The estrogen signaling pathway is pivotal for maintaining female reproductive function and contributes extensively to breast tumorigenesis. Estrogen exerts its effect mainly through binding to its cognate receptors, estrogen receptor (ER) alpha and beta, resulting in recruitment of coactivators and subsequent transcriptional activation of estrogen-dependent genes. Studies over last decade have established the critical roles of nuclear receptor coactivators (including the SRC family of coactivators and CARM1) in estrogen receptor-mediated transcription. Our ongoing studies indicate that CARM1 is a dual-functional nuclear receptor coactivator, as it not only activates transcription by methylating core histone, but also terminates hormone induced transcription by methylating non-histone coactivators and resulting in disassembly of the coactivator complex. One critical question arises about how CARM1 selectively methylates histone and non-histone coactivators in an appropriate order during transcription initiation. Although intensive studies from different laboratories have been focused on identification of CARM1 substrates, very little is known about how the functions of CARM1 are regulated by diverse signals in the cells. We hypothesize that enzymatic activity and substrate specificity of CARM1 are regulated by posttranslational modifications such as phosphorylation and/or CARM1 interacting proteins. The specific aims of this proposal are to: 1. Determine if SRC-3 needs a phosphorylation code to be methylated by CARM1 with in vitro methylation assay. 2. Identify the phosphorylation sites of CARM1 by mass spectrometric analysis. 3. Determine the role of phosphorylation on CARM1 enzymatic activity and substrate specificity. Various assays including in vitro methylation and cell-free chromatin-based in vitro transcription will be used. 4. Investigate the roles of the CARM1 interacting proteins on CARM1 functions by different biochemical and cell biology methods. The SRC-3/AIB1 is an authentic oncogene and its transcript was reported to be over-expressed in 64% of primary breast cancers. In our current study, we found that SRC-3/AIB1 is methylated by CARM1, and methylation of SRC-3 attenuates its coactivator activity. Since CARM1 is the sole methyltransferase responsible for SRC-3 arginine methylation, understanding how CARM1 activity is regulated will not only shed more light on the mechanisms by which nuclear receptors guide transcription, but also will provide further information for design of novel and effective therapy to treat breast tumors.

F32DK078473

2007-04-01

2009-03-31