MODULATORS OF MAD FAMILY FUNCTION, MLX AND MSIN3

MAD FAMILY 功能、MLX 和 MSIN3 的调制器

• 批准号: 2701823
• 负责人: Donald E Ayer
• 金额: $ 19.52万
• 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/2701823
• 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是碱性区螺旋-环- 螺旋亮氨酸拉链（BHLHZip）家族在控制 增殖、分化和细胞死亡。 Myc和Mad 需要与bHLHZip的另一个成员Max二聚化 家庭功能。 Myc：Max复合物是转录的 活化剂，它们的活性与增殖有关。 Mad：Max复合物是转录抑制因子， 与细胞周期退出和分化有关。 因此，疯狂 是Myc的拮抗剂。 本研究的目的是获得一个更好的 了解Mad功能的两种调节剂M1 x和mSin 3。 M1 x是BHLHZip家族的新成员， 被鉴定为Mad 1的结合伴侣。 最像Max 并且可以具有类似的功能。 mSin 3A是一种转录因子， Mad作为转录因子发挥作用所需的辅阻遏物 阻遏物 通过研究这两种蛋白质， Mad的生物活性机制及其对Myc的调节作用 将获得癌基因。 混合活动将在几个 程度. M1 x蛋白将在多种细胞中通过 Western印迹和免疫沉淀。 亚细胞半衰期 与Mad家族蛋白的定位和关联将是 测定 将确定M1 X是否是转录的 激活子或阻遏子及其如何影响转录活性 Myc和Mad M1 x的DNA结合特异性为 通过电泳迁移率变动试验和特异性 化学修饰的接触。 因为M1 x可以用于 抑制细胞生长，M1 x过表达对细胞周期的影响 将通过使用逆转录病毒表达M1 x来检查行为 向量。 mSin 3A是多蛋白复合物的组分。 更好地 理解mSin 3A驱动的转录抑制mSin 3 复合物将通过标准和亲和色谱法纯化 方法. 将对mSin 3A相关蛋白进行微测序 使用这个肽序列，编码它们的cDNA将被 鉴定、克隆和测序。 转录机制 将在体外使用重构的体外抑制剂来研究抑制。 转录系统和纯化的mSin 3A复合物。 到 理解复杂分子间的分子连接性 复合体各组成部分之间的联系将是 使用定向双杂交测定进行研究。