The RNA interference (RNAi) pathway has been implicated in heterochomatin assembly and gene silencing in fission yeast. And now, Grewal, Moazed and colleagues have identified a complex — RITS (RNA-induced initiation of transcriptional gene silencing) — that provides a mechanistic basis for RNAi-mediated targeting of heterochromatin.

Reporting in Science, the authors tested whether Chp1 — a heterochromatin-associated protein that binds centromeric repeats and is required for the heterochromatin-specific methylation of histone H3 at lysine 9 (H3-K9) and the recruitment of the heterochromatin-binding protein Swi6 — might provide the link between RNAi and heterochromatin. They affinity-purified extracts from a yeast strain that produces tagged Chp1 and, in addition to Chp1, identified Ago1, an Argonaute family protein, and an unknown protein, which they named Tas3 (targeting complex subunit 3). The purification profile was identical when tagged Tas3 was used instead, which confirmed that Chp1, Tas3 and Ago1 form a complex — RITS.

Chp1 and Ago1 are required for heterochromatin assembly and gene silencing within heterochromatic regions. Grewal, Moazed and co-workers wanted to know whether Tas3 has similar properties, which it indeed has, as a tas3 deletion strain carrying a reporter gene that was inserted in centromeric repeats showed loss of gene silencing. And, chromatin immunoprecipitation (ChIP) studies showed that, in tas3Δ cells, H3-K9 methylation and recruitment of Swi6 to the reporter gene were abolished.

Next, the authors found that the RITS complex was associated with small RNA molecules of 22–25 nucleotides. However, these small RNA species were absent from RITS that was purified from a strain lacking Dcr1, the fission yeast homologue of the Dicer enzyme, which generates small interfering RNA (siRNA). So, the RITS-associated small RNAs require Dcr1 for their production. Moreover, northern- and Southern-blot analyses established that the siRNAs that are associated with RITS originate from centromeric repeat sequences — providing RITS with target specificity.

To determine the in vivo chromatin localization of RITS, the authors carried out ChIP experiments, which showed that tagged Tas3 localizes to centromeric repeats in wild-type cells, but not in cells that lacked factors involved in the RNAi pathway (Ago1, Dcr1 or RNA-dependent RNA polymerase). This indicates the direct requirement of the RNAi pathway for the targeting of heterochomatin by RITS. The authors propose that RITS uses Dcr1-produced siRNAs to recognize and bind specific chromosome regions to initiate heterochromatin assembly and gene silencing.