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The roles that RNA molecules play in the regulation of gene expression have only recently become apparent. Recent work in this area has uncovered several complex, RNA-mediated networks of gene regulation in eukaryotic systems. One newly discovered mechanism of RNA mediated gene regulation takes place at the level of transcription. In yeast, plant and mammalian systems, small RNAs targeted to gene promoters can result in a repression of transcription. Small RNA mediated transcriptional silencing has been shown to be operative by changes in chromatin structure at the targeted promoter. Specifically, silencing has been observed to correlate with decreases in certain active-state histone modifications, increases in various silent state histone methylation marks, and in some instances, DNA methylation at the targeted promoter. These epigenetic remodeling events represent a more stable, heritable form of gene regulation as opposed to the transitory post-transcriptional regulation observed in traditional RNAi mechanisms. Several recent findings have shed light on this newly discovered link between small RNA molecules and epigenetic regulatory machinery, notably in human cells.
RNA interference (RNAi) is a process by which double-stranded RNA induces homology dependent inhibition of gene expression. 1-3 What is referred to as RNAi in animals is known as quelling in the filamentous fungus Neurospora crassa,4-6 and was initially described in plants where it was termed co-suppression.6 Upon further examination, it became apparent that RNAi is operative in targeted gene suppression by two distinct pathways involving small dsRNAs: transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS).7,8 The better characterized molecular pathway, PTGS, involves siRNAs targeting a gene transcript (mRNA), and in human cells has been shown to be operable in both the cytoplasm and nucleus.9,10 The endogenous equivalent to the PTGS based pathway utilizes micro-RNAs (miRNAs). The miRNA pathway involves the processing of the precursor RNA first by the RNAi related proteins Drosha and Dicer to small single stranded regulatory RNAs, ~21–22 nucleotides long. Once the small RNAs are processed they are then capable of interfering with mRNA expression through interactions with the 3′UTR or by directly targeting the homologous genes mRNA for degradation or translational inhibition,11 these silencing activities appear to take place in cytoplasmic P-bodies.12 Message targeted miRNAs may also function to repress translation by interactions of RISC with the ribosome. 13
The majority of work carried out with small RNAs in human cells has focused on siRNA and/or miRNA directed PTGS of cellular mRNAs, and the determination of complexes involved in this process. However, small RNAs can also be utilized to modulate gene expression by directly inducing epigenetic modifications at targeted gene promoters, resulting in transcriptional gene silencing.
Epigenetics is the study of meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA. 14,15, Most current epigenetic studies focus specifically on chromatin structure and chemical DNA modifications. One common epigenetic modification is DNA methylation, specifically found on CpG dinucleotides. DNA methylation is a robust and long lasting epigenetic mark that is conserved and maintained by the activity of DNA methylatransferase 1 (DNMT1). DNMT1 functions to methylate hemi-methylated DNA during the action of DNA replication and cell division.16 De Novo DNA methylation is mediated by several proteins including DNMT3a and DNMT3b, and can be induced by a multitude of stimuli.17
Another common type of epigenetic regulation of gene expression involves chemical modifications of DNA-bound histones. Histone modifications can be both activating and deactivating, depending on the type of modification and the specific residue modified. The chemical modifications at various residues within histones and the affect these modifications have on gene expression is collectively referred to as the “histone code hypothesis”.18,19 Such modifications are most well characterized on histones 3 and 4 and include phosphorylation, ubiquitination, SUMOylation, acetylation and methylation. Importantly, histone 3 methylation on lysines 9 and 27 (K9 and K27, respectively) has been shown to direct the formation of repressive heterochromatin through the recruitment of heterochromatin protein 1 and polycomb group proteins.20,21
