Introduction
Long non-coding RNAs (LncRNAs) are evolutionarily conserved, eukaryotic RNA molecules greater than 200 nucleotides in length that have no protein-coding capacity. Many LncRNAs are transcribed within the intragenic regions of protein-coding genes, in either sense or antisense orientation, while a subset, known as "lincRNAs" (for large intergenic noncoding RNAs) are transcribed from chromosomal regions occurring between genes (Figure 1). Growing evidence suggests that LncRNAs are important regulators of a surprisingly wide variety of cellular functions, including epigenetic silencing, transcriptional regulation, RNA processing, and RNA modification1-4. In addition, LncRNAs have been associated with human diseases such as cancers, Alzheimer's disease, psoriasis, and heart disease2. Having a better understanding of the functional roles of LncRNAs offers promise to advance our knowledge of cell regulatory and disease mechanisms.
Figure 1. Possible expression patterns of lncRNAs. Blue and green boxes represent exons of protein-coding genes. Angled intervening lines represent introns. Coiled lines represent intergenic genomic regions. Bent arrows represent direction of transcription. lncRNAs can be transcribed either from within protein-coding genes (red) or from intergenic regions of the genome (green). This latter class is also referred to as "long intergenic non coding RNAs" (lincRNAs). lncRNA transcription can also occur in either sense or antisense orientation relative to protein-coding genes.
Regulation of LncRNA gene expression
Recently, increased attention has been focused on the transcriptional regulation of the LncRNA genes themselves. LncRNAs are transcribed by RNA polymerase II, and most of them have the hallmarks typical of pol II-transcribed gene products: 7-methylguanosine capping and polyadenylation. Many LncRNAs are expressed in tissue- and/or developmental specific patterns5. Therefore, the transcription of the LncRNA genes must be tightly controlled. As is typical for RNA polymerase II-transcribed protein-coding genes, LncRNA gene expression can be regulated by several different mechanisms (Figure 2).
Figure 2. LncRNA expression can be regulated by transcription and epigenetic factors. Blue coiled line represents an lncRNA promoter. Red coiled line represents an RNA polymerase II-transcribed, polyadenylated lncRNA (although not all lncRNA transcripts are polyadenylated). Bent arrow indicates direction of transcription. lncRNA transcription has been shown to be positively regulated by several well-characterized transcription factors (such as Oct4, Nanog, and p53). Epigenetic factors can have opposing effects on lncRNA transcription. Cytosine methylation (such as at CpG islands) can repress lncRNA expression, whereas methylation of lysine residues on histones (such as trimethylation of lysine 4 on histone H3) can activate lncRNA transcription.
Control by transcription factors
Recently, LncRNA genes have been shown to be targets of a number of well-characterized transcription factors. Several LncRNA promoters were found to contain putative binding sites for Sox2, Oct4 and Nanog6-8. Treatment of mouse embryonic stem cells (mESCs) with the differentiation factor retinoic acid causes the downregulation of Oct4 and Nanog RNA levels, as well as a concomitant decrease and increase of the LncRNAs AK028326 and AK141205, respectively. Subsequent RNA interference of Oct4 and Nanog led to similar effects on the expression levels of these LncRNAs. Further, Oct4 was found to be necessary for the transcription of no fewer than three LncRNAs in ESCs and induced pluripotent stem cells (iPSCs)8. Similarly, it was recently shown that lincRNA-p21 is one of many LncRNAs whose promoter regions contain canonical binding sites for the tumor suppressor protein p537,9. lincRNA-p21's transcription is directly induced by p53 in response to DNA damage, and loss of this induction leads to a loss of repression of many downstream p53 target genes. lincRNA-p21 was further shown to be necessary for p53's role in DNA damage-dependent apoptosis9. Therefore, the regulation of LncRNA expression is a critical factor associated with the initiation and development of many human cancers.
Control by epigenetic mechanisms
Several groups have further demonstrated that LncRNA expression levels are regulated by epigenetic mechanisms, including histone modificationas well as DNA methylation. In a large screen7, 1,675 mouse lincRNA genes in ESCs, embryonic fibroblasts (MEFs), neural progenitor cells (NPCs), and lung fibroblasts (MLFs) were identified on the basis of harboring a "chromatin signature": Histone H3 Lysine 4 trimethylation in promoter regions, and Histone H3 Lysine 36 trimethylation in the body of the gene. These so-called "K4-K36 domains" are also found in protein-coding genes and are therefore excellent predictors of the occurrence of transcribed units. In another study10, it was shown that the promoters of a subset of LncRNAs expressed in embryonic stem cells (ES cells) occur in regions for Histone H3 Lysine 4 and Histone H3 Lysine 27 trimethylation (H3K4me3 and H3K27me3, respectively). However, Many of these " bivalent marks" were lost in more differentiated NPCs and fibroblasts.
DNA methylation at CpG dinucleotides also plays a significant role in regulating the expression of LncRNA genes. In a lncRNA profiling study, microarray analysis was used to determine which LncRNAs were upregulated in the human colorectal cancer cell line HCT116 following treatment of these cells with the DNA demethylating agent 5'-aza-2'-deozycytidine. It was revealed that the expression of several lncRNAs are significantly upregulated due to the demethylation at the CpG islands in their promoters following exposure to the drug, indicating that the expression of these lncRNAs are controlled by their promoter DNA methylation level., In addition, two groups12,13 found silencing of expression of LncRNA Glt2 (Meg3) in both mouse induced pluripotent stem cells (iPSCs), as well as human hepatocellular cancer (HCC). In the HCC model, MEG3 expression is indirectly controlled by a micro RNA, mir-29a, which regulates the expression of methyltransferases necessary for DNA methylation, and inhibition of DNA methyltransferase activity in HCC cells causes de-repression of MEG3 expression. Together, these results suggest that the mechanism of regulation of the expression of a LncRNA in two disparate systems is conserved.
Summary
In summary, recent experimental results from several groups have revealed the coordinated regulation of expression of the long non-coding RNAs in a plethora of cell types. These studies have uncovered a major role of the epigenetic regulation of the LncRNA genes, opening up new possible therapeutic targets of the pathways involved with this novel class of non-protein coding RNAs, in many diseases including cancer.
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