title: A combinatorial H4 tail library for exploring the histone code.
abstract: Histone modifications modulate chromatin structure and function. A posttranslational modification-randomized, combinatorial library based on the first 21 residues of histone H4 was designed for systematic examination of proteins that interpret a histone code. The 800-member library represented all permutations of most known modifications within the N-terminal tail of histone H4. To determine its utility in a protein binding assay, the on-bead library was screened with an antibody directed against phosphoserine 1 of H4. Among the hits, 59 of 60 sequences were phosphorylated at S1, while 30 of 30 of those selected from the nonhits were unphosphorylated. A 512-member version of the library was then used to determine the binding specificity of the double tudor domain of hJMJD2A, a histone demethylase involved in transcriptional repression. Global linear least-squares fitting of modifications from the identified peptides (40 hits and 34 nonhits) indicated that methylation of K20 was the primary determinant for binding, but that phosphorylation and acetylation of neighboring sites attenuated the interaction. To validate the on-bead screen, isothermal titration calorimetry was performed with 13 H4 peptides. Dissociation constants ranged from 1 mM to 1 microM and corroborated the screening results. The general approach should be useful for probing the specificity of any histone-binding protein.
authors.affiliation: Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706-1532, USA.
authors.forename: Adam L
authors.initials: AL
authors.lastname: Garske
authors.forename: Gheorghe
authors.initials: G
authors.lastname: Craciun
authors.forename: John M
authors.initials: JM
authors.lastname: Denu
mesh_terms: D000595:Amino Acid Sequence; D042421:Histone Code / Q000235:genetics*; D006657:Histones / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D019151:Peptide Library; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006657:Histones; D019151:Peptide Library