RNA and epigenetic silencing: insight from fission yeast

Abstract

Post-translational modifications of histones are critical not only for local regulation of gene expression, but also for higher-order structure of the chromosome and genome organization in general. These modifications enable a preset state to be maintained over subsequent generations and thus provide an epigenetic level of regulation. Heterochromatic regions of the genome are epigenetically regulated to maintain a "silent state" and protein coding genes inserted into these regions are subject to the same epigenetic silencing. The fission yeast Schizosaccharomyces pombe has well characterized regions of heterochromatin and has proven to be a powerful model for elucidation of epigenetic silencing mechanisms. Research in S. pombe led to the breakthrough discovery that epigenetic silencing is not solely a chromatin-driven transcriptional repression and that RNA interference of nascent transcripts can guide epigenetic silencing and associated histone modifications. Over the last 10 years, an eloquent integration of genetic and biochemical studies have greatly propelled our understanding of major players and effector complexes for regulation of RNAi-mediated epigenetic silencing in S. pombe. Here, we review recent research related to regulation of the epigenetic state in S. pombe heterochromatin, focusing specifically on the mechanisms by which transcription and RNA processing interact with the chromatin modification machinery to maintain the epigenetically silent state.

Fig. 1

Heterochromatin and epigenetically silent regions of the genome. (A) Active protein coding genes are generally contained within euchromatin, typically modified by acetylation (Ac) of histone H3 lysine 9 (H3K9) and methylation (Me) of histone H3 lysine 4 (H3K4). In contrast, heterochromatin is generally considered to have a compact structure and can be observed as densely stained regions in nuclei. Heterochromatin is characterized by H3K9 methylation, which is recognized by Heterochromatin Protein 1 (HP1). The upper panel shows one complete 4′6′-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nucleus (center) of a mouse NIH3T3 cell. (B) Schizosaccharomyces pombe chromosomes and heterochromatin. The S. pombe genome is organized across three chromosomes, with heterochromatin found at the telomeres (TEL), centromeres (CEN) and mating type region (MAT). Centromeric regions (lower panel) are arranged with a unique core centromere region (cnt and imr) flanked by heterochromatin covering the pericentromeric outer repeat regions (otr). The otr contains multiple copies of dg and dh repeat sequences that vary in number depending on the chromosome. The upper left panel shows S. pombe cells stained with Hoechst33342.

Fig. 2

Proteins and effector complexes linked to RNAi-mediated epigenetic silencing in Schizosaccharomyces pombe. Complexes containing known RNAi components are shaded in yellow, whereas complexes interacting directly with chromatin are shaded in pink. Heterochromatin Protein 1 (HP1) proteins are also a key component of heterochromatin structure and are included for consistency.

Fig. 3

Schematic model of the self-enforcing loop for RNAi-mediated epigenetic silencing in Schizosaccharomyces pombe. Heterochromatin is shown as DNA (black line) wrapped around nucleosomes (blue circles) modified by histone H3 lysine 9 methylation (H3K9me; red circles “m”). Nascent transcripts generated by RNA polymerase II are targeted by an activated RNAi-induced transcriptional silencing (RITS) complex that also interacts with H3K9me through the Chp1 subunit. RDRC is then recruited for synthesis of dsRNA from the transcripts, which is cleaved into duplex siRNAs by Dcr1 and bound by Argonaute siRNA chaperone (ARC). These siRNAs are then processed into single-stranded siRNAs and loaded back into RITS completing the loop. Catalytically active RITS recruits the CLR4 complex (CLRC) for spreading of epigenetic silencing into adjacent regions through interaction with Stc1, although CLRC can also associate with these regions independent of RNAi and recruit inactive RITS.