The therapeutic potential of RNA directed epigenetic regulation of transcription

Notably, some of these lncRNAs actively modulate gene transcription and epigenetic states. Mechanistically, some of these lncRNAs appear to recruit epigenetic silencing complexes to their homology containing loci. I present here details of this mechanism as well as insights that now allow us to mimic lncRNA function to regulate a genes expression, either epigenetically silencing or de-repressing and activating gene expression. These insights suggest that long-term heritable control of gene transcription can be instilled on target genes in a directed manner. Such insights portend an entirely new therapeutic platform for targeted gene regulation.

Long non-coding RNAs and gene expression

A profound realisation and subsequent paradigm shift is occurring in molecular and cellular biology. This shift in our collective thinking is the result of exciting findings from the ENCODE consortium. ENCODE, a consortium of over 280 scientists, determined that ~80 per cent or greater of the human genome is transcribed and possibly functionally active, despite lacking protein-coding capabilities^1,2. These genome-wide observations resonate with what others and we have observed during the last decade, while studying non-coding RNA directed transcriptional gene regulation, and drastically expand on the inherent complexity of the cell (reviewed in^3,4). Most notably are the recent observations that antisense long lncRNAs⁵ appear to be active transcriptional regulators of particular protein-coding genes^6-9. Many of these regulatory antisense lncRNAs appear to emanate from pseudogenes, which were thought to be evolutionarily remnants in the human genome^8,9.

Non-coding RNA directed discordant regulation

The first observations suggesting lncRNAs might be functional gene regulators were the result of the FANTOM consortium, where deep sequencing of around one per cent of the mouse transcriptome was carried out. In this body of work, it was observed that several genes exhibited transcription in both directionalities and that when some of these transcripts were perturbed, there was an effect on their respective sense or antisense counterparts⁵. Not much later observations in human cells, specifically carried out on the p21 and p15 tumour suppressor genes^6,7, indicated that these genes were also under similar forms of RNA directed regulation as observed in the mouse¹⁰. In these studies it became apparent that transcription for these tumour suppressor genes was occurring in both directions. These bidirectional long antisense non-coding RNA (lncRNAs) were also found to be functional in epigenetically regulating the expression of their sense protein-coding gene counterparts (reviewed in¹¹). Interestingly, a large percentage of those genes which have been found to exhibit bidirectional transcription, in that they express both sense/mRNAs as well as antisense lncRNAs, appeared to be tumour suppressor genes involved in cell cycle regulation (reviewed in¹²). More interesting is the observation that those genes exhibiting bidirectional characteristics, such as tumour suppressor genes, are often times found to be epigenetically silenced in human cancers¹². From these limited studies as well as a recent studies^8,9 much has been gleamed regarding bidirectional genes and discordant regulation of gene transcription.

Mechanistically, this process whereby antisense lncRNAs direct epigenetic silencing complexes to targeted loci in trans appears to involve the lncRNAs interacting with epigenetic protein remodelling complexes and directing them to particular loci in the genome, essentially acting as ‘epigenetic messengers’ (Figure 1). The result of this form of RNA-based transcriptional targeting is stable epigenetic gene silencing (Figure 1b), which has been observed, at least in the case of small RNA guides, to be heritable and passed on to daughter cells¹³. To date, this mechanism of action has been observed and described with several different genes including p21, p15, PTEN, BDNF, and the iPS related gene OCT-4^6-9,14.

Figure 1: LncRNA epigenetic regulatory pathways. Long non-coding RNAs are expressed from many loci in the genome with some that can function in trans to epigenetically regulate gene expression. (A) Some antisense lncRNAs can interact with those proteins involved in chromatin modification such as DNMT3a, EZH29 while other lncRNAs (B) interact with biomodal controlling PRC2 and LSD1 associated complexes15,18, to epigenetically target particular loci in the genome.

