FUNCTIONS OF MSG1 FAMILY TRANSCRIPTION ACTIVATORS

MSG1 家族转录激活剂的功能

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/6377308
• 关键词: 3T3 cells; DNA binding protein; HeLa cells; biological signal transduction; fibroblasts; flow cytometry; gel mobility shift assay; gene expression; genetically modified animals; genotype; immunoprecipitation; laboratory mouse; northern blottings; phenotype; phosphorylation; polymerase chain reaction; protein protein interaction; protein structure function; site directed mutagenesis; southern blotting; tissue /cell culture; transcription factor; transfection; transforming growth factors; western blottings

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.

转录激活因子MSG1家族（MSG1, MRG1, nand SPECK）是小的核蛋白，共享两个保守区域，CR1和CR2；后者是其强大的转录激活活性的必要和充分条件。由于它们明显缺乏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，并作为多亚基转录因子的“反激活亚基”起作用。为了支持这一假设，我们最近发现MSG1以依赖于tgf β信号传导的方式增强了Smad家族信号传感器/ dna结合蛋白介导的转录激活。在本次资助中，我们拟阐明MSG1这种活性的分子机制。为此，我们将从已经产生的MSG1突变敲除小鼠中制备MSG1缺陷的胚胎成纤维细胞，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。我们还将利用纯化蛋白和生化分析（如免疫沉淀或电迁移转移测定）来表征MSG1与SMAD蛋白和转录起始复合物组分在体外的预期物理相互作用。将通过质粒衍生蛋白或内源性蛋白的生化分析来评估MSG1与这些蛋白在体内的复合物形成。为了了解MSG1的生理特性，我们提出在Smad2或Smad4的野生型或杂合突变的遗传背景下，对MSG1缺陷小鼠及其胚胎成纤维细胞的表型进行表征。我们还将描述msg1诱导B16-F10黑色素瘤细胞聚集的分子机制，试图确定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家族蛋白的功能以及Smad2介导的转录是如何被非smad蛋白调控的。