Introduction

RNA interference (RNAi), first described in plants and termed co-suppression (reviewed in Reference 1), is a process in which double-stranded RNA (dsRNA) induces homology-dependent degradation of messenger RNA (mRNA) (2,3,4). RNAi can suppress gene expression via two distinct pathways involving small interfering RNAs (siRNAs): transcriptional gene silencing (TGS) and posttranscriptional gene silencing (PTGS) (5,6). PTGS involves siRNA targeting of mRNA and in human cells is operable in both the cytoplasm and nucleus (7,8).

dsRNAs can also produce TGS in Arabidopsis, Schizosaccharomyces pombe, and mammalian cells. TGS was first observed when doubly transformed tobacco plants exhibited a suppressed phenotype of a transgene. Careful analysis indicated that methylation of the targeted gene was involved in the suppression (9). TGS mediated by dsRNAs was further substantiated in viriod-infected plants (10) and was shown to be due to RNA-dependent

DNA methylation (RdDM). The RdDM requires a dsRNA to target DNA and is subsequently processed to yield short RNAs (10,11). These short dsRNAs happened to include sequences that were identical to genomic promoter regions and in turn proved capable of inducing methylation of the homologous promoter and subsequent TGS. TGS in plants has been shown to be carried out by a generally larger size class of siRNAs, 24–26 nucleotides in length (12,13).

More recently members of the Argonaute protein family in Arabidopsis have been shown to play an essential role in RdDM of promoter DNA and transposon silencing (14). Specifically, Ago4 is known to direct siRNA-mediated silencing, and Ago4 mutants display reactivation of silent SUP alleles, along with a corresponding decrease in both CpNpG DNA and histone H3 Lys-9 methylation (H3K9me + ) (13). Consequently in plants, siRNAs that include sequences with homology to genomic promoter regions are capable of directing the methylation of the homologous promoter and subsequent TGS.

TGS in S. Pombe

Similar to plants, the fission yeast S. pombe also employ TGS via siRNAs to silence heterochromatic regions. However, mechanistically S. pombe lacks the epigenetic mechanism of DNA methylation. Instead, S. pombe utilize Argonaute 1 (Ago1) to direct histone methylation and heterochromatin formation (14). Typically, euchromatin (less condensed and relatively transcriptionally active) is associated with acetylated histones and with histone H3 dimethylation on lysine 4 (H3mLys-4), whereas heterochromatin (condensed and relatively transcriptionally inactive) is associated with histone H3 dimethylation on lysine 9 (H3mLys-9) (15). The acetylation of histone tails by histone acetyltransferasese (HAT) results in relaxing the chromatin and a disruption of histone-DNA interactions and gene activation, while the deacetylation of histones by histone deacetylases (HDACs) result in condensation of the chromatin and transcriptional repression (reviewed in Reference (16). H3K9me+ directly recruits Swi6/HP1 (mammalian heterochromatin protein 1), and this recruitment coincides with spreading H3K9me+ in cis (17). In human cells, HP1 associates with HDAC through interactions with methylated lysines in the histone tail, and this interaction has been shown to mediate transcriptional repression by the recruitment of histone methyltransferase (18). The histone methyl-lysine residues recognized by HP1 also serve as substrates for deacetylation by HDACs subsequently allowing for regulation of HP1 and its association with a variety of transcriptional repressors (19,20,21). Consequently, the removal of the acetyl group from histone H3 Lys-9 by HDAC allows for histone H3 Lys-9 methylation, which is linked to HP1, and transcriptional regulator interactions resulting in epigenetic changes and subsequent regulation of gene expression.

RNAi-mediated TGS in S. pombe operates specifically through H3K9me+ (22). In S. pombe, mutants in dcr1 (Dicer homolog) and ago1 (Argonaute homolog) were shown to be reduced in centromeric repeat H3K9 methylation, which is necessary for centromere function (22). These data denote a link between siRNA-specific targeting of histone modifications to specific genomic sequences that subsequently recruit or interact with Swi6 resulting in regulation of the heterochromatic state (22). Additional investigation demonstrated that the dcr1 processed dsRNAs, which correspond to centormeric repeats in S. pombe, interact with ago1, Chip1 (chromodomain protein), and Tas1 (previously uncharacterized) to form the RITS complex (23). The RITS complex, specifically Ago-1 and Chp1, then associate with chromatin-binding factors Swi6 and Clr4 (Suv39H6 homolog) to silence targeted genomic regions (23). The presence of siRNAs in the RITS complex was shown to require Rdp1, Hrr1 (helicase required for RNA-mediated heterohromatin assembly 1) and Cid12 (a 38-kDa protein involved in mRNA polyadenylation) (24). Consequently, in S. pombe, siRNAs generated from centromeric regions trigger silencing of the corresponding centromeric region. Recently, siRNA-mediated TGS in S. pombe has been shown to also require RNA polymerase II (RNA Pol-II), suggesting that transcription of the homologous target is required to intiate TGS (25).