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

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