Molecular structural study for regulation on the activities of the hematopoietic transcriptional regulatory factors

造血转录调控因子活性调控的分子结构研究

• 批准号: 14380296
• 负责人: OGATA Kazuhiro
• 金额: $ 10.37万
• 依托单位: Yokohama City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380296/
• 关键词: transcriptional regulatory factor; higher-order nucleoprotein; Runxl; c-Myb; C; EBP; allosteric regulation; X-ray crystallography; nuclear magnetic resonance; 転写制御因子高次複合体; 転写因子; エンハンセオソーム; X線結晶構造解析; 原子間力顕微鏡; c-Ets-1

This research project aimed to elucidate a molecular mechanism for transcriptional regulation by multiple transcriptional regulatory proteins. We focused on some hematopoietic transcriptional regulatory proteins, such as Runxl, CBFβ, c-Myb, C/EBP, that play critical roles in blood cell differentiation and are also involved in leukemogenesis, to study their molecular structure-activity relationship for establishment of the fine regulation of specific gene expressions, using molecular structural, biochemical, and cell biological analyses.Firstly, to examine roles of the protein-protein interactions for stabilization of the multi protein-DNA complex, we analyzed dynamic aspects of Runxl in the Runxl-DNA and the Runxl-CBFβ-DNA complexes using NMR relaxation experiments. The obtained data indicated that some loop regions of Runxl including the DNA-interacting loops are conformationally fluctuating on various time scales from nano-to milli-seconds in the free state, and become stabilized upo … More n CBFβ binding. From the measurements of DNA binding affinities with the variously point-mutated Runxl mutants, we have found that the conformational fluctuations in a particular time scale would correlate with the allosteric regulation of the Runxl DNA binding by CBFβ.Secondly, to examine roles of the stereospecificity of the multi protein-DNA assembly for the transactivational regulation, we analyzed structural aspects of the cooperation between c-Myb and C/EBP on the mim-1 promoter, a c-Myb target gene, using X-ray crystallography, atomic force microscopic analyses and thorough biochemical and cell biological experiments. From these analyses, we have found specific c-Myb- C/EBP interactions on a looped DNA. In contrast, we have observed no such stereospecific assembly of Myb and C/EBP on the DNA, when AMV v-Myb, an oncogenic c-Myb mutant, was used instead of c-Myb. These results have provided the first molecular structural evidence of a transactivational cooperation between distantly bound transcriptional regulatory proteins on a promoter DNA in the eukaryotic system. Less

本研究旨在阐明多种转录调控蛋白调控转录的分子机制。本研究以造血细胞分化过程中起关键作用的转录调控蛋白Runxl、CBFβ、c-Myb、C/EBP等为研究对象，通过分子结构、生物化学和细胞生物学分析，研究其分子结构与活性的关系，以期建立特异性基因表达的精细调控机制。为了研究蛋白质-蛋白质相互作用对多蛋白质-DNA复合物稳定性的作用，我们使用NMR弛豫实验分析了Runxl-DNA和Runxl-CBFβ-DNA复合物中Runxl的动力学方面。所获得的数据表明，Runxl的一些环区域，包括DNA相互作用环，在自由状态下在从纳秒到毫秒的各种时间尺度上构象波动，并在一定时间内变得稳定。 ...更多信息 n CBFβ结合。通过对Runxl突变体与DNA结合亲和力的测定，我们发现在特定时间尺度上的构象波动与CBFβ对Runxl DNA结合的变构调节有关。我们使用X射线晶体学、原子力显微镜分析和彻底的生物化学和细胞生物学实验分析了c-Myb和C/EBP在mim-1启动子（c-Myb靶基因）上的合作的结构方面。从这些分析中，我们发现了环状DNA上的特异性c-Myb- C/EBP相互作用。相比之下，我们已经观察到没有这样的立体特异性组装的Myb和C/EBP的DNA，当AMV v-Myb，致癌的c-Myb突变体，被用来代替c-Myb。这些结果提供了第一个分子结构的证据，在真核系统中的启动子DNA上的远距离结合的转录调控蛋白之间的反式激活合作。少

项目成果

Ogata, K.: "Eukaryotic transcription complexes : cooperativity from near and afar"Current Opinion in Structural Biology. 13. 40-48 (2003)

Ogata, K.：“真核转录复合物：近处和远方的协同性”结构生物学的当前观点。

Structural aspects of gene expression by AML1/CBE,c-Myb, AMV v-Myb, C/EBP. and Ets-1

AML1/CBE、c-Myb、AMV v-Myb、C/EBP 基因表达的结构方面。

• 发表时间: 2002
• 期刊: Acta Cryst. A58Supplement
• 作者: Tahirov;T. H.;Sato;K;Inoue-Bungo;T.;Ichikawa-Iwata;E.;Sasaki;M.;Fujikawa;A.;Shiina;M.;Takata;S.;Kimura;K.;Morii;H.;Kumasaka;T.;Yamamoto;M.;Ishii;S.;Ogata;K.
• 通讯作者: K.

構造解析のための試料調製(総論)-タンパク質-DNA複合体を中心として-

用于结构分析的样品制备（概述） - 关注蛋白质-DNA 复合物 -

• 发表时间: 2002
• 期刊: 遺伝子医学 6
• 作者: 緒方一博

Ogata, K.: "c-Myb DNA-binding domain : from molecular structure to functions "Myb Transcription Factors : Their Role in Growth, Differentiation and Diseases", ed.by J.Frampton"Kluwer Academic Publishers B.V.(in press).

Ogata, K.：“c-Myb DNA 结合域：从分子结构到功能“Myb 转录因子：它们在生长、分化和疾病中的作用”，J.Frampton 编辑“Kluwer 学术出版社 B.V.（正在出版）。

特集 : ゲノムからプロテオームヘ-ヒトプロテオミクスの目指すもの- ポストゲノム時代の生化学,構造生物学-生体分子の機能発現機構の解明を目指して-

专题：从基因组到蛋白质组 - 人类蛋白质组学的目标是什么 - 后基因组时代的生物化学和结构生物学 - 旨在阐明生物分子功能表达的机制 -

• 发表时间: 2004
• 期刊: 生化学 76
• 作者: 緒方 一博