Recently it has become clear that in many instances, epigenetic modifications can be induced and directed by small RNA molecules. Small RNA mediated transcriptional gene silencing (TGS) was first observed when doubly transformed tobacco plants exhibited a suppressed phenotype of a transgene that correlated with DNA methylation at the transgene promoter.22 TGS mediated by small RNAs was further substantiated in viriod-infected plants23 and was shown to be the result of the action of RNA-dependent DNA methylation (RdDM). RdDM requires a dsRNA to target the genomic loci.23,24 Recently, members of the Argonaute protein family in Arabidopsis have been shown to play an essential role in RdDM of gene promoter DNA and transposon silencing.25 Specifically, Ago4 is known to direct small RNA mediated silencing, and Ago4 mutants display reactivation of silent alleles, along with a corresponding decrease in both CpNpG DNA and H3K9 methylation. 26 Consequently, in plants siRNAs containing homologous sequences with gene promoter regions are capable of directing DNA and histone methylation of the homologous promoter and sequence specific transcriptional gene silencing. Importantly, RNA directed TGS is a longer-lasting if not permanent mode of silencing as epigenetic modifications are involved in the silencing whereas RNA directed PTGS is more transient as slicing of the target gene transcripts is involved. To date the ability of siRNAs to modulate TGS has been shown in plants (Arabidopsis), yeast (S. Pombe), flies (Drosophila), worms (C. elegans) and most recently in human cells.27
Similarly to plants, the fission yeast S. Pombe also employs small RNA directed TGS. S. Pombe, however, lacks the epigenetic mechanism of DNA methylation. Instead S. Pombe utilizes Argonaute 1 (Ago1) to direct histone methylation and heterochromatin formation.25 RNAi mediated TGS in S. Pombe operates specifically through Histone 3 Lysine-9 methylation (H3K9).28 The result of targeted histone methylation is a compaction of the local DNA with it’s nucleosome and presumably a restriction in RNA polymerase II (RNAPII) activity at the targetted loci that results in transcriptional gene silencing. In S. Pombe mutants in dcr1 (Dicer homolog) and ago1 (Argonaute homolog) were shown to be reduced in centromeric repeat histone 3 lysine 9 di-methylation (H3K9me2), which is necessary for centromere function. 28 These data denote a link between small RNA targeting of specific genomic sequences and the recruitment of Swi6, which results in regulation of the heterochromatic state.28 Recently, RNA-mediated TGS in S. Pombe has been shown to also require some level of RNAPII activity,29 possibly for the expression of an RNA that overlaps the siRNA targeted genomic region. Indeed it has been shown that an RNA/Ago-1 complex can function to slice these gene associated RNAs resulting in TGS of the targeted gene in S. Pombe.30
RNAi mediated TGS appears to be conserved throughout many biological systems. Clearly, the majority of work and detailed characterization of the underlying mechanism involved in TGS has been carried out in S. Pombe and plants. However, other model organisms have demonstrated small dsRNA mediated TGS with slight variations in the underlying theme. In the fungi Neurospora crassa the silencing of homologous sequences has been termed quelling.31 Quelling tends to be used interchangeably in fungi to refer to both PTGS and TGS based mechanisms. Interestingly, in Neurospora both the PTGS and TGS pathways utilize histone 3 lysine 9 dimethylation and yet appear to be distinct from one another.32 In the fruit fly Drosophila, however, the two pathways of RNAi, PTGS and TGS, appear connected via piwi proteins.7 The piwi family of proteins is a composed of several homologous members (piwi/sign/elF2C/rde1/argonaute), which are conserved between many plant and animal species.33-35 Interestingly, in the nematode,C. Elegans, the PAZ-PIWI like protein Rde-1 plays an essential role in RNA mediated silencing in the soma. However, other RNA mediated silencing mechanisms, operative in the germline of C. Elegans, do not appear to require Rde-1.36,37 These data indicate a distinct mechanistic bifurcation in germline compared to somatic cell silencing and RNA mediated modes of gene silencing.
In C. elegans, RNA mediated transcriptional silencing of somatic transgenes has been shown to be the mediated by the ADAR-encoding genes, adr-1 and adr-2, and to be dependent on Rde-1.38 The observed silencing and requirements for adr-1, adr-2 and Rde-1 was characterized by investigation of siRNAs targeted to pre-mRNAs. The silencing corresponded with a decrease in both RNAPII localization and acetylated histones at the targeted genomic region.38 Interestingly, following an RNAi screen in C. elegans, genes encoding RNA-binding, polycomb, chromodomain proteins and histone methyltransferases were detected.38 These data were recapitulated in C. Elegans in an interesting set of experiments which demonstrated long-term transcriptional gene silencing by RNAi. In essence, one dose of siRNA was capable of modulating gene silencing that was inherited indefinitely in the absence of the original siRNA trigger.39 This observed long-term inheritance of siRNA mediated TGS appeared to require hda-4 (a class II histone deacetylase), K03D 10.3 (a histone acetyltransferase of the MYST family), isw1 (a homolog of the Yeast chromatin-remodeling ATPase ISW1), and mrg-1 (a chromodomain protein).39 Taken together these data strongly suggest that RNA mediated TGS in C. elegans contains a convergence of pathways that include epigenetic modifying factors and small RNAs. Overall, when comparisons are made between plants, yeast, worms and flies, one cannot help but notice that indeed RNA is more intricately involved in the regulation of gene expression than has previously been envisioned. This strongly suggests that other organisms might also utilize RNA directed mechanisms to regulate gene expression epigenetically, namely, humans.
