MODULATORS OF MAD FAMILY FUNCTION, MLX AND MSIN3

MAD FAMILY 功能、MLX 和 MSIN3 的调制器

• 批准号: 6180706
• 负责人: Donald E Ayer
• 金额: $ 20.69万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180706
• 关键词: DNA binding protein; cell cycle; chimeric proteins; gel mobility shift assay; gene expression; immunoprecipitation; molecular cloning; protein sequence; protein structure function; transcription factor; western blottings; yeast two hybrid system

Myc and mad are transcription factors of the basic region helix-loop- helix leucine zipper (BHLHZip) family that play a role in controlling proliferation, differentiation and cell death. Myc and Mad both require dimerization with Max, another member of the bHLHZip family for function. Myc:Max complexes are transcriptional activators and their activity is associated with proliferation. Mad:Max complexes are transcriptional repressors and their activity is associated with cell cycle exit and differentiation. Therefore Mad is an antagonist of Myc. The goal of this study is to gain a better understanding two modulators of Mad function, M1x and mSin3. M1x is a new member of the BHLHZip family which we have identified as binding partner for Mad1. It is most similar to Max and may have similar functions. mSin3A is a transcriptional corepressor required by Mad to function as a transcriptional repressor. By studying these two proteins insight into the mechanism of Mad biological activity and its modulation of the Myc oncogene will be gained. Mix activity will be studied on several levels. M1x protein will be examined in a variety of cells by western blotting and immunoprecipitation. The half-life, subcellular localization and association with Mad family proteins will be determined. It will be determined if M1X is a transcriptional activator or repressor and how it effects the transcriptional activities of Myc and Mad. The DNA binding specificity of M1x will be examined by the electrophoretic mobility shift assay and specific contacts by chemical modification. Because M1x can function to inhibit cell growth, the effect of M1x overexpression on cell cycle behavior will be examined by expressing M1x using retroviral vectors. mSin3A is a component of a multiprotein complex. To gain a better understanding of mSin3A driven transcription repression the mSin3 complex will be purified by standard and affinity chromatographic methods. The mSin3A associated proteins will be microsequenced and using this peptide sequence the cDNAs encoding them will be identified, cloned and sequenced. The mechanism of transcription repression will be studied in vitro using a reconstituted in vitro transcription system and the purified mSin3A complex. To understand the molecular connectivity of the complex intermolecular associations between the components of the complex will be investigated using directed two-hybrid assays.

MYC和MAD是碱基区螺旋-环-环的转录因子 螺旋亮氨酸拉链(BHLHZip)家族 增殖、分化和细胞死亡。MYC和Mad都是 需要与bHLHZip的另一成员Max进行二聚化 功能上的家庭。MYC：MAX复合体是转录的 激活剂及其活性与细胞增殖有关。 MAD：MAX复合体是转录抑制因子及其活性 与细胞周期退出和分化有关。因此发疯了 是Myc的敌手。这项研究的目标是获得更好的 了解Mad函数的两个调制器：M1x和mSin3。 M1x是BHLHZip家族的新成员，我们有 确定为Mad1的结合伙伴。它与Max最相似 并且可以具有类似的功能。MSin3A是一种转录 Mad作为转录因子所需的辅抑制子 抑制者。通过对这两种蛋白质的研究，深入了解 Mad生物活性机制及其对Myc的调控 致癌基因将会被获得。混合活动将在以下几个方面进行研究 级别。M1x蛋白将在不同的细胞中进行检测 免疫印迹和免疫沉淀。亚细胞半衰期 Mad家族蛋白的定位和关联将是 下定决心。将确定M1x是否是转录的 激活因子或抑制因子及其对转录活性的影响 Myc和Mad的。M1x的DNA结合特异性将是 通过电泳迁移率改变分析和特异性 通过化学修饰进行接触。因为M1x可以起到 抑制细胞生长，M1x过表达对细胞周期的影响 将通过使用逆转录病毒表达M1x来检查行为 向量。 MSin3A是多蛋白复合体的一个组成部分。为了获得更好的 对mSin3A调控转录抑制基因msin3的理解 络合物将通过标准层析和亲和层析进行纯化 方法：研究方法。将对mSin3A相关蛋白进行微测序 使用这个肽序列，编码它们的cDNA将是 鉴定、克隆和测序。转录的机制 将使用体外重组的细胞在体外研究抑制作用。 转录系统和纯化的mSin3A复合体。至 了解复杂分子间的分子连接性 建筑群各组成部分之间的关联将是 使用定向双杂交分析进行了研究。