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ARS Home » Plains Area » Lincoln, Nebraska » Wheat, Sorghum and Forage Research » Research » Publications at this Location » Publication #158398
Title: LYSINE-8 AND LYSINE-12 ARE BIOTINYLATED ON HUMAN HISTONE H4.

Author
item CAMPOREALE, G - UNI OF NE-LINCOLN
item SCHUBERT, E - UNI OF NE-LINCOLN
item Sarath, Gautam
item CERNY, R - UNI OF NE-LINCOLN
item ZEMPLENI, J - UNI OF NE-LINCOLN

Submitted to: European Journal of Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/16/2004
Publication Date: 6/1/2004
Citation: Camporeale, G., Schubert, E., Sarath, G., Cerny, R., Zempleni, J. 2004. Lysine-8 and lysine-12 are biotinylated on human histone h4. European Journal of Biochemistry. 271:2257-2263.

Interpretive Summary: All higher organisms package their DNA into the cell's nucleus. Packaging of DNA is thus not only critical for cell survival; it is also a means by which information stored in the DNA can be transmitted appropriately. Failure in packaging and or misreading of DNA can lead to numerous pathogenic states including cancer. Packaging of DNA is primarily mediated by a class of cellular proteins called histones. Histones, themselves are modified after their synthesis and association with DNA by a large variety of enzymes leading to a complex maze of modifications, that are collectively called the 'histone code'. Understanding the histone code is major target of current life-science research. We have been studying the modification to histones by the vitamin biotin. Histone modification by biotinylation has been linked to cell-proliferation and DNA damage. This study elucidates the lysine residues of Human histone H4 that are the targets for biotinylation and has utilized synthetic peptides to delineate regions of the H4 protein that are biotinylated. We also used peptides with lysines that were chemically modified with acetyl-, methyl- and formyl- groups to asses their effects on biotinylation and evaluate the degree of cross-talk between these different modifications. Peptides based on the N-terminal sequence of histone H4 were effectively biotinylated; in contrast, peptides based on the C-terminal sequences were not biotinylated. Substitution of lysine-8 or lysine-12 by alanine or arginine decreased biotinylation, suggesting that these lysines are targets for biotinylation; lysine-8 and lysine-12 are also known targets for acetylation. Chemical acetylation or methylation of a given lysine decreased subsequent enzymatic biotinylation of neighboring lysines, suggesting cross-talk among modifications of histones. Substitution of a given lysine (positive charge) by glutamate (negative charge) abolished biotinylation of neighboring lysines, suggesting that the net charge of histones plays a role in biotinylation. An antibody was generated that specifically recognized histone H4 biotinylated at lysine-12. Using this antibody, biotinylated histone H4 was detected in nuclear extracts from human cells. These studies provide evidence that lysine-8 and lysine-12 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. This is the first report that indicates the specificity of biotinylation of histones in-vitro as well as in-vivo. Future studies can now build on these important findings.

Technical Abstract: Folding of DNA into chromatin is mediated by binding to histones such as H4; association of DNA with histones is regulated by covalent modifications of histones, e.g., acetylation, methylation, and biotinylation. Here we sought to identify amino acid residues that are biotinylated in histone H4, and we sought 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 lysine-8 or lysine-12 by alanine or arginine decreased biotinylation, suggesting that these lysines are targets for biotinylation; lysine-8 and lysine-12 are also known targets for acetylation. Chemical acetylation or methylation of a given lysine decreased subsequent enzymatic biotinylation of neighboring lysines, suggesting cross-talk among modifications of histones. Substitution of a given lysine (positive charge) by glutamate (negative charge) abolished biotinylation of neighboring lysines, suggesting that the net charge of histones plays a role in biotinylation. An antibody was generated that specifically recognized histone H4 biotinylated at lysine-12. Using this antibody, biotinylated histone H4 was detected in nuclear extracts from human cells. These studies provide evidence that lysine-8 and lysine-12 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.