Modulators of Mad family Function: MIx and mSin3A

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DESCRIPTION (provided by applicant): The proto-oncoprotein Myc functions as a transcriptional regulator and is deregulated in many human cancers. A complex network of protein-protein and protein-DNA interactions controls Myc activity. Myc does not function on its own, but forms heterodimers with Max; Myc:Max regulates the expression of genes involved in cell division, growth and metabolism. Max has many protein partners including the Mad transcriptional repressors. We have identified a Max analog, Mlx. Like Max, Mlx interacts with Mad family members to repress transcription. Mlx also interacts with a novel family of transcription factors, the Mondo family. Mondo proteins may be functional analogs of Myc. Mondo proteins interact with Mlx and the Mondo:Mlx heterodimer can bind the same CACGTG E box element as Myc:Max and activate transcription. In Drosophila, dMondo and dMyc function in similar genetic pathways. In contrast to the nuclear localization of Myc:Max, Mondo:M1x localizes to the cytoplasm. Two novel domains are required the for cytoplasmic localization of Mondo:Mlx heterodimers: a novel conserved region in the N-terminus of the Mondo family members and a conserved domain in the C-terminus of both Mondo and Mlx proteins. We hypothesize that MondoA:Mlx functions similarly to Myc:Max in controlling cell growth, but its nuclear activity is under tight control by cytoplasmic sequestration. Furthermore, we hypothesize that Mondo:Mlx is released from the cytoplasm and accumulates in the nucleus in response to extracellular signals and that these signals impinge on the regulatory domain at the N-terminus of Mondo. We propose here to determine the proteins associated with MondoA:Mlx (Aim 1), perform structure-function analyses on the novel domains in Mondo and Mlx (Aim 2), investigate the biological function of Mondo:Mlx (Aim 3), and determine the signals that trigger MondoA:Mlx release from the cytoplasm (Aim 4). We propose a multidisciplinary approach using cell biological assays, global transcript analysis, biochemical purification and a novel reverse two-hybrid system.