Observations of RNA mediated TGS in human cells has lagged behind work done in Arabidopsis, S. Pombe and C. Elegans. However, recent studies have revealed that RNA mediated TGS is operative in human cells and that the observed silencing is the result of small RNA directed methylation of histone 3 lysine’s 9 and 27 (H3K9 and H3K27, respectively), and DNA methylation at the targeted promoter.40-45 However, DNA methylation may not be a conserved feature in all cases of small RNA-mediate TGS in human cells46-48 or may be a function of the duration of targeted small RNA exposure to the target loci in the cell.
More recently it has become clear that only the 21 bp antisense (‘guide’) strand of the promoter targeted small RNA is required to modulate TGS of the targeted promoter.43,45 While the role of histone methylation in small RNA mediated TGS is well established in human cells, only recently have components of the RNAi pathway been shown to be implicated in directing the small RNAs to the targeted promoter regions and modulating the silent state chromatin modifications. To date the components of the RNAi pathway shown to be involved in small RNA mediated TGS are the Argonautes (Ago-1 and Ago-2).43,49 Interestingly, time-course experiments suggest that the argonautes are functional in TGS prior to the appearance of histone methylation at the targeted promoter as silent state histone modifications increase at the targeted loci concomitantly will the enrichment of Ago-1 diminishes.43
In addition to Ago 1, the DNA methyltransferase DNMT3a has been implicated in the silencing activity of promoter targeted small RNAs. DMNT3a has been show to elute specifically with small RNAs at the targeted gene promoter regions of some genes.45 One implication of the epigenetic mechanism of transcriptional regulation induced by promoter targeted small dsRNAs is the possibility for long-term heritable changes in expression levels. Recent work from our lab indicates that long-term gene suppression by promoter targeting may in fact be possible. In addition, long term silencing may be achieved after relatively short periods of exposure to the effector small RNA molecule. Interestingly, the time scale for establishment of long-term silencing correlates with the appearance of DNA methylation. This would be predicted as DNA methylation of promoters is a more heritable, long-term form of epigenetic gene silencing than histone modifications alone. Long-term silencing is not, however, associated with a loss of RNA polymerase II (RNAPII) at the targeted promoter, although it appears to remain in an inactive state.
As discussed previously, active RNAPII has been implicated in small RNA-mediated TGS in human cells and shown to co-immunopreciptate with Ago-1 at siRNA targeted gene promoter loci.43 A role for RNAPII in this process is not necessarily intuitive and was initially explained by two competing models. The first was the RNA-DNA model, in which transcription by active RNAPII results in a melting of the DNA duplex at the promoter, making one strand available for hybridization with the small RNA (Fig. 1A). This small RNA would either already be in complex with epigenetic remodeling proteins, or would recruit them to the site upon hybridizing. Supporting this model was the observation that RNAPII has been shown to associate with complete unfolding of 1.85 out of 3 nucleosomes upstream of the transcription start site for the PH05 promoter,50 and the siRNA EF52 target site, shown to initiate TGS in human cells, is ~1 nucleosome upstream of the TATAA transcriptional start site.41,44 Moreover, RNAPII is associated with nearly 60 subunits and a mass in excess of 3 million Da, including the crescent-shaped Mediator, which essentially envelopes the RNA Pol-II core subunits.50 This model seems most plausible when small RNAs are targeted to RNAPII start sites, or TATAA, subsequently resulting in a small RNA/DNA interaction and robust transcriptional silencing.42 However, silencing of regions upstream of the transcriptional start site or TATAA also result in gene silencing but perhaps this silencing is operative via a different mechanism involving an RNA-RNA mediated interaction (Fig. 1B).
In the RNA/RNA model RNAPII synthesizes a low-copy number transcript that corresponds with the targeted promoter to which the small RNA binds (Fig. 1B). Initial support for this model came from the observations that in S. pombe T7-mediated transcribed genomic regions were not susceptible to siRNA-mediated TGS while RNAPII regions were, and that heterochromatin formation in mouse cells involves HP1 proteins and treatment with RNase causes a dispersion of heterochromatin-associated protein (HP1) from pericentromeric foci.51 Recent findings support the RNA-RNA model and incorporate a role for RNAPII, which has been shown to be required for TGS,44 in small RNA mediated TGS (Fig. 1B). This conclusion was reached by the identification of low-copy promoter transcripts spanning several human gene promoters.41 Blocking of these low copy promoter-associated transcripts using complimentary phosphorothioate oligonucleotides (ODNs) results in abrogation of silencing at the small RNA targeted promoter. The characterization of this novel transcript, in all genes that were assessed, and of its role in transcriptional regulation represents a significant step towards understanding the complex and varied roles that RNA molecules play in regulating cellular activities.
