¹, amplification of SAMMSON in 10% of melanoma²²² and loss of ANRIL in >50% of glioblastomas²²³. These regions, however, also harbour proto-oncogenes (MYC, MCL1, MITF) or the tumour suppressor CDKN2A, and are linked with enhancers, which hamper the ability to assess the pathogenic role of lncRNA defects²²⁴. Nonetheless, cancer-associated somatic structural variants such as focal deletions have been reported at the PVT1 promoter region in breast cancer and in large B cell lymphomas, as well as chromosomal rearrangements that separate PVT1 from MYC^74,225. Furthermore, experimental deletions or transcription inhibition of the PVT1 promoter cause high MYC expression and increased cellular proliferation^14,74

In addition to these genetic changes, abnormal expression of many lncRNAs has been linked to cancer progression²²⁴. Well-studied examples are MALAT1 overexpression, which is a strong predictor of metastasis in lung adenocarcinoma²²⁶, and increased HOTAIR expression, which correlates with progression to metastasis and poor outcomes in breast cancer²²⁷. Abnormal expression of such lncRNAs could contribute to oncogenesis regardless of their function in normal physiology. For example, a recent preprint reports that in a mouse model of lung adenocarcinoma, Malat1 overexpression is a driver of metastasis through dysregulation of gene expression and reprogramming of the tumour microenvironment²²⁸. Other studies have shown that increased HOTAIR abundance can alter the stoichiometry of PRCs, resulting in deregulation of gene expression²²⁷. Furthermore, overexpression of cis-acting lncRNAs, such as the imprinted lncRNA H19 (refs. 229–231), the gene-inhibiting lncRNAs PVT1 (refs. 85,232) and MORRBID^233,234, and the gene-activating LINCRNA-P21 (ref. 235) also have oncogenic trans-regulatory activities.

X-chromosome inactivation defects

XCI is essential for development^236–238 and conditional mouse deletions of Xist in the haematopoietic system cause aberrant epigenetic states and oncogenic transformation^239,240. However, recent studies have revealed that XCI is not permanent in all cell lineages, as reversals can be observed in specific adult immune cell subtypes²⁴¹. This reversal has been proposed to underlie the female-specific predisposition for autoimmune diseases through gene dosage increase from the X chromosome, which is known to have a high density of immunity regulating genes^241–245. Another recent study, reported in a preprint, has directly implicated the immunogenicity of XIST ribonucleoprotein complexes in autoimmune disease mechanisms²⁴⁶.

Therapeutic modulation of lncRNAs

Regardless of whether a disease is caused by a lncRNA defect, it is sometimes possible to envision therapeutic targeting of a lncRNA to modulate a disease-relevant process, using sequence-specific RNA degradation, RNA mimetics or genome editing tools that control the transcription of lncRNAs^247,248. A good example is Angelman syndrome, which is an imprinting neurodevelopmental disorder caused by deletions or mutations of the active maternal UBE3A allele. Several clinical trials are underway to activate the silenced paternal UBE3A allele with antisense oligonucleotides that block or cause degradation of the lncRNA UBE3A-ATS, following proof-of-concept studies in model systems^153,248,249.

Another important therapeutic application has been the use of a lncRNA to correct abnormal gene dosage. Insertion of an inducible XIST transgene into one copy of chromosome 21 in induced pluripotent stem cells from an individual with trisomy 21 was shown to cause silencing in cis of the chromosome, and reversal of major transcriptional and other cellular defects²⁵⁰.

Small molecules that target and modulate the activity and stability of lncRNAs represent an additional therapeutic avenue. Compounds have been developed to target the stabilizing 3′-end triple helix structure of MALAT1, given its strong association with metastasis in solid tumours^251–253. Another example is X1, a small molecule that binds XIST A-repeats and displaces PRC2 and SPEN, thereby blocking XCI²⁵⁴. This approach has been proposed to de-repress wild-type alleles in X-linked disorders such as Rett syndrome.

Conclusion and future perspective

Ever since the discovery of lncRNAs, efforts to understand their biological significance have met daunting challenges. Investigations started from a blank slate, with no sense of what type of molecular entities might exist under the lncRNA umbrella, or which experimental tools could be used to elucidate their function. Despite these obstacles, the past years have witnessed major breakthroughs, many of which have come from exhaustive systematic efforts to dissect the function of single lncRNAs. These studies have led to the discovery of a spectrum of lncRNA functions, including essential gene-activating functions, and specialized cis-regulatory feedback mechanisms.

These findings have also raised a long list of pressing new questions. For example, what fraction of the catalogued lncRNAs is functional, and how many lncRNAs belong to the categories identified so far? How many functional lncRNAs act through RNA-dependent mechanisms, as opposed to those that primarily involve the activation of lncRNA promoters or transcription? For lncRNAs with an ascribed function, our knowledge of the underlying mechanisms is fragmented, which surely explains several apparent paradoxes, such as the observation that the transcription of different lncRNAs can lead to either silencing or activation of their antisense genes. Likewise, transcription activation of lncRNAs has been shown to be essential for both cis-activating and inhibiting functions, but the rules that underlie these outcomes are not well understood. Another crucial gap in the field is our lack of knowledge of the major sequence determinants of lncRNA functions, and their vulnerability to disease-causing variation. Understanding the molecular underpinnings of different types of functional lncRNAs, combined with knowledge of which lncRNAs act in disease-relevant processes, holds promise for the development of new therapeutic strategies.

