title: Application of histone modification-specific interaction domains as an alternative to antibodies.
abstract: Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies.
authors.affiliation: Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany;
authors.forename: Goran
authors.initials: G
authors.lastname: Kungulovski
authors.affiliation: Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany;
authors.forename: Ina
authors.initials: I
authors.lastname: Kycia
authors.affiliation: Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany;
authors.forename: Raluca
authors.initials: R
authors.lastname: Tamas
authors.affiliation: Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany;
authors.forename: Renata Z
authors.initials: RZ
authors.lastname: Jurkowska
authors.affiliation: Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany;
authors.forename: Srikanth
authors.initials: S
authors.lastname: Kudithipudi
authors.affiliation: Active Motif, Carlsbad, California 92008, USA;
authors.forename: Chisato
authors.initials: C
authors.lastname: Henry
authors.affiliation: Max-Planck-Genomzentrum Köln, 50829 Köln, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Reinhardt
authors.affiliation: Active Motif, Carlsbad, California 92008, USA;
authors.forename: Paul
authors.initials: P
authors.lastname: Labhart
authors.affiliation: Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany; albert.jeltsch@ibc.uni-stuttgart.de.
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D000090205:DNA Methyltransferase 3A; D057809:HEK293 Cells; D006657:Histones / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D010455:Peptides / Q000378:metabolism; D063150:Polycomb Repressive Complex 1 / Q000235:genetics / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011499:Protein Processing, Post-Translational; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; C503627:CBX7 protein, human; C000717967:DNMT3A protein, human; D006657:Histones; D010455:Peptides; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D063150:Polycomb Repressive Complex 1; D008239:Lysine