K8 and K12 are biotinylated in human histone H4
K8 and K12 are biotinylated in human histone H4
复制标题
DOI：
10.1111/j.1432-1033.2004.04167.x
复制
发表时间：
2004-06-01
期刊：
影响因子：
--
通讯作者：
Zempleni, J
中科院分区：
文献类型：
--
作者：
Camporeale, G;Shubert, EE;Zempleni, J
DNA折叠成染色质是通过与组蛋白（例如H4）结合来介导的; DNA与组蛋白的结合是通过共价组蛋白修饰来调节的。阳离子，e. G.乙酰化、甲基化和生物素化。我们试图确定组蛋白H4中生物素化的氨基酸残基，并确定组蛋白的乙酰化和甲基化是否影响生物素化。使用生物素酶将跨越人组蛋白H4片段的合成肽酶促生物素化。用链霉亲和素-过氧化物酶探测肽结合的生物素。基于组蛋白H4的N-末端序列的肽被生物素酶有效地识别为生物素化的底物;相反，基于C-末端序列的肽不被生物素化。用丙氨酸或精氨酸取代K8或K12降低了生物素化，表明这些赖氨酸是生物素化的靶标; K8和K12也是已知的乙酰化靶标。化学乙酰化或甲基化的一个给定的赖氨酸减少随后的酶生物素化邻近的赖氨酸，与组蛋白modi之间的串扰一致。阳离子。用谷氨酸（负电荷）取代给定的赖氨酸（正电荷）消除了邻近赖氨酸的生物素化，提供了组蛋白的净电荷在生物素化中起作用的证据。产生特异性识别在K12处生物素化的组蛋白H4的抗体。该抗体用于检测人细胞核提取物中的生物素化组蛋白H4。这些研究表明，组蛋白H4中的K8和K12是生物素化的靶点，乙酰化和生物素化竞争相同的结合位点，组蛋白的乙酰化和甲基化影响邻近赖氨酸的生物素化。
Folding of DNA into chromatin is mediated by binding to histones such as H4; association of DNA with histones is regulated by covalent histone modi. cations, e. g. acetylation, methylation, and biotinylation. We sought to identify amino-acid residues that are biotinylated in histone H4, and to determine whether acetylation and methylation of histones affect biotinylation. Synthetic peptides spanning fragments of human histone H4 were biotinylated enzymatically using biotinidase. Peptide-bound biotin was probed with streptavidin-peroxidase. Peptides based on the N-terminal sequence of histone H4 were effectively recognized by biotinidase as substrates for biotinylation; in contrast, peptides based on the C-terminal sequences were not biotinylated. Substitution of K8 or K12 with alanine or arginine decreased biotinylation, suggesting that these lysines are targets for biotinylation; K8 and K12 are also known targets for acetylation. Chemical acetylation or methylation of a given lysine decreased subsequent enzymatic biotinylation of neighboring lysines, consistent with cross-talk among histone modi. cations. Substitution of a given lysine ( positive charge) with glutamate ( negative charge) abolished biotinylation of neighboring lysines, providing evidence that the net charge of histones has a role in biotinylation. An antibody was generated that specifically recognized histone H4 biotinylated at K12. This antibody was used to detect biotinylated histone H4 in nuclear extracts from human cells. These studies suggest that K8 and K12 in histone H4 are targets for biotinylation, that acetylation and biotinylation compete for the same binding sites, and that acetylation and methylation of histones affect biotinylation of neighboring lysines.