Some lncRNAs that are not necessarily antisense to their protein-coding counterpart have been found to modulate epigenetic states and chromatin architecture. One interesting example of lncRNA function is the bimodal regulating capabilities of HOTAIR, which is derived from the HOXC locus and involved in regulating HOXD in trans¹⁵. Similar to another lncRNA, MALAT1, HOTAIR has been observed to be involved in cancer metastasis^16,17. Interestingly, HOTAIR has been found to act as a scaffold for the assembly of histone modification complexes¹⁸ that can control differential epigenetic states at the HOXD locus, severing to recruit either activating or suppressive epigenetic marks (Figure 1c – 1d). It is however unclear to what extent sequence complementarity is required for HOTAIR targeting to the HOXD locus, unlike antisense lncRNA targeting that involves sequence complementarity in the targeting of epigenetic complexes to protein coding loci and active transcription at the target locus^19,20.

There are growing numbers of examples of lncRNA functions involving gene regulation with varying mechanistic themes that for the most part do not rely on epigenetic remodelling of the targeted locus^21,16,18,22. Examples abound ranging from Neat1^23,24, a component of paraspeckles, MALAT-1, a lncRNA involved in subnuclear structure and possibly splicesome function^25,25, and Gomafu, a lncRNA involved in schizophrenia²⁶. Clearly, the varied complexities of lncRNA function and regulatory capabilities are only just now becoming apparent. What is noteworthy is the notion that some lncRNAs are active in regulating particular genes by modulating the local epigenetic environment through epigenetic silencing, a mechanism of action that has been shown to have long lasting and heritable potential²⁷. Such a mechanism of action suggests a deep mechanistic of action that has the potential to potently control gene expression.

Mimicking lncRNA pathways with small RNAs: suppressing or activating genes

Around the time pervasive transcription was being realised in the mouse⁵, it had been observed that small interfering RNAs (siRNAs) could control gene transcription by targeting epigenetic silencing complexes to homology containing loci in human cells²⁸. A year later it was determined that mechanistically only the small antisense RNA (sasRNA) of the siRNAs and DNA methyltransferase 3a (DNMT3a) was required for the observed RNA directed transcriptional gene silencing (TGS)²⁹. Mechanistically, we now know that the sasRNAs interact with a low-copy promoter associated transcript^19,20 and recruit epigenetic silencing complexes consisting of DNMT3a^29-31, Enhancer of Zeste 2 (EZH2)¹³, Histone deacetylase 1 (HDAC-1)³⁰ and Argonatues (AGO)^32,33 (Figure 2a – 2c) to homology containing loci. This mechanism of RNA directed TGS has now been used to target the stable epigenetic silencing of several genes involved in cancer^6,20,34 and diseases such as HIV^35-39.

The discovery that sasRNAs can direct TGS in human cells spurred an investigation into the ability to target different gene promoters. During one such investigation, it was observed that promoter targeting, specifically to Adenosine and Thymine rich regions in promoter, resulted in transcriptional activation of the targeted gene^40-42. Later studies by a different group on the p21 gene, which was observed previously to be activated by siRNAs targeted to the p21 promoter⁴⁰, indicated that the mechanism of activation was the result of the siRNAs targeting an antisense long ncRNA⁶. Such a result, of siRNAs targeting the antisense counterpart, are possible because unlike sasRNAs, siRNAs possess two strands, which have been observed to target both strands of a particular locus, e.g. either sense or antisense transcripts if they are present for a particular targeted mRNA⁴³.

These fortuitous observations of siRNAs targeting an endogenous regulatory lncRNA resulting ultimately in gene activation were some of the first in human cells to indicate that long antisense ncRNAs are functional endogenous modulators of gene expression and epigenetic states (reviewed in⁴⁴). Such a notion presents the intriguing idea that with some level of knowledge of a particular genes endogenous regulatory lncRNA network, one can target the activation of particular genes expression. This targeted gene activation is done by suppressing the endogenous gene regulatory lncRNA, which leads ultimately to de-repression of lncRNA mediated regulation (Figure 2d – 2f). This process is intriguing as it suggests that the mechanism of lncRNA action (Figure 1a and 1b) can be perturbed in a targeted manner using either RNA interference or antisense oligonucleotide targeting of the lncRNA complex (Figure 2d – 2f respectively); resulting in a loss of lncRNA function and ultimately transcriptional activation of gene expression (Figure 2f). Alternatively, if the promoters for particular regulatory lncRNAs are known one can design sasRNAs to target TGS (Figure 2a – 2c) to the lncRNA promoter³⁴. The result of this targeting can be long-term stable epigenetic silencing of the lncRNA and subsequent stable de-repression (up-regulation) of the lncRNA targeted gene, e.g. TGS of the endogenous lncRNA results in a stable loss of the lncRNA repressor at its targeted gene and ultimately gene activation (Figures 2d – 2f). Indeed, both stable long-term TGS¹³ and lncRNA promoter targeting³⁴ have been observed previously suggesting this is a plausible methodology to stably activating a particular genes expression.