Acknowledgements

The authors thank T. Graff, M. Cuenca-Ardura and B. Payer for critical reading of this manuscript. This work was supported by European Research Council (789055) and Spanish Ministry of Science and Innovation (PID2021-122522OB-I00) grants to J.F., and by National Institute of Health (R01CA262286 and R37CA230580) grants to N.D.

Glossary

CpG islands: Genomic regions of 500 nucleotides or longer with >50% CpG dinucleotide repeat content. CpG islands are associated with the transcription start sites of most housekeeping genes and as many as 40% of tissue-specific genes; they are bound by regulatory proteins
CTCF: A zinc-finger transcription factor (TF), also known as CCCTC-binding factor, that binds specific DNA sequences and participates in the formation of chromatin loops that influence gene transcription by defining the boundaries of topologically associated domains (TADs) and bringing enhancers into proximity with promoters
DNA–DNA–RNA triplex: A structure in which single-stranded RNA invades the major groove of double-stranded DNA and binds by forming Hoogsteen hydrogen bonds. DNA–DNA–lncRNA triplexes can be identified by pull-downs with a triplex-specific antibody
Enhancer RNAs: (eRNAs). Non-coding RNAs that are bidirectionally transcribed from enhancer regions, and are typically ≤500 nucleotides and unstable (half-life ≤ 2 min)
Enhancers: Genomic regions that are recognized by transcription factors (TFs) and activate and increase the transcription of genes in cis, sometimes from considerable distances. Active enhancers are flanked by nucleosomes that carry post-translational histone modifications such as histone H3 acetylated at lysine 27 (H3K27ac) and H3 methylated at lysine 4 (H3K4me1)
Expression quantitative trait loci: (eQTL). Genetic loci in which different alleles of a DNA variant influence expression levels of coding or non-coding transcripts
Focal deletions: Cancer-associated genomic deletions smaller than 5 Mb that affect both alleles
Genome-wide association studies: (GWAS). Studies that survey DNA variants genome-wide to identify those showing association with a disease or trait. GWAS have been used to discover susceptibility variants for prevalent polygenic diseases. A large fraction of significant associations are found in non-coding genomic regions, indicating that they are mediated by genetic variants that influence regulatory functions
lncRNAs that act in cis: Long non-coding RNAs (lncRNAs) that act on the same chromosome from which they are transcribed, including the regulation of a neighbouring gene, of multiple genes or of the entire chromosome
Silencers: Genomic regions that are bound by repressive transcription factors (TFs) and decrease the transcription of genes in cis
Splicing quantitative trait loci: Genetic loci in which different alleles influence RNA splicing patterns
Topologically associated domains: (TADs). Genomic regions defined by having a higher frequency of long-range chromatin contacts, such as between genes and their regulatory elements, than the frequency of contacts with elements outside the region
Transcriptional condensates: Chromatin-associated, dynamic nuclear assemblies comprising a heterogeneous mix of RNAs, transcription factors (TFs) and co-regulators that modulate transcriptional output

Footnotes

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Reviews Molecular Cell Biology thanks Takayuki Nojima, Igor Ulitsky and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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PERMALINK

Transcription regulation by long non-coding RNAs: mechanisms and disease relevance

Jorge Ferrer

Nadya Dimitrova

Abstract

Introduction

Transcription activation in cis by lncRNAs

lncRNAs that function as scaffolds

Fig. 1 |. Mechanisms of transcription activation in cis by long non-coding RNAs.

Cis-activation by transcription initiation of lncRNAs

Cis-activation by lncRNA-transcript elongation and processing

Cis-activation through 3D genome reconfiguration

eRNA contribution to enhancer function

lncRNAs as local rheostats of transcription-factor genes

Table 1.

Fig. 2 |. Long non-coding RNAs as cis-acting transcription stabilizers.

Fine-tuning the expression of transcription-factor genes through feedback

Mechanisms of modulation of transcription-factor genes

Dual positive and negative regulatory functions

Signal-induced modulation of transcription-factor genes by lncRNAs

Box 1. A plant long non-coding RNA as a paradigm of environmental switch.

Cis-regulatory lncRNAs as allele-specific repressors

X-chromosome inactivation

Fig. 3 |. Control of X-chromosome inactivation by long non-coding RNAs.

Parent-of-origin allelic repression

Fig. 4 |. Long non-coding RNA-mediated allele-specific repression at imprinted loci.

Transcription regulation by lncRNAs in trans

Global transcription control through nuclear assemblies

Fig. 5 |. Mechanisms of transcription regulation by trans-acting long non-coding RNAs.

Engagement of targets through triplex formation

Dual cis and trans regulation

Roles of transcription-regulating lncRNAs in disease

Mendelian diseases

Fig. 6 |. Involvement of long non-coding RNAs in genetic diseases.

Phenotypic relevance of transcription-stabilizing lncRNAs

Polygenic diseases

lncRNA defects in cancer

X-chromosome inactivation defects

Therapeutic modulation of lncRNAs

Conclusion and future perspective

Acknowledgements

Glossary

Footnotes

References

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