To better understand the biological roles of arginine methylation, our research group uses a number of different biological approaches to interrogate PRMT function. These include mouse gain- and loss-of-function PRMT models, protein microarrays screens for methyl-binding proteins, biochemical screens for PRMT substrates, and screens for chemical inhibitors of PRMT family members.
Knockout Mouse Models
We have generated targeted disruptions of a number of arginine methyltransferase genes in mice in the hopes of unmasking cellular and tissue-specific roles for this post-translational modification.
In-hand we currently have CARM1, PRMT3 and PRMT6 null mice. We are performing transcriptome analysis (RNA-seq) and ChIP-seq on these null mice to identify the repertoire of genes that they regulate. We are also performing double knockouts to investigate redundancy between the different PRMTs.
Gain-of-function Mouse Models
We are also generating gain-of-function transgenic mouse models to determine the effects of PRMT overexpression in vivo. These are transgenic mice that can be activated by crossing to a tissue-specific cre line. The Cre expression removes a lox/STOP/los cassette and activates expression of the PRMT.
We have already generated PRMT6 gain-of-function mice using this approach. We are currently producing CARM1, PRMT1, PRMT5 and TDRD3 transgenic mice using the same approach.
Protein domains are evolutionarily conserved motifs found within proteins that function as signaling units. Certain motifs may serve in recognizing specific post-translational modifications, thereby allowing for the regulation of protein-protein interactions to occur. Our lab has developed a valuable protein array platform to identify protein domains that bind modified peptides. Most of our efforts are focused on identifying protein domains that “read” the histone code.
The Protein Array and Analysis Core has been established as a new facility by the Center for Environmental and Molecular Carcinogenesis. The core provides our protein domain microarrays, a high-throughput technology, to facilitate investigators in identifying novel protein-protein interactions.
Protein Macroarray Screen
Protein macroarray technology is a powerful tool for high throughput parallel screening of enzyme-substrate, protein-ligand or protein-chemical compound, and protein-protein interactions. Using this method, we have successfully identified a number of substrates for CARM1 and PRMT1. We are now utilizing this method to identify proteins that interact with biotinylated small molecules.
Pan Antibody Approach
We are developing and characterizing antibodies that recognize MMA, ADMA, and SDMA motifs. This will allow us to screen for and characterize PRMT substrates. For example, to identify novel substrates for CARM1, we have generated pan-CARM1 substrate antibodies using a mixed antigen pool, which harbors methyl motifs of a number of known CARM1 substrates. These antibodies were then used to enrich for CARM1 methylated proteins, which were then identified by mass spectrometry.
A subset of these putative substrates has been chosen for further evaluation. Functional studies to determine the significance of methylation on these substrates will help us gain a better understanding of CARM1’s role as a transcriptional co-activator.
TAP/MS Approach to Study Protein Complexes
We use a Tandem Affinity Purification (TAP)/Mass Spectrometry technique to study protein-protein interactions. We have generated a number of stable cell lines that express TAP tagged epigenetic regulators. These stable lines include ectopic PRMT6, PRMT7, PHF20 and TDRD3.
The protein of interest with the TAP tag first binds to IgG beads. The protein complex is then released through TEV enzyme digestion of the linker region on the tag. Streptavidin beads are used for the second round of binding. After elution, the protein complex is ready for identification by mass spectrometry.
Small molecules that act as chemical modulators of enzyme functions are valuable tools for probing protein functions and for use as therapeutic agents. Most methyltransferases use the methyl donor S-adenosyl-L-methionine (AdoMet) as a cofactor. Current methyltransferase inhibitors display limited specificity, indiscriminately targeting all enzymes that use AdoMet. We have designed and implemented a high-throughput screen for the identification of small molecules that specifically inhibit protein arginine N-methyltransferase (PRMT) activity. Using this approach, we have identified a lead compound, AMI-1, that inhibits PRMT activity, but not lysine methyltransferases.
We have been working with medicinal chemists to identify AMI-1 analogs that will display better specificity and cell permeability. We are also interested in identifying compounds that bind to the aromatic cage of methyl-binding protein domains. We expect to identify compounds that will block methyl-dependent protein-protein interactions.