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

本研究旨在阐明多种转录调控蛋白的转录调控分子机制。本研究以Runxl、CBFβ、C - myb、C/EBP等造血转录调控蛋白为研究对象，通过分子结构、生化和细胞生物学分析，研究其分子结构-活性关系，建立对特定基因表达的精细调控。首先，为了研究蛋白-蛋白相互作用在稳定多蛋白- dna复合物中的作用，我们使用核磁共振弛豫实验分析了Runxl在Runxl- dna和Runxl- cbf - β- dna复合物中的动态方面。得到的数据表明，Runxl的一些环区，包括dna相互作用环，在从纳米到毫秒的自由状态下，在不同的时间尺度上发生构象波动，并在与cbf - β结合时变得稳定。通过对不同点突变的Runxl突变体的DNA结合亲和力的测量，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。其次，为了研究多蛋白- dna组装的立体特异性在交互激活调控中的作用，我们利用x射线晶体学、原子力显微镜分析和全面的生化和细胞生物学实验，分析了C - myb和C/EBP在C - myb靶基因mim-1启动子上的合作结构。从这些分析中，我们发现了环DNA上特定的C - myb - C/EBP相互作用。相比之下，当使用致癌性C -Myb突变体AMV -Myb代替C -Myb时，我们没有观察到DNA上Myb和C/EBP的这种立体特异性组装。这些结果提供了真核系统中启动子DNA上远结合的转录调节蛋白之间的转激活合作的第一个分子结构证据。少

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