FUNCTIONS OF MSG1 FAMILY TRANSCRIPTION ACTIVATORS

MSG1转录激活因子家族(MSG1、MRG1、Nad SPECK)是一种小核蛋白，具有CR1和CR2两个保守区，后者是其强大转录激活活性的必要条件和充分条件。由于它们明显缺乏DNA结合活性，我们推测它们可能与序列特异的DNA结合蛋白相互作用，并作为多亚基转录因子的“反式激活亚单位”发挥作用。支持这一假设的是，我们最近发现MSG1以一种依赖于TGFbeta信号的方式增强由Smad家族信号转导/DNA结合蛋白介导的转录激活。在这项授权中，我们建议阐明MSG1这一活性的分子机制。为此，我们将从我们已经建立的Msg1突变基因敲除小鼠中制备Msg1缺陷的胚胎成纤维细胞，并利用它们通过基于转染的分析来表征MSG1对Smads介导的转录激活的促进作用。我们还将使用纯化的蛋白质和生化分析，如免疫沉淀或电迁移率改变分析，在体外表征MSG1与SMAD蛋白和转录起始复合体组件的预期物理相互作用。在体内，MSG1与这些蛋白质的复合体的形成将通过对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，这些小鼠的遗传背景是Smad2或Smad4的野生型或杂合突变。我们还将研究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(S)。阐明MSG1的生理特性和分子作用机制将有助于深入了解MSG1家族蛋白的功能，以及Smad2介导的转录如何受到非Smad蛋白的调控。

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.