Uncovering the transcription factor networks in early human cell specification

描述（由申请人提供）：揭示早期人类细胞特化中的转录因子网络。 在原肠胚形成期间，多能性外胚层细胞产生三个胚层--内胚层、中胚层和外胚层。在发育后期，这些层形成了我们身体中几乎所有不同的组织和器官。然而，建立从外胚层到三个胚层的过渡的分子机制在很大程度上仍然未知。一类被称为转录因子（TF）的蛋白质可以结合特定的DNA元件并激活或抑制基因表达。TF将成纤维细胞“去分化”为诱导的多能干细胞（1，2）或将细胞直接编程为各种谱系（3）的能力使其成为在发育期间调节细胞转变的可能候选者。 为了更深入地了解指导早期人类细胞特化的调控事件，需要对TF结合及其动力学进行更全面的研究。目前对TF在发育中的研究主要集中在稳态条件下单一因子的调节作用，但许多TF以动态和合作的方式介导信号级联下游的基因表达，这是每种细胞类型所独有的（4，5）。在我的博士后研究期间，我的目标是揭示细胞特化背后的分子事件。首先，我将确定超过30个TF，DNA甲基化，染色质标记和RNA表达在多个决策时间点，在人类胚胎干细胞（ES）分化为内胚层，中胚层和外胚层的全基因组动态。这种全面的TF结合动力学图谱将使我能够剖析建立细胞身份的主调节因子、辅因子和信号蛋白之间的组合和时间相互作用。其次，我将结合联合收割机这些动态测量，以产生一个临时模型的网络，控制细胞的身份在不同的胚层和解剖TF占用和表观遗传状态之间的相互作用，在调节发展。第三，我将使用网络中关键TF的选定RNAi扰动来验证和重申我的多维模型预测。这些数据的结合将使我能够揭示建立细胞命运的原则和参与者。 拟议的研究将大大提高我们对调控电路及其在人类早期发育细胞分化过程中的作用的理解，这反过来将提高我们获得治疗方法的能力，例如体外产生心肌细胞，胰岛和神经元。这有望对防治脊髓损伤、青少年糖尿病和帕金森病等多种疾病产生重大影响。

DESCRIPTION (provided by applicant): Uncovering the transcription factor networks in early human cell specification. During gastrulation, pluripotent epiblast cells give rise to the three germ layers-- endoderm, mesoderm, and ectoderm. Later in development, these layers form nearly all the different tissues and organs in our body. However the molecular mechanisms that establish the transition from epiblast to the three germ layers are still largely unknown. A class of proteins known as transcription factors (TFs) can bind specific DNA elements and activate or repress gene expression. The capacity of TFs to "de-differentiate" fibroblast cells to induced pluripotent stem cells (1, 2) or to directly program cells into various lineages (3) makes them likely candidates for regulating cellular transitions during development. To gain a deeper understanding of the regulatory events that guide early human cell specification, a more comprehensive study of TF binding and their dynamics is needed. Current research on TFs in development mainly focuses on the regulatory role of single factors in steady state conditions, yet many TFs mediate gene expression downstream of signaling cascades in a dynamic and cooperative fashion that is unique to each cell type (4, 5). During my postdoctoral studies I aim to uncover the molecular events underling cellular specification. First, I will determine the genome-wide dynamics of over 30 TFs, DNA methylation, chromatin marks, and RNA expression at multiple decision time-points, during differentiation of human embryonic stem (ES) cells into endoderm, mesoderm, and ectoderm. Such comprehensive maps of TF binding dynamics will allow me to dissect the combinatorial and temporal interactions between master regulators, cofactors, and signaling proteins that establish cell identity. Second, I will combine these dynamic measurements to generate a provisional model of the network that controls cell identity in the different germ layers and dissect the interplay between TF occupancy and epigenetic state in the regulation of development. Third, I will validate and reiterate my multi-dimensional model predictions using selected RNAi perturbations of critical TFs in the network. The combination of this data will allow me to uncover the principles and players that establish cell fate. The proposed research will greatly enhance our understanding of regulatory circuits and their roles during cell differentiation in early human development, which will, in turn, improv our ability to derive therapeutic approaches, such as in vitro generation of cardiomyocytes, pancreatic islets, and neurons. This promises to have significant impact in combating number of diseases, such as spinal cord injury, juvenile diabetes, and Parkinson's disease.