c-Myc (Myc) is a DNA-binding transcription factor that heterodimerizes with an obligatory partner, Max, to regulate transcription of genes important for cell proliferation, differentiation, and apoptosis. Myc is highly regulated and is required for normal embryonic development. Deregulated Myc expression is associated with disorders characterized by excess cell proliferation, including cancer and cardiovascular disease. All known functions of Myc require its N-terminal transcription regulatory domain (TAD). However, the cofactors and molecular mechanisms that mediate Myc TAD functions have remained elusive. Thus, elucidating the mechanisms by which Myc regulates transcription is of general medical relevance. Recent observations, including our own, suggest possible roles of histone acetyltransferases (HATs) in regulating Myc functions. We have purified a multisubunit transcription coactivator complex, STAGA, which contains the human HAT GCN5, TRRAP, and other transcription cofactors. STAGA associates with Myc in vivo and in vitro via direct physical interactions with the TAD domain. We postulate that Myc recruits HAT complexes, including STAGA, to its regulatory DNA sequences on target genes and that this allows transcription activation through mechanisms that we propose to investigate.
In Specific Aim 1 we will analyze the role of chromatin acetylation, STAGA, and specific STAGA/GCN5-interacting coactivators in transcription regulation by Myc. We will use both cell-free (in vitro) and tissue culture (in vivo) transcription assays, including modified chromatin immunoprecipitation (CHIP) protocols to analyze the function of the Myc-coactivator/HAT associations.
In Specific Aim 2 we will address the possible regulation of Myc:Max functions by p300 and the role(s) of Myc:Max acetylation.
In Specific Aim 3 we will characterize the Myc-STAGA/p300 protein interface. We will map the region(s) of Myc TAD that directly interact with STAGA and p300 by using GST pull-down and co-immunoprecipitation assays. We will identify the STAGA subunit(s) that contacts Myc TAD in crosslinking experiments. This proposal constitutes the first phase of our long-term goal to understand the role of coactivators and acetylation in regulation of transcription and cell proliferation by Myc.
