Studies on the Mechanism of RNAi-dependent Heterochromatin Assembly

Abstract

Assembly of heterochromatin at centromeric DNA regions in the fission yeast Schizosaccharomyces pombe involves an intimate interplay between chromatin modifying complexes and components of the RNAi pathway. The RNA-induced transcriptional silencing (RITS) complex, containing Chp1, Ago1, Tas3, and centromeric siRNAs, localizes to centromeric DNA repeats and is required for the assembly and maintenance of heterochromatin. RITS brings together two types of molecular recognition modules: a chromodomain protein, which binds to lysine 9 methylated histone H3 (H3K9), and Argonaute, which binds to specific sequences by siRNA-directed base-pairing interactions. The RNA-directed RNA polymerase complex (RDRC), composed of Rdp1, the Hrr1 helicase, and the Cid12 Poly(A) polymerase family member, synthesizes doublestranded RNA and creates the substrate for Dicer to generate siRNAs. RDRC physically associates with RITS, and both complexes localize to noncoding centromeric RNAs and centromeric DNA repeats, suggesting that recognition of nascent RNA transcripts may be involved in localization of these complexes to specific chromosome regions. In support of this possibility, tethering of the RITS complex to the transcript of the normally euchromatic ura4 + gene results in siRNA generation and RNAi-and heterochromatin-dependent silencing of the ura4 + gene. Finally, silencing of a subset of endogenous and transgene promoters within heterochromatic DNA domains occurs by RNAi-dependent degradation of nascent transcripts by a mechanism that we have termed co-transcriptional gene silencing (CTGS).

Dicer independent small RNAs associate with telomeric heterochromatin

“…Direct roles of small RNAs in the propagation of heterochromatin have been established in several eukaryotic systems, including Tetrahymena, fission yeast, and Drosophila (Moazed et al 2006;Zofall and Grewal 2006;Liu et al 2007). However, the case remains unresolved in mammalian cells, in large part due to the difficulty of detecting small RNAs homologous to heterochromatin regions, especially in somatic cells.…”

“…Extensive studies, mostly in fission yeast, demonstrate that small RNA-dependent processes are central for the formation of constitutive heterochromatin (Moazed et al 2006;Grewal and Elgin 2007). In this pathway, siRNAs generated by a Dicer homolog (Dcr1) associate with an Argonaut homolog (Ago1) and help to recruit histonemodifying activities to chromatin regions with homologous sequences.…”

Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

“…In S. pombe, siRNAs recruit the RITS complex to repeated DNAs to establish heterochromatin structure (Moazed et al, 2006;Noma et al, 2004), and siRNAs are produced from rDNA repeats (Cam et al, 2005). These demonstrations point to the intriguing probability that dcr-2 and the siRNA mechanism preferentially direct Su(var)3-9 to methylate H3K9 at rDNA, leaving the rasiRNA mechanism to regulate Su(var)3-9 methyltransferase activity in other repeated DNAs, e.g., 5S…”

“…and H3K9 methylation (Janicki et al, 2004), suggesting that a high density of repeats is sufficient for 'heterochromatinization.' This process is likely RNAimediated and directed by double-strand RNAs (dsRNAs) transcribed from these repeats (Grewal and Jia, 2007;Moazed et al, 2006).…”

“…RNAs transcribed from repeated DNAs form doublestranded RNAs (dsRNAs) that are processed by dicer to produce small interfering RNAs (siRNAs). These siRNAs recruit the RITS (RNA-induced transcriptional gene silencing) complex to heterochromatic regions, where the RITS complex recruits Su(var)3-9/clr4 (Su(var)3-9 from now on), which methylates H3K9 residues and establishes heterochromatin (Grewal and Jia, 2007;Moazed et al, 2006). Intriguingly, recruitment of the RITS complex to heterochromatin also requires Su(var)3-9 binding (Noma et al, 2004), demonstrating a cooperative relationship that is essential for both Su(var)3-9 and the RITS complex.…”