Project Summary Arginine methylation is a common posttranslational modification (PTM), which is catalyzed by a family of nine arginine methyltransferases – the PRMTs. This PTM has been clearly implicated in a number of cellular processes, including transcriptional regulation. In the context of transcriptional regulation, PRMT4/CARM1 functions as a coactivator with a large number of transcription factors. The mechanisms behind its coactivator functions are unclear. In this grant proposal, we will follow-up on two fundamental discoveries that we made in the past funding cycle: 1) that CARM1 methylates the MED12 component of Mediator and a transcription factor NF1; and 2) that CARM1 overexpression in the mammary gland causes hyperplasia and increased ductal out- growth. These two Aims are linked to each other, as they allow us to characterize two prominent CARM1 substrates in vitro and in cells, and then investigate their importance relaying the CARM1 signal in an in vivo overexpression mouse model. Together, these Aims speak to the overarching goal of this grant, which is “The Functional Analysis of the Coactivator CARM1”. Our working hypothesis is that when CARM1 is overexpressed in disease states, it promotes cell proliferation by regulating the Mediator complex and NFI transcription factors. A detailed understanding of the spectrum of CARM1 substrates, the “readers” of CARM1-generated methylation marks, and the examination of physiological defects observed in the CARM1 overexpression mouse models will help us develop a holistic view of the biological roles of this arginine methyltransferase enzyme.