Kinetochores are proteinaceous assemblies that mediate the interaction of chromosomes with the mitotic spindle. The 180 kDa Ndc80 complex is a direct point of contact between kinetochores and microtubules. Its four subunits contain coiled coils and form an elongated rod structure with functional globular domains at either end. We crystallized an engineered "bonsai" Ndc80 complex containing a shortened rod domain but retaining the globular domains required for kinetochore localization and microtubule binding. The structure reveals a microtubule-binding interface containing a pair of tightly interacting calponin-homology (CH) domains with a previously unknown arrangement. The interaction with microtubules is cooperative and predominantly electrostatic. It involves positive charges in the CH domains and in the N-terminal tail of the Ndc80 subunit and negative charges in tubulin C-terminal tails and is regulated by the Aurora B kinase. We discuss our results with reference to current models of kinetochore-microtubule attachment and centromere organization.
Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription. Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation and transcriptional repression in a variety of species. H3K9me3 has long been regarded as a 'permanent' epigenetic mark. In a search for proteins and complexes interacting with H3K9me3, we identified the protein GASC1 (gene amplified in squamous cell carcinoma 1), which belongs to the JMJD2 (jumonji domain containing 2) subfamily of the jumonji family, and is also known as JMJD2C. Here we show that three members of this subfamily of proteins demethylate H3K9me3/me2 in vitro through a hydroxylation reaction requiring iron and alpha-ketoglutarate as cofactors. Furthermore, we demonstrate that ectopic expression of GASC1 or other JMJD2 members markedly decreases H3K9me3/me2 levels, increases H3K9me1 levels, delocalizes HP1 and reduces heterochromatin in vivo. Previously, GASC1 was found to be amplified in several cell lines derived from oesophageal squamous carcinomas, and in agreement with a contribution of GASC1 to tumour development, inhibition of GASC1 expression decreases cell proliferation. Thus, in addition to identifying GASC1 as a histone trimethyl demethylase, we suggest a model for how this enzyme might be involved in cancer development, and propose it as a target for anti-cancer therapy.
PERSPECTIVES were cultured in different glucose concentrations. The amplitude of circadian oscillations in gene expression correlated to glucose concentrations only in wild-type cells, but not in the absence of AMPK. In mouse liver, the accumulation and nuclear localization of AMPK, as well as the phosphorylation of known AMPK target proteins, oscillated in a circadian manner. Thus, perturbation of nutrient availability-and consequently, of AMPK activity-alters output of the circadian clock. Although AMPK is an attractive candidate for coupling metabolic and circadian cycles, additional regulators are likely involved. Thus, the ratio of oxidized nicotinamide ade-nine dinucleotide phosphate (NADP +) to its reduced form (NADPH)-which, like the AMP/ATP ratio, constitutes a diagnostic signature of a cell's metabolic state-has been proposed to affect circadian gene expression through diverse mechanisms. At least in vitro, the binding of the heterodimeric core clock transcription factors CLOCK-BMAL1 and NPAS2-BMAL1 to their cognate DNA sequences (so-called E-boxes) is enhanced by NADPH and impaired by NADP + (6). The transcriptional regulatory protein peroxisome proliferator-activated receptor γ (P PA R γ) coactivator 1α (PGC-1α), a well-known mediator of glucose and lipid metabolism, has been proposed to be another important player in connecting metabolism to circadian gene expression. This transcriptional coacti-vator associates with nuclear receptors of the ROR family and thereby modulates the transcription of the clock genes Bmal1 and Rev-erbα. Finally, the NAD +-dependent protein deacetylase sirtuin 1 infl uences the stability and activity of the core clock components PER2 and BMAL1, respectively (7, 8). Why are metabolic processes under tight circadian control? A simple explanation arises from the necessity to separate incompatible enzymatic processes within the same cell. Because complete spatial separation of anabolic and catabolic processes is frequently impossible, these have to be gated to different time windows. This necessity is well illustrated by the temporal sequestration of oxida-tive and reductive phases in yeast by an ultra-dian respiratory clock. For example, DNA is replicated exclusively in the reductive phase, when the concentration of genotoxic reactive oxygen species generated by mitochondrial respiration is minimal (9). In a yeast mutant in which the reductive phase is too short to allow for the completion of DNA synthesis, the mutation rate increases dramatically (10). In mammals, the master pacemaker in the SCN is phase-entrained primarily by light-dark cycles and thus cannot readily adapt to altered feeding rhythms. Hence, when food availability changes, nutrient-dependent synchronization cues must dominate the more direct signals from the SCN to maintain proper homeostasis of metabolism in peripheral tissues (1). This could explain the multitude of metabolic phase entrainment cues that synchronize the circadian core clock machinery in metabolically active peripheral organs. A major challen...
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