To better understand the biological roles of arginine methylation, the Bedford 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
The lab has generated targeted disruptions of a number of arginine methyltransferase genes in mice in order to unmask cellular and tissue-specific roles for this post-translational modification. Mouse models include CARM1, PRMT3 and PRMT6 null mice. The group is performing transcriptome analysis (RNA-seq) and ChIP-seq on these null mice to identify the repertoire of genes that they regulate. In addition, they are creating double knockout animals to investigate redundancy between the different PRMTs.
Gain-of-function Mouse Models
The Bedford lab is also generating gain-of-function transgenic mouse models to observe the effects of PRMT overexpression in vivo. These models are developed with transgenic mice bearing genes 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. They have already generated PRMT6 gain-of-function mice using this approach, and are now developing CARM1, PRMT1, PRMT5 and TDRD3 transgenic mice.
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. The lab has developed a valuable protein array platform to identify protein domains that bind modified peptides. Most of the core's microarrays are designed to identify protein domains that “read” the histone code.
The Protein Array and Analysis Core (PAAC) was originally established and developed as a new facility with the aid of the Center for Environmental and Molecular Carcinogenesis at MD Anderson. The core is now supported by a grant from Cancer Prevention and Research Institute of Texas (CPRIT RP 180804). The core provides 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, the Bedford lab has identified a number of substrates for CARM1 and PRMT1 and is extending this method to identify proteins that interact with biotinylated small molecules.
Pan Antibody Approach
The Bedford group is also developing and characterizing antibodies that recognize MMA, ADMA, and SDMA motifs in order to screen for and characterize PRMT substrates. For example, to identify novel substrates for CARM1, they 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
The lab employs a Tandem Affinity Purification (TAP)/Mass Spectrometry technique to study protein-protein interactions and has generated a number of stable cell lines that express TAP tagged epigenetic regulators. These stable lines include ectopic PRMT6, PRMT7, PHF20 and TDRD3.
In this method, the TAP-tagged protein of interest is first bound to IgG beads. The bound 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. The Bedford lab has 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, they have identified a lead compound, AMI-1, that inhibits PRMT activity, but not lysine methyltransferases.
The lab collaborates with medicinal chemists to identify AMI-1 analogs that will display better specificity and cell permeability. The lab is also invested in identifying compounds that bind to the aromatic cage of methyl-binding protein domains and expects to identify compounds that will block methyl-dependent protein-protein interactions.