The RITS complex plays a central role in the formation of heterochromatin.It acts as a switch that integrates the sequence specificity of the RNAi effector protein Argonaute with sensing the H3K9 methyl marks on chromatin through its chromodomain protein Chp113
. Once recruited to a specific location, it serves as a platform for binding of ClrC and RDRP, which lead to robust heterochromatin formation and gene silencing. The structure of Chp1 bound to Tas3 presented here shows that these two RITS subunits interact tightly to form a solid bridge between chromatin substrate and the RNAi machinery. This structure together with the structures of the Chp1 chromodomain13
- the attachment point to chromatin, and the α-helical motif on the C terminus of Tas320
- a multimerization module, most likely represent most, if not all, of the structured domains of the Chp1-Tas3 complex. Long, unstructured regions connect these domains, allowing the Chp1-Tas3 backbone to span great distances between attachment sites, thereby possibly crosslinking neighboring strands of chromatin fibers. Combining all the available information suggests cooperation between Ago1 and the Chp1-Tas3 complex in spreading on chromatin, thereby setting up a robust, polymeric platform that supports the enforcement of heterochromatin ().
Context-specific functions of RITS subunits at centromeres and subtelomeres
In addition to the role of the RITS complex in both RNAi and Clr4-mediated repression of transcription, the identification of a PIN domain in Chp1 provides important clues for the unique function of the Chp1-Tas3 complex in RNAi-independent silencing of telomeric transcripts. Although the PIN domain of Chp1 itself does not appear to possess endonuclease activity, the elevation of transcripts without change in Pol II occupancy in Chp1 mutants is clear evidence for an important role of Chp1 and Tas3 in post-transcriptional gene silencing (PTGS) at the subtelomere, much of it mediated by the PIN domain. The PIN domain may be involved in recruiting nuclease activities to subtelomeric regions, much like the PIN domain of the exosome subunit Rrp44/Dis3 was demonstrated to tether Rrp44 to the rest of the core exosome30,39
. In addition, it has been previously reported that subtelomeric tlh
transcripts are regulated by the S. pombe
TRAMP complex and the exosome6
. It will be interesting to determine whether the PIN domain of Chp1 is similarly involved in recruitment of the exosome to subtelomeric regions for processing of tlh
transcripts. Since mutation of the basic region at the neck of the PIN domain yields the same loss of subtelomeric transcript regulation, it is possible that the basic region is mediating protein-protein interactions. Or it might be involved in nucleic acid binding such as binding of the tlh
transcripts themselves, thus presenting substrates to an RNA degradation pathway, the exosome, for example, that is recruited through the PIN domain. Given that Chp1 localizes to all sites of heterochromatin, and that the exosome is also involved in regulation of centromeric transcripts6
, the question still remains as to why Chp1's PIN domain plays no role in the regulation of centromeric transcripts. One possibility is that Ago1 and the RNAi pathway dominate PTGS at the centromere, thereby masking the activity of the PIN domain. The picture thus emerging suggests that the various modules of the RITS complex play context-specific roles (). It would be of great interest to elucidate how the various functions of RITS are specifically regulated.
It is curious that fungal EST1 proteins, in contrast to their metazoan counterparts, do not feature a PIN domain. Since we present evidence for a subtelomeric silencing role of Chp1's PIN domain, there exists a possibility that Chp1 provides a functionality that has been incorporated into the telomeric EST1 proteins in higher organisms. It is intriguing that proteins bearing both a chromodomain and a PIN domain are conserved only within fungi, and that tlh
genes (as their name implies) are restricted to subtelomeric domains in fungi43
. Future experiments will address whether the physical link between RNAi and heterochromatin observed in the Chp1-Tas3 structure, as well as the RNAi-independent role of Chp1's PIN domain in processing tlh
transcripts is conserved amongst organisms of the fungal kingdom.