FUNCTIONS OF MSG1 FAMILY TRANSCRIPTION ACTIVATORS

MSG1 家族转录激活剂的功能

基本信息

批准号：
6173591
负责人：
TOSHIHIRO SHIODA
金额：
$ 28.85万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-07-01 至 2002-06-30
项目状态：
已结题

项目摘要

The MSG1 family of transcriptional activators (MSG1, MRG1, nad SPECK) are small nuclear proteins that share two conserved regions, CR1 and CR2; the latter is necessary and sufficient for their strong transcriptional activating activity. Since they apparently lack DNA- binding activity, we hypothesize that they may interact with sequence- specific DNA-binding proteins and function as "transactivating subunits" of multi-subunit transcription factors. Supporting this hypothesis, we recently have discovered that MSG1 enhances transcriptional activation mediated by the Smad family signal transducer/DNA-binding proteins in a manner dependent of TGFbeta signaling. In this grant, we propose to elucidate the molecular mechanisms of this activity of MSG1. For this purpose, Msg1-deficient embryonic fibroblasts will be prepared from Msg1-mutant knockout mice, which we have already generated, and the enhancing effect of MSG1 on Smads-mediated transcriptional activation will be characterized using them by transfection-based analyses. We will also characterize expected physical interactions of MSG1 with the SMAD proteins and components of the transcription initiation complex in vitro using purified proteins and biochemical analyses, such as immunoprecipitation or electromobility shift assay. In vivo complex formation of MSG1 with such proteins will be evaluated by biochemical analyses of plasmid-derived proteins or endogenous proteins. To understand the physiological properties of MSG1, we propose to characterize the phenotypes of the Msg1-deficient mice and their embryonic fibroblasts, with genetic backgrounds of wild type or heterozygous mutations of Smad2 or Smad4. We will also characterize molecular mechanisms of MSG1-induced aggregation of B16-F10 melanoma cells, attempting to identify target gene(s) of MSG1-enhanced transcriptional activation. Elucidation of the physiological properties and the molecular mechanisms of action of MSG1 will provide insights as to how the MSG1 family proteins function as well as how the Smad2- mediated transcription is regulated by non-Smad proteins.

MSG1 转录激活因子家族（MSG1、MRG1、nad SPECK）是共享两个保守区域 CR1 和 CR2 的小核蛋白；后者对于其强大的转录激活活性是必要且充分的。由于它们显然缺乏DNA结合活性，我们假设它们可能与序列特异性DNA结合蛋白相互作用并充当多亚基转录因子的“反式激活亚基”。支持这一假设的是，我们最近发现 MSG1 以依赖于 TGFbeta 信号传导的方式增强 Smad 家族信号转导器/DNA 结合蛋白介导的转录激活。在这笔资助中，我们建议阐明 MSG1 这种活性的分子机制。为此，将从我们已经制备的 Msg1 突变型敲除小鼠中制备 Msg1 缺陷型胚胎成纤维细胞，并通过基于转染的分析来表征 MSG1 对 Smads 介导的转录激活的增强作用。我们还将使用纯化的蛋白质和生化分析（例如免疫沉淀或电迁移率测定）在体外表征 MSG1 与 SMAD 蛋白和转录起始复合物成分的预期物理相互作用。 MSG1 与此类蛋白质的体内复合物形成将通过质粒衍生蛋白质或内源蛋白质的生化分析来评估。为了了解 MSG1 的生理特性，我们建议表征 Msg1 缺陷小鼠及其胚胎成纤维细胞的表型，这些小鼠具有野生型或 Smad2 或 Smad4 杂合突变的遗传背景。我们还将表征 MSG1 诱导 B16-F10 黑色素瘤细胞聚集的分子机制，试图识别 MSG1 增强转录激活的靶基因。阐明 MSG1 的生理特性和分子作用机制将有助于了解 MSG1 家族蛋白如何发挥作用以及非 Smad 蛋白如何调节 Smad2 介导的转录。