A TR‐FRET‐Based Functional Assay for Screening Activators of CARM1

Abstract

Epigenome 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 (R) Methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that overexpression of CARM1 by two-fold in MCF7 breast cancer cells increased the expression of ERα-target genes involved in differentiation and reduced cell proliferation, leading to the hypothesis that activating CARM1 by chemical activators may 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 Förster resonance energy transfer (TR-FRET) assay using poly (A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen® TR-FRET assay utilizes MCF7 cells expressing GFP-PABP1 fusion protein via BacMam gene delivery system, methyl-PABP1 specific antibody and terbium-labeled secondary antibody. This assay has been validated to reflect 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.

CARM1 Methylates Chromatin Remodeling Factor BAF155 to Enhance Tumor Progression and Metastasis

“…For instance, normal developmental functions were maintained in genetically engineered CARM1 hypomorphic mice with only 25% of the wild-type (WT) CARM1 level (Kim et al, 2010). We recently showed that knocking down 90% of endogenous CARM1 in MCF7 cells only slightly reduces methylation of PABP1 (Zeng et al, 2013). These results imply that even greatly depleted CARM1 catalytic activity and substrate methylation are sufficient to maintain major biological functions.…”

O-GlcNAcylation of co-activator-associated arginine methyltransferase 1 regulates its protein substrate specificity

“…For more potent CARM1 substrates, such as histone H3 and PABP1, the C-terminus of CARM1 may be dispensable for their methylation. For example, we have shown that even when CARM1 was reduced to 10 % of endogenous level in MCF7 cells, CARM1-mediated methylation of PABP1 was not strikingly affected [61]. Co-crystal structures of full-length CARM1 with some substrates will help to elucidate whether the role of the C-terminus in regulating CARM1 substrate binding is substrate specific.…”

Assessing the transport of receptor-mediated drug-delivery devices across cellular monolayers

“…Advantages of collecting binding data include the ability to use cells during the growth phase, enabling for faster experiments and larger volumes, and flexibility in culture equipment. High-throughput screening methods have been extensively used for receptor-ligand binding data[57–59], and would ideally be used to screen for more advantageous ligand/cell pairings. Transport studies, despite being limited to specialized Transwell® trays and requiring longer culture periods, provide more direct data for drug-delivery studies, and can be tested alongside passive transport for comparison.…”