K8 and K12 are biotinylated in human histone H4.
Folding of DNA into chromatin is mediated by binding to histones such as H4; association of DNA with histones is regulated by covalent histone modifications, 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 modifications. 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.
Camporeale
Gabriela
G
Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE 68583-0806, USA.
Shubert
Elizabeth E
EE
Sarath
Gautam
G
Cerny
Ronald
R
Zempleni
Janos
J
D000107:Acetylation; D019943:Amino Acid Substitution; D000906:Antibodies; D019921:Biotinylation; D002851:Chromatography, High Pressure Liquid; D006657:Histones / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008239:Lysine / Q000378:metabolism*; D013058:Mass Spectrometry; D008745:Methylation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism
D000906:Antibodies; D006657:Histones; D010455:Peptides; D008239:Lysine