Automated Identification of Core Regulatory Genes in Human Gene Regulatory Networks

10.1371/journal.pcbi.1004504PLoS Computational Biology119e100450

Use of synthetic peptides for identifying biotinylation sites in human histones

Posttranslational modifications of histones play an important role in the regulation of chromatin structure and, hence, gene regulation. Recently, we have identified a novel modification of histones: binding of the vitamin biotin to lysine residues in histones H2A, H3, and H4. Here, we describe a procedure to identify those amino acids that are targets for biotinylation in histones. Briefly, the following analytical sequence is used to identify biotinylation sites: (i) short peptides (<20 amino acids in length) are synthesized chemically; amino acid sequences in the peptides are based on the sequence in a given region of a given histone; (ii) peptides are incubated with biotinidase or holocarboxylase synthetase to conduct enzymatic biotinylation; and (iii) biotin in peptides are probed using streptavidin peroxidase. Amino acid substitutions (e.g., lysine-to-alanine substitutions) in synthetic peptides can be used to corroborate identification of biotinylation sites.Fil: Camporeale, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Nebraska - Lincoln; Estados UnidosFil: Chew, Yap Ching. Universidad de Nebraska - Lincoln; Estados UnidosFil: Kueh, Alice. Universidad de Nebraska - Lincoln; Estados UnidosFil: Sarath, Gautam. Universidad de Nebraska - Lincoln; Estados UnidosFil: Zempleni, Janos. Universidad de Nebraska - Lincoln; Estados Unido