Protein arginine N-methyltransferase 2 plays a noncatalytic role in the histone methylation activity of PRMT1.

Protein arginine N-methyltransferases are a family of epigenetic enzymes responsible for monomethylation or dimethylation of arginine residues on histones. Dysregulation of protein arginine N-methyltransferase activity can lead to aberrant gene expression and cancer. Recent studies have shown that PRMT2 expression and histone H3 methylation at arginine 8 are correlated with disease severity in glioblastoma multiforme, hepatocellular carcinoma, and renal cell carcinoma. In this study, we explore a noncatalytic mechanistic role for PRMT2 in histone methylation by investigating interactions between PRMT2, histone peptides and proteins, and other PRMTs using analytical and enzymatic approaches. We quantify interactions between PRMT2, peptide ligands, and PRMT1 in a cofactor- and domain-dependent manner using differential scanning fluorimetry. We found that PRMT2 modulates the substrate specificity of PRMT1. Using calf thymus histones as substrates, we saw that a 10-fold excess of PRMT2 promotes PRMT1 methylation of both histone H4 and histone H2A. We found equimolar or a 10-fold excess of PRMT2 to PRMT1 can improve the catalytic efficiency of PRMT1 towards individual histone substrates H2A, H3, and H4. We further evaluated the effects of PRMT2 towards PRMT1 on unmodified histone octamers and mononucleosomes and found marginal PRMT1 activity improvements in histone octamers but significantly greater methylation of mononucleosomes in the presence of 10-fold excess of PRMT2. This work reveals the ability of PRMT2 to serve a noncatalytic role through its SH3 domain in driving site-specific histone methylation marks.

Protein arginine N-methyltransferase activity determination with filter binding and phosphor screening (FBAPS) assay.

We developed a cost-effective assay to measure protein arginine N-methyltransferase (PRMT) activity in a medium-throughput manner by combining P81 filter binding and phosphor screening (FBAPS). Recombinantly-expressed PRMT1 and coactivator-associated arginine methyltransferase 1 (CARM1) were used to develop the FBAPS assay using GST fusions of glycine- and arginine-rich (GAR) protein and polyA binding protein 1 (PABP1(437-488)) as substrates, respectively, and radiolabelled S-adenosyl-L-[methyl-14C]-methionine as cofactor. Methylation reactions were spotted onto P81 filter paper in a dot blot apparatus and radioactive signals were measured both by phosphor imaging and liquid scintillation counting. Kinetic parameters (KM, kcat) for enzymes and substrates were determined, and IC50 values were obtained for well-characterized inhibitors. FBAPS yielded kinetic parameters with no statistically significant difference to what was obtained using liquid scintillation counting. The IC50 values obtained by the FBAPS assay for PRMT1 and CARM1 were comparable to values reported in literature. The FBAPS assay is a modification to the P81 filter binding assay with a dot blot apparatus that allows for processing of samples in a multi-well format, moderately increasing throughput. Signal detection by phosphor imaging offers an affordable and quantitative method that can be used to screen several inhibitors simultaneously against PRMT enzymes with high accuracy.