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: Hao Zeng, Jiacai Wu, Mark T Bedford, Gianluca Sbardella, F Michael Hoffmann, Kun Bi, Wei Xu
• Journal: Chembiochem : a European journal of chemical biology (Chembiochem) Vol. 14 Issue 7 Pg. 827-35 (May 10 2013) ISSN: 1439-7633 [Electronic] Germany
• PMID: 23585185 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical References

• Protein-Arginine N-Methyltransferases
• coactivator-associated arginine methyltransferase 1

Topics

• Breast Neoplasms (enzymology)
• Enzyme Activation (drug effects)
• Female
• Fluorescence Resonance Energy Transfer
• Humans
• MCF-7 Cells
• Protein-Arginine N-Methyltransferases (metabolism)
• Time Factors