Consortia for High-Throughput-Enabled Structural Biology Partnerships (U01)

这一建议的主要目标是揭示潜在的分子机制的形成和功能。 胚胎干细胞和诱导多能性细胞所必需的关键转录组件 (诱导多能干细胞)。利用生物信息学和高通量实验方法，我们 将准备控制细胞多能性的关键转录因子的明确结构域并对其进行分析 在他们的功能协会中。数以千计的组件将通过生化和生物物理进行评估 方法确定X射线结晶学研究的关键靶点。我们的目标是确定 大约100个稳定的多组分转录组件的三维结构。他们中的每一个 将代表控制特定转录的复杂转录机制的部分图像 多能细胞的景观。我们希望，对这些结构的深思熟虑的分析将使我们能够 在这些局部图像之间建立适当的联系，并重建功能的一般模型 这一转录机制的关键参与者。我们将证明这种模式和遵守的监管措施是合理的。 利用诱导性多能性细胞进行突变和功能研究中识别的转录复合体内的相互作用。 实验将在多个地点进行：卫理公会医院研究所(休斯顿)， 加州大学格拉德斯通心血管疾病研究所生物化学和生物物理系 (加州大学旧金山分校)和PSI实验室的X射线结晶学。拟议的结构和功能研究将推动 我们对控制细胞命运的基本机制的一般知识，包括那些潜在的自我更新， 癌症的分化和发病机制。这项研究的结果也将提供更有效的 分子工具，可精确控制细胞编程和重新编程。累积的 结构和功能数据将立即提供给生化和临床研究人员，以及 因此，这将对干细胞研究以及再生医学产生重大影响。

The major goal of this proposal is to reveal molecular mechanisms underlying formation and function of critical transcriptional assemblies essential to embryonic stem (ES) cells and cells with induced pluripotency (induced pluripotent stem (iPS) cells). Using bioinformatics and high throughput experimental methods, we will prepare defined domains of critical transcriptional factors controlling cell pluripotency and analyze them in their functional associations. Thousands of assemblies will be evaluated by biochemical and biophysical methods to identify the critical ones to be targeted by X-ray crystallography. We aim for determination of three-dimensional structures for about 100 stable multi-component transcriptional assemblies. Each of them will represent a partial image of complicated transcriptional machinery controlling the specific transcriptional landscape of pluripotent cells. We expect that thoughtful analyses of these structures will enable us to establish the proper connections between these partial images and reconstruct a general model for function of critical participants of this transcriptional machinery. We will justify this model and the observed regulatory nteractions within identified transcriptional complexes in mutational and functional studies using iPS cells. The experiments are to be done at multiple sites: The Methodist Hospital Research Institute (Houston), Department of Biochemistry and Biophysics at UCSF, the Gladstone Institute of Cardiovascular Disease (UCSF) and X-ray crystallography by the PSI labs. The proposed structural and functional studies will propel our general knowledge ofthe basic mechanisms controlling cell fate, including those underlying self renewal, differentiation and pathogenesis of cancer. The results of this research will also provide more efficient molecular tools allowing precise control over cell programming and reprogramming. The accumulated structural and functional data would be immediately available to biochemical and clinical researchers, and therefore, would have a major impact on stem cell research as well as regenerative medicine.