Study of molecular structural and dynamical aspects of transcriptional regulatory factors

本项目的目标是阐明增强体形成的分子调控机制，特别是与基因增强子区域结合的转录因子的稳定性，我们研究了一些含有转录调控因子Runx 1的复合物，Runx 1在造血中起着关键作用，并参与急性白血病。Runxl的DNA结合活性通过非DNA结合异二聚体配偶体CBFβ变构增强。以前，当比较Runx 1-DNA复合物与Runx 1-CBFβ-DNA复合物的晶体结构时，我们表明在CBFβ结合后Runx 1没有显著的结构变化。它促使我们关注Runx 1的分子波动。核磁共振的磁弛豫测量表明，在没有CBFβ的情况下，Runx 1的某些区域发生了明显的波动，而在结合CBFβ后，Runx 1的波动变得减少。我们认为，分子功能的调节是通过改变分子结构来实现的。 ...更多信息 Runxl还与另一种转录调节因子Etsl一起参与T细胞抗原受体α链（TCRα）的转录调节。Etsl的DNA结合活性由ETS结构域的N-末端侧翼区域（称为外显子VII）控制。外显子VII抑制Ets 1的DNA结合活性，而该抑制通过相邻放置的Runx 1释放。Etsl不能在外显子VII磷酸化后结合增强子，即使Runxl结合增强子。我们利用X射线晶体学、EMSA和荧光素酶报告基因分析研究了TCRα增强子中Runxl和Etsl组成的复合物的分子细节。我们确定了Etsl的哪些区域负责与Runxl的协同DNA结合以及外显子VII磷酸化的影响。可能需要进行进一步的结构分析，以揭示监管制度的总体方面。我们还将研究Runxl在Etsl结合时的波动。少

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 alteri … More ng 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. Less