Structural analyses of the chromatin remodelling enzymes INO80-C and SWR-C.

INO80-C and SWR-C are conserved members of a subfamily of ATP-dependent chromatin remodelling enzymes that function in transcription and genome-maintenance pathways. A crucial role for these enzymes is to control chromosomal distribution of the H2A.Z histone variant. Here we use electron microscopy (EM) and two-dimensional class averaging to demonstrate that these remodelling enzymes have similar overall architectures. Each enzyme is characterized by a dynamic 'tail' domain and a compact 'head' that contains Rvb1/Rvb2 subunits organized as hexameric rings. EM class averages and mass spectrometry support the existence of single heterohexameric rings in both SWR-C and INO80-C. EM studies define the position of the Arp8/Arp4/Act1 module within INO80-C, and we find that this module enhances nucleosome-binding affinity but is largely dispensable for remodelling activities. In contrast, the Ies6/Arp5 module is essential for INO80-C remodelling, and furthermore this module controls conformational changes that may couple nucleosome binding to remodelling.

Budding yeast Pch2, a widely conserved meiotic protein, is involved in the initiation of meiotic recombination.

Budding yeast Pch2 protein is a widely conserved meiosis-specific protein whose role is implicated in the control of formation and displacement of meiotic crossover events. In contrast to previous studies where the function of Pch2 was implicated in the steps after meiotic double-strand breaks (DSBs) are formed, we present evidence that Pch2 is involved in meiotic DSB formation, the initiation step of meiotic recombination. The reduction of DSB formation caused by the pch2 mutation is most prominent in the sae2 mutant background, whereas the impact remains mild in the rad51 dmc1 double mutant background. The DSB reduction is further pronounced when pch2 is combined with a hypomorphic allele of SPO11. Interestingly, the level of DSB reduction is highly variable between chromosomes, with minimal impact on small chromosomes VI and III. We propose a model in which Pch2 ensures efficient formation of meiotic DSBs which is necessary for igniting the subsequent meiotic checkpoint responses that lead to proper differentiation of meiotic recombinants.

HP1 proteins form distinct complexes and mediate heterochromatic gene silencing by nonoverlapping mechanisms.

HP1 proteins are a highly conserved family of eukaryotic proteins that bind to methylated histone H3 lysine 9 (H3K9) and are required for heterochromatic gene silencing. In fission yeast, two HP1 homologs, Swi6 and Chp2, function in heterochromatic gene silencing, but their relative contribution to silencing remains unknown. Here we show that Swi6 and Chp2 exist in nonoverlapping complexes and make distinct contributions to silencing. Chp2 associates with the SHREC histone deacetylase complex (SHREC2), is required for histone H3 lysine 14 (H3K14) deacetylation, and mediates transcriptional repression by limiting RNA polymerase II access to heterochromatin. In contrast, Swi6 associates with a different set of nuclear proteins and with noncoding centromeric transcripts and is required for efficient RNAi-dependent processing of these transcripts. Our findings reveal an unexpected role for Swi6 in RNAi-mediated gene silencing and suggest that different HP1 proteins ensure full heterochromatic gene silencing through largely nonoverlapping inhibitory mechanisms.

A cullin E3 ubiquitin ligase complex associates with Rik1 and the Clr4 histone H3-K9 methyltransferase and is required for RNAi-mediated heterochromatin formation.

The assembly of heterochromatin in fission yeast and metazoans requires histone H3-lysine 9 (-K9) methylation by the conserved Clr4/Suv39h methyltransferase. In fission yeast, H3-K9 methylation requires components of the RNAi machinery and is initiated by the RNA-Induced Transcriptional Silencing (RITS) complex. Here we report the purification of a novel complex that associates with the Clr4 methyltransferase, termed the CLRC (CLr4-Rik1-Cul4) complex. By affinity purification of the Clr4-associated protein Rik1, we show that, in addition to Clr4, Rik1 is associated with the fission yeast E3 ubiquitin ligase Cullin4 (Cul4, encoded by cul4(+)), the ubiquitin-like protein, Ned8, and two previously uncharacterized proteins, designated Cmc1 and Cmc2. In addition, the complex contains substochiometric amounts of histones H2B and H4, and the 14-3-3 protein, Rad24. Deletion of cul4(+), cmc1(+), cmc2(+) and rad24(+) results in a complete loss of silencing of a ura4(+) reporter gene inserted within centromeric DNA repeats or the silent mating type locus. Each of the above deletions also results in accumulation of noncoding RNAs transcribed from centromeric repeats and telomeric DNA regions, and a corresponding loss of small RNAs that are homologous to centromeric repeats, suggesting a defect in the processing of noncoding RNA to small RNA. Based on these results, we propose that the components of the Clr4-Rik1-Cul4 complex act concertedly at an early step in heterochromatin formation.

A role for Ddc1 in signaling meiotic double-strand breaks at the pachytene checkpoint.

The pachytene checkpoint prevents meiotic cell cycle progression in response to unrepaired recombination intermediates. We show that Ddc1 is required for the pachytene checkpoint in Saccharomyces cerevisiae. During meiotic prophase, Ddc1 localizes to chromosomes and becomes phosphorylated; these events depend on the formation and processing of double-strand breaks (DSBs). Ddc1 colocalizes with Rad51, a DSB-repair protein, indicating that Ddc1 associates with sites of DSB repair. The Rad24 checkpoint protein interacts with Ddc1 and with recombination proteins (Sae1, Sae2, Rad57, and Msh5) in the two-hybrid protein system, suggesting that Rad24 also functions at DSB sites. Ddc1 phosphorylation and localization depend on Rad24 and Mec3, consistent with the hypothesis that Rad24 loads the Ddc1/Mec3/Rad17 complex onto chromosomes. Phosphorylation of Ddc1 depends on the meiosis-specific kinase Mek1. In turn, Ddc1 promotes the stable association of Mek1 with chromosomes and is required for Mek1-dependent phosphorylation of the meiotic chromosomal protein Red1. Ddc1 therefore appears to operate in a positive feedback loop that promotes Mek1 function.