Many questions remain regarding small RNA mediated TGS in human cells. Perhaps the most pressing issue is the identification of an endogenous trigger for this process in human cells. To date all examples of small RNA directed TGS in human cells has been the result of transfecting small RNAs designed a priori to target a particular gene promoter loci. Thus, essentially two scenarios are plausible, either (1) the mechanism which is being employed to transcriptionally silence gene promoter loci is a vestigial mechanism, or (2) the mechanism is endogenously active at some stage in cell development. In either instance, directed control of gene transcription is feasible with small antisense RNAs targeted to particular regions of RNAPII gene promoters. If this particular mechanism proves to be vestigial, then there is little doubt this pathway should be capitalized on for therapeutic benefit as off-target effects, which often result from targeting physiologically relevant pathways, would be limited. Clearly, the identification of an endogenously expressed, promoter targeted small RNA which mediates TGS in human cells would solidify this phenomenon as a physiologically relevant process.
In addition to the suppressive activities of promoter targeted small RNAs, evidence is mounting which suggests that small RNAs are involved in many additional aspects of transcriptional regulation. One such finding is the observation that small RNAs targeted to promoter regions low in GC content and high in complexity can activate certain genes.52,43 Activation in these cases seemed to be mediated by histone modifications, and in one instance53 was shown to confer long-term activation. Importantly, this work demonstrated that promoter targeted small RNA molecules do not inherently function in only an inhibitory manner. Another interesting recent finding is that a liver specific miRNA is involved in the positive regulation of hepatitis C replication in human liver.54
However, in other instances gene activation by small RNAs was shown to be the result of off-target effects.55 In this example, a small RNA was found to activate HIV-1 LTR transcription. Upon examination it became apparent that the small RNA was targeting a non-coding RNA, C10orf76, which appeared to exhibit a cell wide regulatory effect. When C10orf76 was suppressed by RNAi, increased cell-wide gene expression was observed.55 While the mechanism of small RNA directed transcriptional activation are not completely understood, these observations provide yet another example of RNA mediated gene regulation. Clearly, these recent findings point to the existence of a complex genetic regulatory network mediated by RNA.
With the discovery of RNAi came an explosion of new small RNA species such as microRNAs (miRNAs) and piwi associated RNAs (piRNAs).56 The observation and characterization of these small RNA species has significantly changed the perception of how gene expression is controlled.57 It has also become apparent that several non-coding RNAs (ncRNA)58-60 are present within the cell and that the majority of the entire human genome appears to be transcribed.61,62 It is also very intriguing that ncRNA expression profiles do not appear to be conserved between various organisms,63 suggesting a role for ncRNAs in the differential aspects of various species. NcRNAs have been shown to be involved in X inactivation, dosage compensation, imprinting and polycomb mediated silencing.62 Interestingly, many of the ncRNAs that have been detected have been in the antisense orientation relative to gene transcription58,64 contain some homology to gene promoter loci.65 These observations support earlier notions that sequence specific ncRNAs might be involved in the regulation of gene promoter activity. Indeed, such an eventuality has been observed in S. cerevisiae where the ncRNA, SRG1 has been observed to interfere with SER3 gene expression.66,67
There are many implications from this work in the fields of evolutionary biology and medicine. One prominent evolutionary theory states that at some point in Earth’s evolutionary history, organisms may have existed which relied entirely on RNA for the storage of information as well as the completion of enzymatic cellular processes. RNA is well suited for these functions as the molecule is able to encode information as well as act enzymatically, as a ribozyme, to catalyze necessary chemical reactions.68 As our understanding of the various roles that RNA plays in gene regulation expands, the prospect that all cellular processes once relied on RNA molecules seems more and more possible.
As aberrant gene expression is the cause of many human diseases, the ability to specifically modulate human gene expression holds exciting potential for the development of novel therapeutics. In fact, adaptations of the PTGS and TGS pathways of RNAi are currently being investigated as potential treatments for diseases ranging from AIDS to cancer. In addition to gene therapy applications, it is also possible that previously uncharacterized factors involved in these forms of genetic regulation may prove to be feasible targets for the development of new therapeutics.