Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association

The amino-terminal histone tails are subject to covalent post-translational modifications such as acetylation, methylation, and phosphorylation. In the histone code hypothesis, these exposed and unstructured histone tails are accessible to a repertoire of regulatory factors that specifically recognize the various modified histones, thereby generating altered chromatin structures that mediate specific biological responses. Here, we report that lysine (Lys) 79 of histone H3, which resides in the globular domain, is methylated in eukaryotic organisms. In the yeast Saccharomyces cerevisiae, Lys 79 of histone H3 is methylated by Dot1, a protein shown previously to play a role in telomeric silencing. Mutations of Lys 79 of histone H3 and mutations that abolish the catalytic activity of Dot1 impair telomeric silencing, suggesting that Dot1 mediates telomeric silencing largely through methylation of Lys 79. This defect in telomeric silencing might reflect an interaction between Sir proteins and Lys 79, because dot1 and Lys 79 mutations weaken the interaction of Sir2 and Sir3 with the telomeric region in vivo. Our results indicate that histone modifications in the core globular domain have important biological functions.     

Heat shock protein 90 mediates macrophage activation by Taxol and bacterial lipopolysaccharide

Taxol, a plant-derived antitumor agent, stabilizes microtubules. Taxol also elicits cell signals in a manner indistinguishable from bacterial lipopolysaccharide (LPS). LPS-like actions of Taxol are controlled by the lps gene and are independent of binding to the known Taxol target, beta-tubulin. Using biotin-labeled Taxol, avidin-agarose affinity chromatography, and peptide mass fingerprinting, we identified two Taxol targets from mouse macrophages and brain as heat shock proteins (Hsps) of the 70- and 90-kDa families. Geldanamycin, a specific inhibitor of the Hsp 90 family, blocked the nuclear translocation of NF-kappaB and expression of tumor necrosis factor in macrophages treated with Taxol or with LPS. Geldanamycin did not block microtubule bundling by Taxol or macrophage activation by tumor necrosis factor. Thus, Taxol binds Hsps, and Hsp 90 helps mediate the activation of macrophages by Taxol and by LPS.     

Antithrombin Chicago, amino acid substitution of arginine 393 to histidine

Antithrombin Chicago is a functionally inactive antithombin variant whose inheritance is associated with thrombotic disease. The variant antithrombin was isolated from plasma of the propositus by chromatography on heparin-Sepharose, followed by passage through thrombin-Sepharose to remove the normal antithrombin component that is present. A pool of fragments (“CNBr pool 4”) containing the reactive site region was prepared from the reduced and S-carboxymethylated variant by cleavage with cyanogen bromide followed by reverse-phase HPLC. Sequential treatment of CNBr pool 4 with trypsin and V8 protease produced peptides whose molecular masses were then determined by fast atom bombardment mass spectrometry. The variant protein digests were characterised by a reduction of a peptide of mass 1086, corresponding to the normal antithrombin sequence Ala382-Arg393. However, they contained a peptide of mass 1748, which arises when Arg393 is replaced by His in the sequence Ala382-Arg399. It is concluded that the functional and clinical abnormalities of antithrombin Chicago are all probably caused by a single amino acid substitution, Arg393 to His.