Gene regulation by non-coding RNAs in the 3D genome architecture

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Appropriate gene expression is essential for producing the correct amount of proteins at the right time, which is critical for living organisms. In the three-dimensional (3D) space of the nucleus, genomes are folded into higher order chromatin structures that are intimately associated with epigenetic factors, including histone modifications and nuclear long non-coding RNAs (lncRNAs). LncRNAs regulate transcription for both activation and repression, either in cis or in trans. Many ncRNAs are expressed in development-specific, differentiation-specific, and disease-specific manners, suggesting that they are critical regulators for organ generation and maintenance. In this review, we mainly describe the following ncRNAs: Xist, involved in X chromosome inactivation, Firre, which serves as a platform for trans-chromosomal associations, and UMLILO and ELEANORS, which co-regulate genes involved in the immune response and breast cancer, respectively. These ncRNAs are gene regulators in the context of the 3D genome structure.

Introduction

In eukaryotes, the genomic DNA is packaged into chromatin, in which the fundamental repeating unit is a nucleosome consisting of 180 bp DNA and four histone proteins, H2A, H2B, H3, and H4 [1]. The array of nucleosomes are folded into multiple layers, from lower to higher order, involving hundreds of kbs of chromatin loops, approximately 1 Mb of TADs (topologically associated domains), mega-bases of A and B compartments, and individual chromosome territories [2, 3, 4, 5, 6, 7, 8]. Chromatin loops contain long-range chromosomal contacts and local chromatin loops, such as in enhancer–promoter interactions. TAD is a self-interacting chromatin region that compartmentalizes genomes. Enhancers can co-regulate genes within the TAD, but not outside of it. Disruption of the TAD boundaries leads to impaired gene expression, and corresponds to certain diseases [9,10]. The A and B compartments are much larger chromatin domains, and roughly correspond to euchromatin with active histone marks and heterochromatin with repressive histone marks, respectively [5,11]. This finding implies that the 3D genome structures originating from chromatin interactions play a key role in the regulation of gene expression.

Over 40 years ago, chromatin was found to cofractionate with RNAs, thus suggesting the presence of chromatin-associated RNAs [12, 13, 14]. More recent experiments with the Drosophila cell line have demonstrated that chromatin is increasingly endonuclease-resistant when cellular RNAs are hydrolyzed with RNaseA [15]. In this case, small nucleolar RNAs bind to chromatin though their associated proteins, and this is responsible for the chromatin inaccessibility. The possible involvement of other less-abundant RNAs remains to be investigated. These indicate that nuclear RNAs may facilitate the formation of an open euchromatin structure, and regulate gene expression under certain circumstances.

Recent high throughput sequence analyses have revealed that the genome is pervasively transcribed [16]. It is estimated that over 100 000 RNAs lacking protein coding potential, referred to as non-coding RNAs (ncRNAs), exist in cells [16]. ncRNAs with lengths longer than 200 nt are long ncRNAs (lncRNAs), and some play key roles in development. One of the best-studied examples is the Xist RNA, which is involved in X-chromosome inactivation (XCI) in mammalian females, as described below. Xist is produced from the unique locus, Xic (X chromosome inactivation center), which contains a cluster of ncRNA genes including RepA, Tsix, Xite, Jpx, Ftx, and Tsx. These ncRNAs are involved in the regulation of Xist expression and function, as well as XCI. This implies that ncRNAs are important cellular regulatory factors.

In this review article, we discuss the recent work on the ncRNAs that are involved in gene regulation, mainly through modulating higher order chromatin structures and epigenetic marks. We also consider the significance of ncRNAs in mammalian development, immunity, and cancer.

Section snippets

Xist functions in X chromosome inactivation during female early embryonic development

During early development in female mammals, one of the two X chromosomes (XX) is silenced as dosage compensation, relative to males with only one X chromosome (XY). This is referred to as X-chromosome inactivation (XCI). The X chromosome carries over 1000 genes essential for development and cell viability, and their overexpression due to XCI failure is potentially harmful [17,18]. The key regulator of XCI is the Xist (X-inactive–specific transcript) RNA, a 17 kb lncRNA expressed from the

Other ncRNAs that recruit repressive and active factors to chromatin

Several genes are expressed from only the maternal or paternal chromosome, in a phenomenon referred to as genomic imprinting. In addition to DNA methylation and histone modifications, ncRNAs are involved in this process. The Airn (Antisense Igf2r RNA non-coding) ncRNA is expressed only from the paternal allele, and required for the paternal-specific silencing of the multiple neighboring imprinted genes, Slc22a3, Slc22a2, and Igf2r, in the mouse placenta [36, 37, 38]. As with Xist, Airn forms an

Firre serves as a platform for trans-chromosomal associations

The long-range chromatin interaction analyses identified a genomic region that interacts with the X-linked macrosatellite region, DXZ4. It is the Firre (Functional intergenic repeating RNA element) locus that abundantly produces the Firre lncRNA, primarily from the active X chromosome [40,41]. Firre forms RNA clouds in the nucleus, and serves as a platform for trans-chromosomal associations. Firre has 156 nt repeats, termed the repeating RNA domain (RRD), and they bind to the nuclear-matrix

UMLILO primes immune-genes for robust transcription in trained immunity

For an enhanced innate immune response, or trained immunity, immune-related gene promoters are primed for robust transcription. The active histone mark H3K4me3 is accumulated at their promoters, before immune stimulations. IPLs (Immune-gene priming lncRNAs) are a collection of lncRNAs expressed from the TAD containing the TNF (tumor-necrosis factor) responsive genes, and regulate them in cis [43••]. Among them is the UMLILO (Upstream Master LncRNA of the Inflammatory chemokine Locus) lncRNA,

ELEANORS delineate the active TAD and the long-range chromatin interactions in breast cancer recurrence

Gene expression profiles are remodeled in cancers. For example, the ESR1 gene is upregulated when ER (estrogen receptor)-positive breast cancer acquires endocrine therapy resistance. In this recurrence process, estrogen is deprived due to the therapy, and a cluster of lncRNAs, ELEANORS (ESR1 locus enhancing and activating noncoding RNAs), are produced from the TAD including the ESR1 gene, termed the ELEANOR TAD. ELEANORS remain at their own transcription sites, form the RNA cloud, and activate

Conclusion and perspectives

In this review, we have described examples of lncRNAs that are involved in the 3D genome structure and gene regulation. The modes of action for lncRNAs are diverse, and they participate in transcription activation or repression, by recruiting epigenetic modifiers, organizing nuclear substructures, co-regulating multiple genes in the same TAD, and mediating long-range chromatin interactions. LncRNAs are also involved in many different events, including development, immune responses, and

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We apologize to authors whose work could not be cited due to space constraints. We would like to thank the members of the Saitoh laboratory (The Cancer Institute of JFCR) for helpful discussions. This work was supported by JSPS KAKENHI Grant Numbers JP17H05013 [to H.T.], JP19K23736 [to T.Y.], JP18H05531 [to N.S.], and JP18K19310 [to N.S.], and by grants from the Takeda Science Foundation [to N.S.], The Vehicle Racing Commemorative Foundation [to N.S.], and a Research Grant of the Princess

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