Structures of Protein Complexes Regulating Transcription in Enbryonic Stem Cells

描述(申请人提供)：本提案的主要目的是揭示胚胎干细胞(ES细胞)和具有诱导多能性的细胞(诱导多能干细胞)所必需的关键转录组件的形成和功能的分子机制。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，并分析它们的功能关联。数以千计的组件将通过生化和生物物理方法进行评估，以确定X射线结晶学要针对的关键组件。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。它们中的每一个都代表了复杂的转录机制的部分图像，这些机制控制着多能细胞的特定转录格局。我们希望，对这些结构的深入分析将使我们能够在这些部分图像之间建立适当的联系，并重建这一转录机制的关键参与者的功能的一般模型。我们将在使用iPS细胞的突变和功能研究中证明这一模型和所观察到的转录复合体中的调控相互作用是合理的。这些实验将在多个地点进行：卫理公会医院研究所(休斯顿)、加州大学旧金山分校的生物化学和生物物理系、格拉斯顿心血管疾病研究所(UCSF)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，包括那些潜在的自我更新，分化和癌症发病机制的一般认识。这项研究的结果还将提供更有效的分子工具，允许对细胞编程和重新编程进行精确控制。积累的结构和功能数据将立即提供给生化和临床研究人员，因此，将对干细胞研究和再生医学产生重大影响。

DESCRIPTION (provided by applicant): 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 interactions 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 of the 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.