FUNCTIONS OF MSG1 FAMILY TRANSCRIPTION ACTIVATORS

MSG1 家族转录激活剂的功能

基本信息

批准号：
6377308
负责人：
TOSHIHIRO SHIODA
金额：
$ 29.72万
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转录激活剂家族（MSG1，MRG1，NAD Speck）是小的核蛋白，共有两个保守区域CR1和CR2。后者对于强大的转录激活活性是必要的，足以满足。由于它们显然缺乏DNA结合活性，因此我们假设它们可能与序列特异性的DNA结合蛋白相互作用，并且起作用是多生育转录因子的“反式激活亚基”。在支持这一假设的情况下，我们最近发现，MSG1增强了由SMAD家族信号传感器/DNA结合蛋白介导的转录激活，其方式取决于TGFBETA信号传导。在这项赠款中，我们建议阐明MSG1活性的分子机制。为此，将从我们已经生成的MSG1突变敲除小鼠中制备MSG1缺陷型胚胎成纤维细胞，并且MSG1对SMADS介导的转录激活的增强效应是通过基于转染的分析来表征的。我们还将使用纯化的蛋白质和生化分析（例如免疫沉淀或电动性转移测定法）在体外进行了MSG1与转录起始复合物的SMAD蛋白和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对质粒衍生蛋白或内源性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缺陷型小鼠的表型及其胚胎成纤维细胞，具有野生型的遗传背景或Smad2或Smad4的杂合突变。我们还将表征MSG1诱导的B16-F10黑色素瘤细胞聚集的分子机制，试图鉴定MSG1增强转录激活的靶基因。阐明生理特性和MSG1作用的分子机制将提供有关MSG1家族蛋白如何功能以及SMAD2-介导的转录如何受非SMAD蛋白调节的见解。