A TR‐FRET‐Based Functional Assay for Screening Activators of CARM1. Issue 7 (12th April 2013)

• Record Type: Journal Article
• Authors: Zeng, Hao; Wu, Jiacai; Bedford, Mark T.; Sbardella, Gianluca; Hoffmann, F. Michael; Bi, Kun; Xu, Wei
• Is Part Of: Chembiochem. Volume 14:Issue 7(2013)
• Page Start: 827
• Page End: 835
• Publication Date: 2013-04-12
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201300029
• Languages: English
• ISSNs: 1439-4227

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <p>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.</p> </abstract>