title: Fluorescence-based methods for screening writers and readers of histone methyl marks.
abstract: The histone methyltransferase (HMT) family of proteins consists of enzymes that methylate lysine or arginine residues on histone tails as well as other proteins. Such modifications affect chromatin structure and play a significant regulatory role in gene expression. Many HMTs have been implicated in tumorigenesis and progression of multiple malignancies and play essential roles in embryonic development and stem cell renewal. Overexpression of some HMTs has been observed and is correlated positively with various types of cancer. Here the authors report development of a continuous fluorescence-based methyltransferase assay in a 384-well format and its application in determining kinetic parameters for EHMT1, G9a, PRMT3, SETD7, and SUV39H2 as well as for screening against libraries of small molecules to identify enzyme inhibitors. They also report the development of a peptide displacement assay using fluorescence polarization in a 384-well format to assay and screen protein peptide interactions such as those of WDR5 and EED, components of MLL and EZH2 methyltransferase complexes. Using these high-throughput screening methods, the authors have identified potent inhibitors and ligands for some of these proteins.
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Abdellah
authors.initials: A
authors.lastname: Allali-Hassani
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Wasney
authors.forename: Alena
authors.initials: A
authors.lastname: Siarheyeva
authors.forename: Taraneh
authors.initials: T
authors.lastname: Hajian
authors.forename: Cheryl H
authors.initials: CH
authors.lastname: Arrowsmith
authors.forename: Masoud
authors.initials: M
authors.lastname: Vedadi
mesh_terms: D043383:Adenosylhomocysteinase / Q000378:metabolism; D000595:Amino Acid Sequence; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005453:Fluorescence; D057166:High-Throughput Screening Assays; D006649:Histocompatibility Antigens / Q000032:analysis / Q000378:metabolism; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D063151:Polycomb Repressive Complex 2; D011484:Protein-Arginine N-Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D054852:Small Molecule Libraries
chemical_list: C491488:EED protein, human; D004791:Enzyme Inhibitors; D006649:Histocompatibility Antigens; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D012097:Repressor Proteins; D054852:Small Molecule Libraries; C502547:WDR5 protein, human; C455847:EHMT1 protein, human; D000076983:Histone Methyltransferases; C413164:PRMT3 protein, human; D011484:Protein-Arginine N-Methyltransferases; C445405:EHMT2 protein, human; D011495:Histone-Lysine N-Methyltransferase; C470730:KMT5A protein, human; D063151:Polycomb Repressive Complex 2; C540312:SETD7 protein, human; C515701:SUV39H2 protein, human; D043383:Adenosylhomocysteinase