RNA-directed Transcriptional Gene Silencing Research Articles

IDN2 has a role downstream of siRNA formation in RNA-directed DNA methylation

In plants, a particular class of short interfering (si)RNAs can serve as a signal to induce cytosine methylation at homologous genomic regions. If the targeted DNA has promoter function, this RNA-directed DNA methylation (RdDM) can result in transcriptional gene silencing (TGS). RNA-directed transcriptional gene silencing (RdTGS) of transgenes provides a versatile system for the study of epigenetic gene regulation. We used transcription of a nopaline synthase promoter (ProNOS)-inverted repeat (IR) to provide a RNA signal that triggers de novo cytosine methylation and TGS of a homologous ProNOS copy in trans. Utilizing a ProNOS-NPTII reporter gene showing high sensitivity to silencing in this two component system, a forward genetic screen for EMS-induced no rna-directed transcriptional silencing (nrd) mutations was performed in Arabidopsis thaliana. Three nrd mutant lines were found to contain one novel loss-of-function allele of idn2/rdm12 and two of nrpd2a/nrpe2a. IDN2/RDM12 encodes a XH/XS domain protein that is able to bind double-stranded RNA with 5′ overhangs, while NRPD2a/NRPE2a encodes the common second-largest subunit of the plant specific DNA-dependent RNA polymerases IV and V involved in silencing processes. Both idn2/rdm12 and nrpd2a/nrpe2a release target transgene expression and reduce CHH context methylation at transgenic as well as endogenous RdDM target regions to similar extents. Nevertheless, accumulation of IR-derived siRNA is not affected, allowing us to present a refined model for the pathway of RdDM and RdTGS that positions function of IDN2 downstream of siRNA formation and points to an important role for its XH domain.

Genetic analysis of RNA-mediated gene silencing in Arabidopsis thaliana

In plants, a particular class of small non-coding RNAs, short interfering RNAs, can serve as a signal to induce cytosine methylation at homologous genomic DNA regions in the nucleus. If the targeted DNA regions have promoter function, this RNA-directed DNA methylation (RdDM) can result in transcriptional gene silencing (TGS). RNA-directed transcriptional gene silencing of transgenes provides a versatile system for the study of epigenetic gene regulation in plants. In our experimental setup in Arabidopsis thaliana, transcription of a promoter-inverted repeat provides a RNA signal that triggers de novo cytosine methylation and TGS of a homologous nopaline synthase promoter (proNOS) in trans. Utilising this two component transgene system in a forward-genetic screen for “suppressor of silencing” mutations, we were able to identify new candidates for factors involved in RdDM of transgenic as well as endogenous target regions.

Small RNA-Mediated Epigenetic Myostatin Silencing

Myostatin (Mstn) is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

RNA-directed transcriptional gene silencing and activation in human cells.

The overt loss or uncontrolled gain of gene expression is found at some level in virtually every malady afflicting humans. From cancer to HIV-1, the uncontrolled expression or loss of gene expression is prevalent in human diseases. Approaches toward the specific control of gene expression at the transcriptional level could have the potential to revert or reduce disease pathologies. Over the last several years, researchers have developed methodologies that utilize small antisense non-coding RNAs to specifically silence transcription. Only recently has the endogenous molecular pathway usurped by the introduction of these small RNAs to regulate transcription in human cells been defined. Observations suggest that long antisense non-coding RNAs function as the endogenous epigenetic regulators of transcription in human cells, thus explaining why small antisense RNAs were observed early on to silence transcription via directed epigenetic changes at the target loci. The mechanism of action whereby small regulatory RNAs can either turn gene transcription on or off will be discussed as evidence that one day it may be possible to develop therapeutics to regulate gene transcription and ameliorate particular disease conditions.

Promoter-targeted siRNAs Induce Gene Silencing of Simian Immunodeficiency Virus (SIV) Infection In Vitro

RNA interference is a conserved process by which sequence-specific double-stranded RNA is converted into small interfering double-stranded RNAs (siRNAs) that can induce gene silencing via two pathways: post-transcriptional gene silencing and transcriptional gene silencing (TGS). We previously reported TGS of human immunodeficiency virus-1 (HIV-1) could be induced by siRNAs targeting regions within its 5'-long-terminal repeat (5'LTR) promoter region. Here we show that promoter-targeted siRNAs can also induce silencing of simian immunodeficiency virus (SIV) replication by similar mechanisms. Suppression of productive infection was achieved in two different cell lines: a CD4, CCR5, CXCR4 expressing HeLa cell line (MAGIC-5) and in a human lymphoid cell line (CEMx174). HpaII digestion demonstrated induction of methylation at a CpG site within the SIV promoter region following siRNA-induced suppression. Both 5-azacytidine (5-AzaC) and trichostatin A (TSA), inhibitors of DNA methyltransferases (DNMTs) and histone deacetylation, respectively, partially reversed the silencing effect. Furthermore, using chromatin immunoprecipitation (ChIP) assays we found enrichment in the region of the LTR of heterochromatin markers dimethylated histone 3 lysine 9 (H3K9) and trimethylated histone 3 lysine 27 (H3K27) in the siRNA silenced cultures. Together, these results strongly suggest certain siRNAs targeting the promoter region of SIV can effect viral silencing through the induction of epigenetic changes.

Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells

siRNAs targeted to gene promoters can direct epigenetic modifications that result in transcriptional gene silencing in human cells. It is not clear whether the antisense strand of the siRNAs bind directly to DNA or to a sense-stranded RNA transcript corresponding to the known promoter region. We present evidence that an RNA polymerase II expressed mRNA containing an extended 5' untranslated region that overlaps the gene promoter is required for RNA-directed epigenetic modifications and transcriptional silencing of the RNA-targeted promoter. These promoter-associated RNAs were detected by their hybridization to the antisense strand of the complementary promoter-directed siRNA. Antisense phosphorothioate oligodeoxynucleotides were used to degrade the promoter-associated RNA transcripts, the loss of which abrogated the effect of siRNA-mediated transcriptional gene silencing, as well as the complexing of the siRNA with the silent state histone methyl mark and the promoter-associated RNA. These data demonstrate that low-copy promoter-associated RNAs transcribed through RNAPII promoters are recognized by the antisense strand of the siRNA and function as a recognition motif to direct epigenetic silencing complexes to the corresponding targeted promoters to mediate transcriptional silencing in human cells.

RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for maintenance.

The association between DNA methylation and gene silencing has long been recognized; however, signals that initiate de novo methylation are largely unknown. In plants, recognition of RNAs that are inducers of posttranscriptional gene silencing (PTGS) can result in sequence-specific DNA methylation, and the aim of this work was to investigate whether heritable epigenetic changes can occur by this mechanism and if the Met1 methyltransferase is required. RNA-directed DNA methylation (RdDM) was initiated in 35S-GFP transgenic plants following infection with plant RNA viruses modified to carry portions of either the 35S promoter or the GFP coding region. Targeting of the promoter sequence resulted in both methylation and transcriptional gene silencing (TGS) that was inherited independently of the RNA trigger. Targeting the coding region also resulted in methylation; however, this was not inherited. Expression of Met1 was suppressed in order to investigate its role in initiation and maintenance of RdDM. Initiation of RdDM was found to be Met1-independent, whereas maintenance of methylation and TGS in the subsequent generations in the absence of the RNA trigger was Met1-dependent. Maintenance of methylation associated with systemic PTGS was also found to be Met1-independent. RNA-triggered events can lead to heritable changes in gene expression, and it is possible that initiation of other epigenetic phenomena such as trans-silencing and paramutation may have an RNA component.