Mechanisms of Transcription Regulation by c-Myc

描述（由申请人提供）： c-Myc（Myc）是一种DNA结合转录因子，其与专性伴侣Max异二聚化以调节对细胞增殖、分化和凋亡重要的基因的转录。Myc是高度调节的，是正常胚胎发育所必需的。Myc表达失调与以过度细胞增殖为特征的疾病相关，包括癌症和心血管疾病。Myc的所有已知功能都需要其N-末端转录调节结构域（N-terminal transcription regulatory domain，NRT）。然而，介导Myc γ功能的辅因子和分子机制仍然是难以捉摸的。因此，阐明Myc调控转录的机制具有普遍的医学意义。最近的观察，包括我们自己的，表明组蛋白乙酰转移酶（HAT）在调节Myc功能的可能作用。我们已经纯化了一个多亚基转录辅激活因子复合物，STAGA，其中包含人HAT GCN 5，TRRAP和其他转录辅因子。STAGA通过与Myc结构域的直接物理相互作用在体内和体外与Myc结合。我们假设，Myc招募HAT复合物，包括STAGA，其调节DNA序列的靶基因，这使得转录激活通过机制，我们建议调查。在具体目标1中，我们将分析染色质乙酰化，STAGA和特定的STAGA/GCN 5相互作用的辅激活因子在Myc转录调控中的作用。我们将使用无细胞（体外）和组织培养（体内）转录测定，包括修改染色质免疫沉淀（CHIP）协议来分析Myc-辅激活因子/HAT协会的功能。在具体目标2中，我们将讨论p300对Myc：Max功能的可能调节以及Myc：Max乙酰化的作用。在具体目标3中，我们将表征Myc-STAGA/p300蛋白界面。我们将使用GST下拉和免疫共沉淀分析来定位Myc γ直接与STAGA和p300相互作用的区域。我们将鉴定在交联实验中接触Myc γ的STAGA亚基。这个提议构成了我们长期目标的第一阶段，即了解辅激活因子和乙酰化在Myc转录和细胞增殖调控中的作用。

DESCRIPTION (provided by applicant): 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.