RNA Interference (RNAi)-Dependent and RNAi-Independent Association of the Chp1 Chromodomain Protein with Distinct Heterochromatic Loci in Fission Yeast

Abstract

The establishment of centromeric heterochromatin in the fission yeast Schizosaccharomyces pombe is dependent on the RNA interference (RNAi) pathway. Dicer cleaves centromeric transcripts to produce short interfering RNAs (siRNAs) that actively recruit components of heterochromatin to centromeres. Both centromeric siRNAs and the heterochromatin component Chp1 are components of the RITS (RNA-induced initiation of transcriptional gene silencing) complex, and the association of RITS with centromeres is linked to Dicer activity. In turn, centromeric binding of RITS promotes Clr4-mediated methylation of histone H3 lysine 9 (K9), recruitment of Swi6, and formation of heterochromatin. Similar to centromeres, the mating type locus (Mat) is coated in K9-methylated histone H3 and is bound by Swi6. Here we report that Chp1 associates with the mating type locus and telomeres and that Chp1 localization to heterochromatin depends on its chromodomain and the C-terminal domain of the protein. Another protein component of the RITS complex, Tas3, also binds to Mat and telomeres. Tas3 interacts with Chp1 through the C-terminal domain of Chp1, and this interaction is necessary for Tas3 stability. Interestingly, in cells lacking the Argonaute (Ago1) protein component of the RITS complex, or lacking Dicer (and hence siRNAs), Chp1 and Tas3 can still bind to noncentromeric loci, although their association with centromeres is lost. Thus, Chp1 and Tas3 exist as an Ago1-independent subcomplex that associates with noncentromeric heterochromatin independently of the RNAi pathway.

ACKNOWLEDGMENTS

We thank Kathy Gould for the TAP tagging and pREP81 vectors, Jurg Bahler for the 13xmyc cassette, Robin Allshire for strains and the anti-Cnp1 antibody, and Keith Gull for the anti-TAT1 antibody. We gratefully acknowledge the support and advice of Paul Brindle, John Cleveland, Gerry Zambetti, Jim Ihle, and Evan Parganas and thank John Cleveland, Paul Brindle, Paul Mead, Katsumi Kitagawa, Pascal Bernard, and Jill Lahti for critical reading of the manuscript. J.P. also thanks Paul Perry for advice on setting up the microscopy system and Tom Lawrence and Wendy Bickmore for support and encouragement.

This work was supported in part by Cancer Center Support Grant Developmental funds 2 P30 CA 21765-25 and 2 P30 CA 21765-26 and the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital.