SPECIFIC ANTIBODIES REVEAL ORDERED AND CELL-CYCLE-RELATED USE OF HISTONE-H4 ACETYLATION SITES IN MAMMALIAN-CELLS
SPECIFIC ANTIBODIES REVEAL ORDERED AND CELL-CYCLE-RELATED USE OF HISTONE-H4 ACETYLATION SITES IN MAMMALIAN-CELLS
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DOI：
10.1111/j.1432-1033.1989.tb14530.x
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发表时间：
1989-01-15
期刊：
影响因子：
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通讯作者：
FELLOWS, G
中科院分区：
文献类型：
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作者：
TURNER, BM;FELLOWS, G
在兔中用对应于四乙酰化H4的18个N-末端残基的合成肽（即在位置5、8、12和16处具有乙酰赖氨酸）产生对乙酰化形式的组蛋白H4（H4）特异的抗体。通过抑制试验测定特异性，使用4种额外的肽，每种肽仅在单个位点乙酰化。使用的抗血清（R6）的乙酰化位点在赖氨酸-5的特异性，我们估计的比例的赖氨酸-5位点乙酰化的单，二和三乙酰化形式的H4从随机生长的人HL-60细胞。如果乙酰化是随机的（即25%、50%和75%）或如果位点使用遵循所有H4分子的设定顺序（即从0%跳到100%），则获得的值（分别为7%、29%和61%）与预期值不同。来自第二只动物（R5）的抗体优先与在Lys-12处乙酰化的肽结合，并且在几种细胞类型中也与单乙酰化的H4结合相对较弱。与此相反，单乙酰化的H4中期HeLa细胞标记更强烈的抗血清，表明在赖氨酸-5和赖氨酸-12显着的乙酰化。我们得出结论：（1）在来自各种哺乳动物细胞类型的单乙酰化H4中，Lys-5和Lys-12位点未得到充分利用，因此Lys-8和/或Lys-16首先被乙酰化，（2）可能有一个以上的位点使用顺序，（3）位点使用存在中期特异性偏移。这些结果表明H4乙酰化在哺乳动物细胞染色质结构的调节中发挥作用。
Antibodies specific for the acetylated forms of histone H4 (H4) were produced in rabbits with a synthetic peptide corresponding to the 18 N-terminal residues of tetra-acetylated H4 (i.e. with acetyllysine at positions 5, 8, 12 and 16). Specificity was determined by inhibition assays using four additional peptides, each acetylated at only a single site. Using an antiserum (R6) specific for the acetylation site at Lys-5 we have estimated the proportion of Lys-5 sites acetylated in the mono-, di- and tri-acetylated forms of H4 from randomly growing human HL-60 cells. The values obtained (7%, 29% and 61% respectively) differ from those expected if acetylation were random (i.e. 25%, 50% and 75%) or if site usage followed a set order for all H4 molecules (i.e. a jump from 0% to 100%). Antibodies from a second animal (R5) bound preferentially to peptides acetylated at Lys-12 and also bound to mono-acetylated H4 relatively weakly in several cell types. In contrast, monoacetylated H4 from metaphase HeLa cells labelled more strongly with both antisera, indicating significant acetylation at Lys-5 and Lys-12. We conclude that (1) the sites at Lys-5 and Lys-12 are under-used in mono-acetylated H4 from a variety of mammalian cell types and Lys-8 and/or Lys-16 are therefore the first to be acetylated, (2) more than one order of site usage is possible and (3) there is a metaphase-specific shift in site usage. These results suggest that H4 acetylation plays a role in the modulation of chromatin structure in mammalian cells.