Siegel T Nicolai, Kawahara Taemi, Degrasse Jeffrey A, Janzen Christian J, Horn David, Cross George A M
Laboratory of Molecular Parasitology, The Rockefeller University, New York, USA.
Mol Microbiol. 2008 Feb;67(4):762-71. doi: 10.1111/j.1365-2958.2007.06079.x. Epub 2007 Dec 30.
Post-translational histone modifications have been studied intensively in several eukaryotes. It has been proposed that these modifications constitute a 'histone code' that specifies epigenetic information for transcription regulation. With a limited number of histone-modifying enzymes, implying less redundancy, Trypanosoma brucei represents an excellent system in which to investigate the function of individual histone modifications and histone-modifying enzymes. In this study, we characterized the acetylation of lysine 4 of histone H4 (H4K4), the most abundant acetylation site in T. brucei histones. Because of the large sequence divergence of T. brucei histones, we generated highly specific antibodies to acetylated and unmodified H4K4. Immunofluorescence microscopy and Western blots with sorted cells revealed a strong enrichment of unmodified H4K4 in S phase and suggested a G1/G0-specific masking of the site, owing to non-covalently binding factors. Finally, we showed that histone acetyltransferase 3 (HAT3) is responsible for H4K4 acetylation and that treatment of cells with the protein synthesis inhibitor cycloheximide led to an almost instantaneous loss of unmodified H4K4 sites. As HAT3 is located inside the nucleus, our findings suggest that newly synthesized histone H4 with an unmodified K4 is imported rapidly into the nucleus, where it is acetylated, possibly irreversibly.
翻译后组蛋白修饰已在几种真核生物中得到深入研究。有人提出，这些修饰构成了一种“组蛋白密码”，用于指定转录调控的表观遗传信息。由于组蛋白修饰酶数量有限，意味着冗余性较低，布氏锥虫是研究单个组蛋白修饰和组蛋白修饰酶功能的理想系统。在本研究中，我们对组蛋白H4赖氨酸4（H4K4）的乙酰化进行了表征，H4K4是布氏锥虫组蛋白中最丰富的乙酰化位点。由于布氏锥虫组蛋白的序列差异很大，我们制备了针对乙酰化和未修饰H4K4的高度特异性抗体。免疫荧光显微镜检查和对分选细胞的蛋白质免疫印迹显示，未修饰的H4K4在S期高度富集，并表明由于非共价结合因子，该位点在G1/G0期有特异性掩盖。最后，我们表明组蛋白乙酰转移酶3（HAT3）负责H4K4的乙酰化，并且用蛋白质合成抑制剂环己酰亚胺处理细胞会导致未修饰的H4K4位点几乎瞬间丧失。由于HAT3位于细胞核内，我们的研究结果表明，新合成的具有未修饰K4的组蛋白H4会迅速导入细胞核，在那里被乙酰化，可能是不可逆的。
