ABSTRACT
In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.
Subject(s)
Cell Cycle Proteins/metabolism , Centromere/metabolism , Heterochromatin/metabolism , Histones/metabolism , Schizosaccharomyces pombe Proteins/genetics , Amino Acid Sequence , Argonaute Proteins , Binding Sites , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/metabolism , Crystallography, X-Ray , Lysine/metabolism , Methylation , Models, Molecular , Molecular Sequence Data , Mutation , Protein Structure, Tertiary , RNA, Small Interfering/metabolism , RNA-Binding Proteins , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
The maintenance of centromeric heterochromatin in fission yeast relies on the RNA interference-dependent complexes RITS (RNA-induced transcriptional silencing complex) and RDRC (RNA-directed RNA polymerase complex), which cooperate in a positive feedback loop to recruit high levels of histone H3 K9 methyltransferase activity to centromeres and to promote the assembly and maintenance of centromeric heterochromatin. However, it is unclear how these complexes are targeted to chromatin. RITS comprises Chp1, which binds K9-methylated histone H3; Ago1, which binds short interfering (siRNAs); the adaptor protein Tas3, which links Ago1 to Chp1; and centromeric siRNAs. We have generated mutants in RITS to determine the contribution of the two potential chromatin-targeting proteins Chp1 and Ago1 to the centromeric recruitment of RITS. Mutations in Tas3 that disrupt Ago1 binding are permissive for RITS recruitment and maintain centromeric heterochromatin, but the role of Tas3's interaction with Chp1 is unknown. Here, we define the Chp1 interaction domain of Tas3. A strain expressing a tas3 mutant that cannot bind Chp1 (Tas3(Delta)(10-24)) failed to maintain centromeric heterochromatin, with a loss of centromeric siRNAs, a failure to recruit RITS and RDRC to centromeres, and high levels of chromosome loss. These findings suggest a pivotal role for Chp1 and its association with Tas3 for the recruitment of RITS, RDRC, and histone H3 K9 methyltransferase activity to centromeres.
Subject(s)
Carrier Proteins/physiology , Cell Cycle Proteins/physiology , Centromere/ultrastructure , Chromosomes, Fungal/ultrastructure , Heterochromatin/ultrastructure , Schizosaccharomyces pombe Proteins/physiology , Schizosaccharomyces/metabolism , Argonaute Proteins , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Genes, Mating Type, Fungal/genetics , Genomic Instability , Heterochromatin/metabolism , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/metabolism , Multiprotein Complexes/metabolism , Protein Interaction Mapping , Protein Methyltransferases , Protein Structure, Tertiary , Protein Transport , RNA, Fungal/metabolism , RNA, Small Interfering/metabolism , RNA-Binding Proteins , Schizosaccharomyces/genetics , Schizosaccharomyces/ultrastructure , Schizosaccharomyces pombe Proteins/genetics , Telomere/ultrastructure , Transcription, GeneticABSTRACT
The establishment and maintenance of centromeric heterochromatin in fission yeast require the RITS complex. Comprised of centromeric siRNAs, the chromodomain protein Chp1, Argonaute (Ago1), and Tas3, RITS couples the cellular RNAi pathway with assembly of constitutive heterochromatin. However, the mechanisms governing RITS-dependent establishment versus maintenance of centromeric heterochromatin remain unresolved. Here, we report that a mutant Tas3 protein that cannot bind Ago1 supports the maintenance of centromeric heterochromatin but cannot mediate efficient de novo establishment from cells transiently depleted for the histone H3 lysine 9 methyltransferase Clr4. In contrast, centromeric heterochromatin efficiently assembles in mutant cells transiently depleted for dicer. This mutant therefore allows ordering of the events leading to establishment of centromeric heterochromatin and places lysine 9 methylation of histone H3 upstream of dicer function.
