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

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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