Recent evidence, indicates that the Brg1 and hBrm chromatin remodeling complexes are regulated by phosphorylation during mitosis. In addition, Brg1 and Mi-2 complexes appear to be involved in the control of B and T cell growth and differentiation through their interaction with the lymphoid specific transcription factors, Ikaros and Aiolos, in a cell cycle dependent manner. The major goal of this proposal is to understand the mechanisms that modulate the activity of these chromatin remodeling complexes, and to determine the role that these complexes play during B and T cell growth and development.
The first aim of the research described here is to understand how phosphorylation modulates the activity of these complexes. The residues important for this regulation will be identified by peptide mapping and site directed mutagenesis, and their effect on the activity of the complex will evaluated using chromatin remodeling and disruption assays. The initial experiments will focus on the highly conserved subunits that form the active catalytic core of the complex. Based on the results which show that ERK1 can phosphorylate Brg1 and hBrm complexes in vitro, the role of the MAP kinase signaling pathway in the regulation of the activity of these complexes will be examined using cell lines that express either Flag-tagged hSWI/SNF subunits. A similar approach will be used to determine whether this growth regulated kinase pathwayj is also involved in the regulation of the Mi-2 complex both in vitro and in vivo. Brg1 and hBrm are highly related, and during mitosis both proteins become phosphorylated; however, only hBrm is targeted for degradation. To understand the mechanism that underlies this regulation, deletion mutations and domain swapping experiments will be used to identify the sequences that are responsible for the mitotic degradation of hBrm. The second part of this research proposal is aimed at addressing the role of the Brg1 and Mi-2 chromatin remodeling complexes in vivo, and to examine their effects on transcription of genes that are important for B and T cell specification and differentiation. Transgenic mice that express tetracycline inducible dominant negative forms of Brg1 and Mi-2 will be generated and used to examine the levels of genes that are growth and developmentally regulated. Since Ikaros proteins can interact with these chromatin remodeling complexes, genes that are regulated by Ikaros proteins will also be examined. Ikaros proteins bind DNA either as homo or heterodimers, and mutations that affect either their dimerization or DNA binding domain result in leukemias and lymphomas. To elucidate the mechanism by which mutant proteins cause cancer, the effects of the oncogenic Ikaros proteins on the activity of Brg1 and Mi-2 complexes will be examined both in vivo and in vitro. These studies will help shed some light on the role played by chromatin remodelers in the control of cell growth and development, and will provide a strong basis to establish a model system that can be used to examine the effects of chromatin remodelers and their regulators on transcription of genes that are important for cell cycle progression and differentiation.
