title: An acetyltransferase assay for CREB-binding protein based on reverse phase-ultra-fast liquid chromatography of fluorescent histone H3 peptides.
abstract: CREB-binding protein (CBP) is a lysine acetyltransferase that regulates transcription by acetylating histone and non-histone substrates. Defects in CBP activity are associated with hematologic malignancies, neurodisorders, and congenital malformations. Sensitive and quantitative enzymatic assays are essential to better characterize the pathophysiological features of CBP. We describe a sensitive nonradioactive method to measure purified and immunopurified cellular CBP enzymatic activity through rapid reverse phase-ultra-fast liquid chromatography (RP-UFLC) analysis of fluorescent histone H3 peptide substrates. The applicability and biological relevance of the assay are supported by kinetic, inhibition, and immunoprecipitation studies. More broadly, this approach could be easily adapted to assay other lysine acetyltransferases or methyltransferases.
authors.affiliation: Unité de Biologie Fonctionnelle et Adaptative (BFA), CNRS UMR 8251, Université Paris Diderot, Sorbonne Paris Cité, 75013 Paris, France.
authors.forename: Romain
authors.initials: R
authors.lastname: Duval
authors.affiliation: Centre National de la Recherche Scientifique CNRS - Université Paris Diderot, Sorbonne Paris Cité, Epigenetics and Cell Fate, UMR 7216 CNRS, Paris, France.
authors.forename: Lauriane
authors.initials: L
authors.lastname: Fritsch
authors.affiliation: Unité de Biologie Fonctionnelle et Adaptative (BFA), CNRS UMR 8251, Université Paris Diderot, Sorbonne Paris Cité, 75013 Paris, France.
authors.forename: Linh-Chi
authors.initials: LC
authors.lastname: Bui
authors.affiliation: Unité de Biologie Fonctionnelle et Adaptative (BFA), CNRS UMR 8251, Université Paris Diderot, Sorbonne Paris Cité, 75013 Paris, France.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Berthelet
authors.affiliation: INSERM UMR_S1131, Institut Universitaire d'Hématologie, Université Paris Diderot, Sorbonne Paris Cité, 75010 Paris, France.
authors.forename: Fabien
authors.initials: F
authors.lastname: Guidez
authors.affiliation: Unité de Biologie Fonctionnelle et Adaptative (BFA), CNRS UMR 8251, Université Paris Diderot, Sorbonne Paris Cité, 75013 Paris, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Mathieu
authors.affiliation: Unité de Biologie Fonctionnelle et Adaptative (BFA), CNRS UMR 8251, Université Paris Diderot, Sorbonne Paris Cité, 75013 Paris, France.
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Dupret
authors.affiliation: INSERM UMR_S1131, Institut Universitaire d'Hématologie, Université Paris Diderot, Sorbonne Paris Cité, 75010 Paris, France; Service de Biologie Cellulaire, Assistance Publique des Hôpitaux de Paris (AP-HP), Hôpital Saint Louis, 75010 Paris, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Chomienne
authors.affiliation: Centre National de la Recherche Scientifique CNRS - Université Paris Diderot, Sorbonne Paris Cité, Epigenetics and Cell Fate, UMR 7216 CNRS, Paris, France.
authors.forename: Slimane
authors.initials: S
authors.lastname: Ait-Si-Ali
authors.affiliation: Unité de Biologie Fonctionnelle et Adaptative (BFA), CNRS UMR 8251, Université Paris Diderot, Sorbonne Paris Cité, 75013 Paris, France. Electronic address: fernando.rodrigues-lima@univ-paris-diderot.fr.
authors.forename: Fernando
authors.initials: F
authors.lastname: Rodrigues-Lima
mesh_terms: D000595:Amino Acid Sequence; D050882:CREB-Binding Protein / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006657:Histones / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D006657:Histones; D010446:Peptide Fragments; D050882:CREB-Binding Protein
keywords: CREB-binding protein; Fluorescent peptide; HPLC; Histone acetyltransferase; Histone methyltransferases; SETD2