title: A TR-FRET-based functional assay for screening activators of CARM1.
abstract: Epigenetics is an emerging field that demands selective cell-permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator-associated arginine methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that twofold overexpression of CARM1 in MCF7 breast cancer cells increased the expression of ERα-target genes involved in differentiation and reduced cell proliferation, thus leading to the hypothesis that activating CARM1 by chemical activators might be therapeutically effective in breast cancer. Selective, potent, cell-permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell-based, time-resolved (TR) FRET assay that uses poly(A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen TR-FRET assay uses MCF7 cells expressing GFP-PABP1 fusion protein through BacMam gene delivery system, methyl-PABP1 specific antibody, and terbium-labeled secondary antibody. This assay has been validated as reflecting the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR-FRET platform serves as a generic tool for functional screening of cell-permeable, chemical modulators of CARM1 for elucidation of its in vivo functions.
authors.affiliation: Graduate Program in Cellular and Molecular Biology, McArdle Laboratory for Cancer Research and Carbone Comprehensive Cancer Center, University of Wisconsin, 1400 University Ave, Madison, WI 53706, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Zeng
authors.forename: Jiacai
authors.initials: J
authors.lastname: Wu
authors.forename: Mark T
authors.initials: MT
authors.lastname: Bedford
authors.forename: Gianluca
authors.initials: G
authors.lastname: Sbardella
authors.forename: F Michael
authors.initials: FM
authors.lastname: Hoffmann
authors.forename: Kun
authors.initials: K
authors.lastname: Bi
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
mesh_terms: D001943:Breast Neoplasms / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D061986:MCF-7 Cells; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism*; D013997:Time Factors
chemical_list: D011484:Protein-Arginine N-Methyltransferases; C119966:coactivator-associated arginine methyltransferase 1