RNA interference and heterochromatin assembly

Abstract

In most eukaryotes, histone and DNA modifications are responsible for the silencing of genes integrated in heterochromatic sequences, as well as the silencing of pericentromeric repeats and transposable elements themselves. But the mechanisms that guide these modifications to heterochromatin during the cell cycle have been elusive. RNA interference takes advantage of heterochromatic transcription to process small RNAs and recruit enzymes required for both histone and DNA modifications, and is one such mechanism that has been identified. The processes are best understood in fission yeast and plants, but recent work in mammalian cells, especially in the germline, suggests these mechanisms may be highly conserved.

Figure 1.

Position effect variegation in Drosophila and fission yeast. In Drosophila, the white gene w[m4h] is silenced by heterochromatin, causing white sectors on an otherwise red eye. Silencing is enhanced in males. In fission yeast, loss of ade6 silencing when integrated near heterochromatic sequences gives rise to white sectors on red colonies.

Figure 2.

Centromeric heterochromatin in the fission yeast S. pombe. The three centromeres of S. pombe each include a central region (green rectangles) flanked by large inverted innermost repeats (red rectangles). These are flanked by tandem copies of outermost elements (blue arrows, orange arrows, white rectangles and grey rectangles) that are composed of dg and dh repeats. Centromeres also contain clusters of tRNA genes (yellow rectangles) at the boundaries between heterochromatin and euchromatin. Reporter genes integrated into centromere 1 (ade6 or ura4) are silenced by RNAi-mediated heterochromatic silencing.

Figure 3.

Model for heterochromatin assembly and spreading at S. pombe centromeric outer repeats. Heterochromatic centromere sequences (yellow arrow) are transcribed by RNA Polymerase II. These centromere transcripts are targeted by RITS via siRNA loaded Ago1. Association of RITS with centromere heterochromatin is strengthened by binding of Chp1 to H3mK9. RITS activity can recruit both CLRC, via interactions with Stc1, and RDRC resulting in spreading of H3mK9 and amplification of siRNAs, respectively (see text for details). dsRNA generated either by bi-directional transcription from centromere promoters (black arrows) or by RDRC activity is recognized and processed by Dicer (Dcr1). The resulting centromere siRNAs are then loaded onto Ago1 first in the ARC complex and then in RITS.

Figure 4.

Cell cycle regulation of heterochromatin transcription and assembly. Pericentromeric heterochromatin is silent through most of the cell cycle (G2) but is transcriptionally activated when Swi6 (the HP-1 homolog) is evicted from methylated histone H3 lysine-9 by phosphorylation of histone H3 serine-10. Transcription continues during G1 and S phase, when transcripts are processed by RNAi and converted into siRNA. Replication during early S phase replaces approximately half of the parental nucleosomes with freshly assembled nucleosomes that lack H3K9me2. The RITS complex promotes H3K9 methylation and H3K4 demethylation, by the CLRC complex, which recruits Swi6 and silences heterochromatin in the subsequent G2. Thus transient expression and siRNA production during S phase promotes epigenetic inheritance of heterochromatic modifications.