The Aurora kinase Ipl1p plays a crucial role in regulating kinetochore-microtubule attachments in budding yeast, but the underlying basis for this regulation is not known. To identify Ipl1p targets, we first purified 28 kinetochore proteins from yeast protein extracts. These studies identified five previously uncharacterized kinetochore proteins and defined two additional kinetochore subcomplexes. We then used mass spectrometry to identify 18 phosphorylation sites in 7 of these 28 proteins. Ten of these phosphorylation sites are targeted directly by Ipl1p, allowing us to identify a consensus phosphorylation site for an Aurora kinase. Our systematic mutational analysis of the Ipl1p phosphorylation sites demonstrated that the essential microtubule binding protein Dam1p is a key Ipl1p target for regulating kinetochore-microtubule attachments in vivo.

The plasticity of aging suggests that longevity may be controlled epigenetically by specific alterations in chromatin state. The link between chromatin and aging has mostly focused on histone deacetylation by the Sir2 family1,2, but less is known about the role of other histone modifications in longevity. Histone methylation plays a crucial role during development and in maintaining stem cell pluripotency in mammals3. Regulators of histone methylation have been associated with aging in worms4,5,6,7 and flies8, but characterization of their role and mechanism of action has been limited. Here we identify the ASH-2 trithorax complex9, which trimethylates histone H3 at lysine 4 (H3K4), as a regulator of lifespan in C. elegans in a directed RNAi screen in fertile worms. Deficiencies in members of the ASH-2 complex–ASH-2 itself, WDR-5, and the H3K4 methyltransferase SET-2 extend worm lifespan. Conversely, the H3K4 demethylase RBR-2 is required for normal lifespan, consistent with the idea that an excess of H3K4 trimethylation–a mark associated with active chromatin–is detrimental for longevity. Lifespan extension induced by ASH-2 complex deficiency requires the presence of an intact adult germline and the continuous production of mature eggs. ASH-2 and RBR-2 act in the germline, at least in part, to regulate lifespan and to control a set of genes involved in lifespan determination. These results suggest that the longevity of the soma is regulated by an H3K4 methyltransferase/demethylase complex acting in the C. elegans germline.

The orderly deposition of histones onto DNA is mediated by conserved assembly complexes, including chromatin assembly factor-1 (CAF-1) and the Hir proteins . CAF-1 and the Hir proteins operate in distinct but functionally overlapping histone deposition pathways in vivo . The Hir proteins and CAF-1 share a common partner, the highly conserved histone H3/H4 binding protein Asf1, which binds the middle subunit of CAF-1 as well as to Hir proteins . Asf1 binds to newly synthesized histones H3/H4 , and this complex stimulates histone deposition by CAF-1 . In yeast, Asf1 is required for the contribution of the Hir proteins to gene silencing . Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 comprise the HIR complex, which copurifies with the histone deposition protein Asf1. Together, the HIR complex and Asf1 deposit histones onto DNA in a replication-independent manner. Histone deposition by the HIR complex and Asf1 is impaired by a mutation in Asf1 that inhibits HIR binding. These data indicate that the HIR complex and Asf1 proteins function together as a conserved eukaryotic pathway for histone replacement throughout the cell cycle.

A B S T R A C T PurposeWith three available chemotherapy drugs for advanced colorectal cancer (CRC), response rate (RR) and survival outcomes have improved with associated morbidity, accentuating the need for tools to select optimal individualized treatment. Pharmacogenetics identifies the likelihood of adverse events or response based on variants in genes involved in drug transport, metabolism, and cellular targets. Patients and MethodsGermline DNA was extracted from 520 patients on the North American Gastrointestinal Intergroup N9741 study. Three study arms were evaluated:, and IROX (IRN ϩ oxaliplatin). Information on adverse events, response, and disease-free survival was available. Thirty-four variants in 15 candidate genes for analysis based on previous associations with adverse events or outcome were assessed. Genotyping was performed using pyrosequencing. ResultsAll variants were polymorphic. The homozygous UGT1A1*28 allele observed in 9% of patients was associated with risk of grade 4 neutropenia in patients on IROX (55% v 15%; P ϭ .002). Deletion in GSTM1 was associated with grade 4 neutropenia after FOLFOX (28% v 16%; P ϭ .02). Patients with a homozygous variant genotype for GSTP1 were more likely to discontinue FOLFOX because of neurotoxicity (24% v 10%; P ϭ .01). The presence of a CYP3A5 variant was significantly associated with RR on IFL (29% v 60%; P ϭ .0074). Most previously published genotype-toxicity or -efficacy relationships were not validated in this study. ConclusionThis study provides a platform to evaluate pharmacogenetic predictors of response or severe adverse events in advanced CRC. Pharmacogenetic studies can be conducted in multicenter trials, and our findings demonstrate that with continued research, clinical application is practical.