Project 1: Defining the Logic of Genome Organization In Pluripotent Cells

描述 该提案的目标是揭示控制三维（3D） 在多能细胞中以及在重编程为IPS细胞状态期间，基因组的结构。染色体的空间组织是后生动物生物学的一个迷人的问题，但留下了许多未回答的问题，特别是破解驱动遗传基因座共定位的机制的挑战。 基于分化的细胞数据，细胞核中染色体相互作用的3D网络被认为对于维持细胞身份是重要的，并且通过基因及其调控区的空间聚类以及通过将基因组聚集在相同的亚核结构允许其协调表达和调节表观遗传状态来影响基因表达。多能细胞基因组的3D结构基本上未被研究。然而，最近一项关于DNA复制时机的研究表明，基因组的大规模重组与多能细胞的承诺相一致， 分化，在胚层规格之前。这一过程的逆转似乎是重编程的最后步骤之一，与重编程因子与多能性基因靶标的结合以及全局染色质结构和DNA复制模式的变化有关。基于这些发现，我们假设，只有通过揭示多能细胞的空间组织之间的结构和功能关系，才能真正理解多能细胞和重编程过程中的基因组调控。 基因组和线性基因组特征，如染色质和表达状态以及与转录因子的关联;以及多能核结构的建立代表了重编程的障碍。为了开始解决多能细胞中的3D基因组相互作用，我的实验室已经成功建立了4C-seq方法来识别整个基因组中物理上接近Oct 4位点的区域，这表明该位点与早期复制的高度表达的基因相互作用，这些基因被自身在Oct 4位点富集的多能性转录因子结合。基于这一广泛的工作，我们在重编程和多能细胞染色质方面的专业知识，沿着P01内部的特定合作以及与P01生物信息学核心的紧密联系，我们处于有利地位，可以揭示人类和小鼠胚胎干细胞（ES）中3D基因组相互作用的分子机制，并研究分化细胞基因组如何在人类细胞重编程过程中重组;具有以下目标：

Description The goal of this proposal is to reveal the mechanisms that govern the three-dimensional (3D) architecture of the genome in pluripotent cells and during reprogramming to the IPS cell state. The spatial organization of chromosomes is a fascinating problem of metazoan biology, but leaves many unanswered questions, particularly the challenge to decipher the mechanisms driving the co-localization of genetic loci. Based on differentiated cell data, the 3D network of chromosomal interactions in the nucleus is thought to be important for the maintenance of cell identity and affect gene expression by spatial clustering of genes and their regulatory regions and by congregating groups of genes at the same sub-nuclear structure allowing their coordinated expression and modulation of epigenetic states. The 3D structure of the pluripotent cell genome is basically unstudied. However, a recent study of DNA replication timing, to which my lab contributed, suggested that a large-scale reorganization of the genome coincides with the commitment of pluripotent cells to differentiation, prior to germ layer specification. The reversal of this process appears to be one of the final steps of reprogramming, linked to the binding of the reprogramming factors to pluripotency gene targets and changes in global chromatin structure and DNA replication patterns. Based on these findings, we hypothesize that a true understanding of genome regulation in pluripotent cells and during reprogramming can only be obtained by revealing the structural and functional relationships between the spatial organization of the genome and linear genomic features such as chromatin and expression states and association with transcription factors; and that the establishment of the pluripotent nuclear architecture represents a road block to reprogramming. In an effort to begin to address 3D genomic interactions in pluripotent cells, my laboratory has successfully established the 4C-seq method to identify regions throughout the genome that are physically close to the Oct4 locus, which revealed that this locus interacts with early replicating, highly expressed genes that are bound by pluripotency transcription factors that themselves are enriched at the Oct4 locus. Based on this extensive work, our expertise in reprogramming and pluripotent cell chromatin, along with specific collaborations within the P01 and strong ties to the P01 Bioinformatics Core, we are well positioned to unveil molecular mechanisms governing the 3D genomic interactions in human and mouse embryonic stem (ES) cells and to study how the differentiated cell genome is reorganized during human cell reprogramming; with these Aims: