Grant amount：\4420000 （ Direct Cost: \3400000 、 Indirect Cost：\1020000 ）

本研究は、遺伝子転写を正に調節するヌクレオソーム修飾酵素MMSETの異常な活性亢進によって多発性骨髄腫(MM)や急性リンパ性白血病(ALL)が引き起こされるメカニズムの解明を目的とした、クライオ電子顕微鏡によるMMSET-ヌクレオソーム複合体の高解像度での立体構造決定を行う。
これまでの予備実験において低解像度(約8Å)のMMSET-ヌクレオソーム複合体の立体構造が得られていた。本年度はさらなる高解像度のデータ取得を目的として、試料緩衝液の塩濃度、試料濃度、試料グリッド作製条件などを含めた試料調製方法全般の最適化や、予備実験で用いていた200kVの加速電圧を持つクライオ電子顕微鏡Talos Arcticaから、より高出力(300kV)の Titan Kriosに測定装置を変更してデータを取得し解析を行った。その結果、アミノ酸側鎖が観測可能な解像度(2.8Å)まで解像度が向上した。得られた高解像度の立体構造から、MMSETはヌクレオソームと複合体を作ることによって単体構造から大きく構造変化を起こしていること、ヌクレオソーム単体構造と比較してＤＮＡが一部はがれていること、またDNAがはがれたヌクレオソーム部分にMMSETが入れ替わるように結合していることがわかった。
本研究で用いたMMSETはヒストンH3の36番目のリジンをメチル化する活性が異常亢進をしているガン化変異体(1099番目のグルタミン酸がリジンに変異)である。得られた複合体の高解像度の立体構造から、なぜ１アミノ酸の変異によって酵素の活性が異常亢進しているのかを解明するために、様々なアミノ酸変異体の作製及び活性の測定や、分子動力学的な手法を用いたシミュレーションを現在行っている。

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Grant amount：\105600000 （ Direct Cost: \105600000 ）

Multiple transcription factors (TFs) regulate transcription by forming TFs-DNA complexes. A cell signaling causes chemical modification of transcription factors, which modulates transcriptional activities. Besides, effect of chemical modification of transcription factors on their activity in the context of TFs-DNA complexes, however, is largely unknown. In this sturdy, we showed that the phosphorylation of a transcription factor Ets1 "differentially" modulates Ets1-containing TFs-DNA complexes. In addition, we clarified the underlying mechanism by which the selective modulation of Ets1-containing TFs-DNA complexes is achieved using NMR analyses of allosteric regulation of Runx1 by CBFβ and crystallographic analyses of multiprotein-DNA complex comprised of Ets1, Runx1 and CBFβ formed on the TCRα gene enhancer.

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Grant amount：\15300000 （ Direct Cost: \15300000 ）

The goal of this project is to elucidate molecular mechanisms for regulating the enhanceosome formation, particularly, stabilization of transcription factors bound to an enhancer region of a gene.
We investigated some complexes containing a transcriptional regulatory factor, Runx1, which plays a pivotal role in hematopoiesis and is involved in acute leukemias. The DNA binding activity of Runxl is enhanced allosterically by non-DNA binding heterodimeric partner, CBFβ. Previously, we showed that there was no significant structural change of Runxl upon binding of CBFβ when a crystal structure of Runx1-DNA complex was compared with that of Runx1-CBFβ-DNA complex. It promoted us to focus on molecular fluctuations of Runx1. Magnetic relaxation measurements using NMR demonstrated that some regions of Runx1 are significantly fluctuated without CBFβ, while the fluctuations of Runx1 became reduced upon binding of CBFβ. We proposed that regulations of molecular functions are accomplished by altering molecular fluctuations.
Runxl is also involved in transcriptional regulation of the T cell antigen receptor a chain (TCRα) together with another transcriptional regulatory factor, Etsl. The DNA binding activity of Etsl is controlled by an N-terminal flanking region of ETS domain, referred exon VII. The exon VII inhibits the DNA binding activity of Ets1 while the inhibition is released by an adjacently placed Runxl. Etsl is not capable of binding the enhancer upon phosphorylation of exon VII even if Runxl binds to the enhancer. We investigated the molecular details of the complex composed of Runxl and Etsl in the TCRα enhancer using X-ray crystallography, EMSA and the luciferase reporter gene assay. We identified which regions of Etsl are responsible for cooperative DNA binding with Runxl and the impact of the exon VII phosphorylation. Further structural analyses might be required to unveil overall aspect of the regulatory systems. We will also study about fluctuations of Runxl upon Etsl binding.

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Grant amount：\16200000 （ Direct Cost: \16200000 ）

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 upon 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.

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Grant type：Competitive

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