Figure 2: Mimicking and perturbing lncRNA pathways with small regulatory RNAs. (A) Exogenously introduced sasRNAs can target epigenetic silencing to homology containing targeted loci28,29,33, a pathway that miRNAs may also utilise in the nucleus47. (B) This form of targeting requires DNMT3a, HDAC-1, EZH2 and AGO and transcription at the targeted locus (reviewed in 35), and (C) ultimately results in stable epigenetic silencing. (D-E) Suppression of endogenous lncRNA targeted epigenetic silencing can lead to de-repression/activation of the lncRNAs targeted genes expression (reviewed in 44). Regulatory lncRNAs can be targeted with (D) antisense oligonucleotides or RNAi, resulting in (E) conformational changes or degredation of the lncRNA and (F) ultimately de-repression of the lncRNA targeted locus and gene activation.

Collectively, the observations over the last decade portend a new paradigm where it is now possible to specifically modulate a genes expression by mimicking or perturbing endogenous ncRNA directed epigenetic pathways to either turn a gene down or up in a relatively long-term manner. While such a realisation offers an entire new therapeutic reality it is not without its limitations and pitfalls.

Conclusion

Emerging data from ENCODE¹ studies suggest that transcription is pervasive⁴⁵ in both directionalities¹⁰. Indeed, a new-world cellular order is upon us whereby an entire level of complexity has emerged that is as equally fascinating as it is complex. With an understanding of this non-coding RNA world and its intricacies come new paradigms in molecular medicine.

One such example is presented here whereby an appreciation for one molecular mechanism, ncRNA direct epigenetic silencing^11,35, juxtaposed with current antisense technologies⁴⁶, suggests it is now possible to modulate stable gene silencing of a targeted locus. The key to this molecular targeting approach is knowledge of the targeted genes functions; genes in this case can be both protein-coding and/or non-coding. Thus targeting depending on the target it is now possible to molecularly control a gene by specifically activating or suppressing its expression. Such RNA directed epigenetic targeting does however also have the potential to stable modulate a genes expression¹³, suggesting that perturbing lncRNA pathways in this manner can result in long-term heritable epigenetic changes. Indeed such transgenerational epigenetic heritable traits have been observed in model organisms as well as humans (reviewed in¹¹) and may be a key aspect in cellular evolutionary process, suggesting that such superlative control over gene expression may also come at the cost of altering the evolutionary trajectories of the treated cell. While an intriguing notion much remains to be determined with regards to ncRNA mediated epigenetic regulatory networks, as we are only now, just, seeing the proverbial non-coding RNA light.

Acknowledgements: The project was supported by the National Institute of Allergy and Infectious Disease (NIAID) P01 AI099783-01 to KVM and the National Cancer Institute (NCI) R01 CA151574, National Institutes of Health (NIH) R01 CA153124 to KVM and Peter K Vogt.

Biography

KEVIN MORRIS received his BS from Humboldt State University and PhD from the University of California Davis. During his post-doctoral training at the University of California San Diego, he determined that non-coding RNAs are capable of epigenetically modulating gene transcription in human cells. The Morris lab has since determined the mechanistic underpinnings of non-coding RNA mediated regulation of gene transcription in human cells. Emerging evidence from the lab suggests a role for longer forms of non-coding RNAs, some emanating from pseudogenes, are functional endogenous effectors involved in epigenetically modulating gene expression human cells. The lab is currently interested in utilising the recently described endogenous non-coding RNA pathway in human cells to epigenetically modulate gene expression in those genes involved in several diseases ranging from HIV to cystic fibrosis and cancer.

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