RNA-mediated epigenetic regulation of gene expression

Abstract

Diverse classes of RNA, ranging from small to long non-coding RNAs, have emerged as key regulators of gene expression, genome stability and defence against foreign genetic elements. Small RNAs modify chromatin structure and silence transcription by guiding Argonaute-containing complexes to complementary nascent RNA scaffolds and then mediating the recruitment of histone and DNA methyltransferases. In addition, recent advances suggest that chromatin-associated long non-coding RNA scaffolds also recruit chromatin-modifying complexes independently of small RNAs. These co-transcriptional silencing mechanisms form powerful RNA surveillance systems that detect and silence inappropriate transcription events, and provide a memory of these events via self-reinforcing epigenetic loops.

Figure 1. The ‘nascent transcript’ model and a self-reinforcing epigenetic loop in S. pombe

In Schizosaccharomyces pombe, the RNA-induced transcriptional silencing (RITS) complex establishes a physical connection between small interfering RNAs (siRNAs) and heterochromatin by targeting a nascent transcript, and forms the basis of a self-sustaining feedback mechanism that couples siRNA production to chromatin modification. A siRNA-targeted centromeric long non-coding (lncRNA) bound to the RITS complex becomes a template for double-stranded RNA (dsRNA) synthesis by the RNA-dependent RNA polymerase complex (RDRC, which is composed of Rdp1, Hrr1 and Cid12) and generation by Dicer 1 (Dcr1) of new siRNAs, leading to further targeting of the RITS complex after passage of Argonaute (Ago) through the ARC (Ago siRNA chaperone) complex. The Chp1 subunit of the RITS complex anchors the complex onto nucleosomes with histone H3 lysine 9 (H3K9) methylation, and the RITS complex recruits the Clr4–Rik1–Cul4 (CLRC, of which Clr4 is the methyltransferase) complex via Rik1 and Stc1 to promote the further spread of H3K9 methylation. The heterochromatin protein 1 (HP1) homologue Swi6 binds to methylated H3K9 and promotes RDRC recruitment and siRNA biogenesis via the silencing factor Ers1. Swi6, and particularly the other HP1 protein Chp2, help to restrict RNA polymerase II (Pol II) access by recruiting the Snf2–histone deacetylase repressor complex (SHREC). The TRAMP non-canonical poly(A) polymerase and the exosome also contribute to silencing. Together, the RITS complex and the nascent lncRNA transcript provide a hub for the assembly of machineries that make siRNAs, modify histones and silence gene expression. me2, dimethylation.

Figure 2. A self-reinforcing loop linking siRNAs to DNA and histone methylation in A. thaliana

Elaborate feedback between small RNAs and DNA and histone methylation underlies a robust silencing pathway at sites of asymmetrical cytosine methylation in the Arabidopsis thaliana genome. Two plant-specific polymerases transcribe the critical RNAs. RNA polymerase IV (Pol IV) transcripts are processed by the RNA-dependent RNA polymerase RDR2 and the Dicer protein DCL3 into 24-nucleotide (nt) small interfering (siRNAs), while Pol V transcripts act as their targets. The Argonaute protein AGO4, the siRNA-dependent recruitment of which to Pol V transcripts is reinforced by interactions with the GW domains of Pol V and an associated elongation factor KTF1, in turn recruits the CHH DNA methyltransferase DRM2. RDM1 associates with the Pol V–AGO4–DRM2 complex and may link siRNA amplification to pre-existing DNA methylation. Meanwhile, another DNA methyltransferase that targets CHG sites for maintenance, CMT3, is recruited directly to methylated histone H3 lysine 9 (H3K9). Silencing by DNA methylation is augmented by H3K9 methylation, which is deposited by the enzymes KYP, SUVH5 and SUVH6. These methylation events are coupled to one another and to siRNA activity in several ways. KYP is recruited directly to methylated DNA, where it methylates neighbouring histones, and the H3K9 methylation reader SHH1 recruits Pol IV to promote siRNA generation, while the DNA methylation readers SUVH2 and SUVH9 recruit Pol V to promote AGO4 targeting and further DNA methylation. Thus, the different methylation readers, RNA polymerases and AGO4 act together to create self-reinforcing interactions between pre-existing DNA methylation and siRNA amplification. Erasure of DNA methylation by mutations in either the histone deacetylase HDA6 or the maintenance DNA methyltransferase MET1 results in loss of siRNA biogenesis, emphasizing the importance of these self-enforcing interactions. Altogether, the A. thaliana pathway for DNA methylation at asymmetrical sites is one of the most remarkable examples of a recurring theme in epigenetic regulation by small RNAs: self-reinforcing feedback loops. SRA, SET and RING finger-associated; SWD, SAWADEE domain.

Figure 3. Small-RNA-driven transcriptional silencing of gene expression in C. elegans

a | In Caenorhabditis elegans, exogenous double-stranded RNA (dsRNA) is processed into primary small interfering RNAs (siRNAs) that are loaded onto the Argonaute (AGO) protein RDE-1 and amplified by RNA-dependent RNA polymerases (RdRPs) to give rise to secondary siRNAs called 22G-RNAs. When loaded with 22G-RNAs, the somatic AGO protein NRDE-3 translocates to the nucleus, where it targets nascent RNA transcripts and silences corresponding genes, acting in concert with the silencing factor NRDE-2. Gene expression is halted by NRDE-2 during the elongation phase of transcription, and silencing involves histone H3 lysine 9 trimethylation (H3K9me3) and recruitment of the heterochromatin protein 1 (HP1)-like protein HPL-2. b | In the germ line, small-RNA-directed transcriptional silencing is mediated not by NRDE-3 but by a different AGO protein, HRDE-1, which also acts through NRDE-2, H3K9 methylation and HPL-2. HRDE-1 receives 22G-RNA inputs both from the pathway that responds to exogenous dsRNA and from the PIWI-interacting RNA (piRNA), or 21U-RNA, pathway that scans the transcriptome for foreign RNAs. 21U-RNA-programmed PRG-1 promotes the RdRP-dependent generation of 22G-RNAs, which are loaded onto HRDE-1. In both cases, HRDE-1 maintains a persistent, transgenerational memory of silenced genes in the germ line. Meanwhile, another AGO protein called CSR-1 binds to 22G-RNAs that represent the full complement of endogenously expressed RNAs, and protects the corresponding loci from possible silencing by HRDE-1. Thus, the 22G-RNAs bound by CSR-1 and HRDE-1 transmit a germline memory of ‘self’ and ‘non-self’ RNAs, respectively, to be appropriately licensed for expression or silenced. HMTase, histone methyltransferase; Pol II, RNA polymerase II.

Figure 4. RNAs, both short and long, represent an alternative to DNA-binding proteins as specificity determinants for epigenetic regulation of gene expression

Enzymes (E) that catalyse methylation of histone tails or cytosine bases in DNA are recruited to chromatin by distinct mechanisms. a | Sequence-specific DNA-binding proteins recruit histone- or DNA-modifying enzymes to chromatin. b | Small RNAs target an Argonaute (AGO) or PIWI protein to a nascent transcript through base-pairing interactions to recruit modifying enzymes. c | Long RNAs act as scaffolds for RNA-binding proteins to recruit chromatin-modifying complexes. In all cases, the binding of the enzyme or the recruiting factors (for example, AGO–PIWI complexes in part b and RNA-binding protein complexes in part c) to chromatin may be enhanced by interactions with pre-existing modifications, which self-reinforce the epigenetic state. Pol II, RNA polymerase II.