RESUMO
Pericentromeric heterochromatin formation is mediated by repressive histone H3 lysine 9 methylation (H3K9Me) and its recognition by HP1 proteins. Intriguingly, in many organisms, RNAi is coupled to this process through poorly understood mechanisms. In Schizosaccharomyces pombe, the H3-K9 methyltransferase Clr4 and the heterochromatin protein 1 (HP1) ortholog Swi6 are critical for RNAi, whereas RNAi stimulates H3K9Me. In addition to the endoribonuclease Dcr1, RNAi in S. pombe requires two interacting protein complexes, the RITS complex, which contains an Argonaute subunit, and the RDRC complex, which contains an RNA-dependent RNA polymerase subunit. We previously identified Ers1 (essential for RNAi-dependent silencing) as an orphan protein that genetically acts in the RNAi pathway. Using recombinant proteins, we show here that Ers1 directly and specifically interacts with HP1/Swi6. Two-hybrid assays indicate that Ers1 also directly interacts with several RNAi factors. Consistent with these interactions, Ers1 associates in vivo with the RITS complex, the RDRC complex, and Dcr1, and it promotes interactions between these factors. Ers1, like Swi6, is also required for RNAi complexes to associate with pericentromeric noncoding RNAs. Overexpression of Ers1 results in a dominant-negative phenotype that can be specifically suppressed by increasing levels of the RDRC subunit Hrr1 or of Dcr1, further supporting a functional role for Ers1 in promoting the assembly of the RNAi machinery. Through the interactions described here, Ers1 may promote RNAi by tethering the corresponding enzyme complexes to HP1-coated chromatin, thereby placing them in proximity to the nascent noncoding RNA substrate.
