Non-random acetylation of histone H4 by a cytoplasmic histone acetyltransferase as determined by novel methodology.
Non-random acetylation of histone H4 by a cytoplasmic histone acetyltransferase as determined by novel methodology.
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发表时间：
1994-07
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通讯作者：
R. Sobel;R. G. Cook;C. Allis
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作者：
R. Sobel;R. G. Cook;C. Allis
在活跃的DNA复制和染色质组装期间，新合成的组蛋白H4以二乙酰化形式沉积。在四膜虫中，一对特定的残基，赖氨酸4和11，已经显示在体内经历这种修饰（Chicoine，L. G.，舒尔曼岛G.，里奇曼河，库克河G.，和Allis，C. D.（1986）J.Biol.Chem.261，1071 - 1076）。据推测，该反应至少部分由B型组蛋白乙酰转移酶（HAT）催化，所述细胞质酶显示对游离的、非染色质结合的H4的强烈偏好。为了研究哪些赖氨酸是来自其他生物体的H4中的优选乙酰化位点，从果蝇胚胎制备细胞质HAT B活性，并用于乙酰化来自几个物种的H4。当H4或合成，NH2-末端肽四膜虫被用作未封闭的基板，直接微序列分析表明，[3H]乙酸优先纳入赖氨酸11很少，如果有的话，在其他保守的，乙酰化赖氨酸纳入。果蝇H4在体外乙酰化后使用不使内部赖氨酸脱乙酰化的条件进行化学去封闭。直接序列分析验证了正确的NH2-末端序列的果蝇H4和证明，[3H]乙酸掺入优先发生在赖氨酸12，类似于赖氨酸11在四膜虫的残基。这些数据显示果蝇HAT B体外活性对赖氨酸11/12的显著偏好，并为以下断言提供支持：该活性的功能是乙酰化新的H4，至少部分用于体内沉积和染色质组装。由于大多数H4，如果蝇，被封锁在其氨基末端的乙酰苏氨酸或乙酰丝氨酸，我们的研究结果表明，这种去封闭和微测序策略可用于研究乙酰化位点利用H4和推测其他核心组蛋白NH2末端封闭与这些残基。
During periods of active DNA replication and chromatin assembly, newly synthesized histone H4 is deposited in a diacetylated form. In Tetrahymena, a specific pair of residues, lysines 4 and 11, has been shown to undergo this modification in vivo (Chicoine, L. G., Schulman, I. G., Richman, R., Cook, R. G., and Allis, C. D. (1986) J. Biol. Chem. 261, 1071-1076). Presumably, this reaction is catalyzed, at least in part, by histone acetyltransferases (HAT) of the B type, cytoplasmic enzymes displaying strong preference for free, non-chromatin-bound H4. To investigate which lysines are preferred acetylation sites in H4 from other organisms, a cytoplasmic HAT B activity was prepared from Drosophila embryos and used to acetylate H4 from several species. When H4 or synthetic, NH2-terminal peptides from Tetrahymena were used as unblocked substrates, direct microsequence analyses showed that [3H]acetate was preferentially incorporated at lysine 11 with little, if any, incorporation at other conserved, acetylatable lysines. Drosophila H4 was chemically deblocked following its acetylation in vitro using conditions that do not deacetylate internal lysines. Direct sequence analysis verified the correct NH2-terminal sequence of Drosophila H4 and demonstrated that [3H]acetate incorporation occurred preferentially on lysine 12, the residue analogous to lysine 11 in Tetrahymena. These data show remarkable preference for lysine 11/12 by the Drosophila HAT B activity in vitro and provide support for the assertion that this activity functions to acetylate new H4, at least in part, for deposition and chromatin assembly in vivo. Since most H4s, like Drosophila, are blocked at their amino termini by an acetylthreonine or acetylserine, our results demonstrate that this deblocking and microsequencing strategy can be used to study acetylation site utilization in H4 and presumably other core histones NH2 terminally blocked with these residues.