RNAi-dependent formation of heterochromatin and its diverse functions

Abstract

Expression profiling of eukaryotic genomes has revealed widespread transcription outside the confines of protein-coding genes, leading to production of antisense and non-coding RNAs (ncRNAs). Studies in Schizosaccharomyces pombe and multicellular organisms suggest that transcription and ncRNAs provide a framework for the assembly of heterochromatin, which has been linked to various chromosomal processes. In addition to gene regulation, heterochromatin is crucial for centromere function, cell fate determination as well as transcriptional and posttranscriptional silencing of repetitive DNA elements. Recently, heterochromatin factors have been shown to suppress antisense RNAs at euchromatic loci. These findings define conserved pathways that probably have major impact on the epigenetic regulation of eukaryotic genomes.

Figure 1. Heterochromatin-mediated transcriptional silencing and degradation of repeat RNAs

RNAi factors RITS, Rdp1-containing RNA-dependent RNA polymerase complex (RDRC) and Dicer (Dcr1) that process centromeric transcripts into siRNAs are required for heterochromatin assembly. Heightened Pol II transcription of repeats during S-phase stimulates recruitment of heterochromatin factors including Clr4 complex subunit Rik1 and RITS subunit Ago1, as well as H3K36 methylation (blue lollipop) by Set2 implicated in heterochromatic silencing and recruitment of HDAC Clr6 [28,31]. siRNA-bound Ago1 is believed to specify RITS targeting to nascent repeat transcripts. siRNAs also facilitate recruitment of Clr4 to methylate H3K9. H3K9me (red lollipop) recruits HP1 proteins (Chp2 and Swi6) but also establishes feedback loop by stabilizing chromatin association of RITS (via Chp1 chromodomain) and Clr4 (via Clr4 chromodomain). Chromatin-bound RITS, RDRC and Dicer process heterochromatic transcripts into siRNAs to mediate posttranscriptional silencing in cis (cis-PTGS). Clr4 bound to H3K9me could modify adjacent nucleosomes, creating additional binding sites for chromodomain protein. This process, along with oligomerization of Swi6, is believed to facilitate heterochromatin spreading. Chp2 and Swi6 provide platform for targeting HDAC transcriptional gene silencing (TGS) factors SHREC and Clr6. Swi6 also recruits an anti-silencing factor Epe1 (see text for details).

Figure 2. Targeting a “toolkit” of repressors to different chromosomal addresses

(A) Heterochromatin-dependent and –independent targeting of silencing activities to repeat loci. Chp2 and Swi6 recruit HDACs across domains containing dg and dh repeats targeted by RNAi. However, RNAi and heterochromatin are dispensable for silencing Tf2 retrotransposons and their remnants (such as LTRs). CENP-Bs target SHREC and Clr6 to silence full-length Tf2 elements and solo LTRs often associated with gene promoters. (B) Redundancy in silencing mechanisms at the mating-type locus. A 20-kb heterochromatic domain containing donor mating-type cassettes mat2 and mat3 is flanked by boundary elements that prevent heterochromatin spreading. cenH sharing homology to dg and dh repeats serves as an RNAi-dependent heterochromatin nucleation center. In addition to HP1 proteins targeting HDACs across heterochromatic domain, silencer elements (REII, REIII and CENP-B silencer) recruit SHREC and Clr6 via DNA binding proteins (such as CENP-Bs Abp1 and Cbh1, and ATF/CREB family proteins Atf1 and Pcr1) bound to these sites.