Genomic-wide Analysis of Oct 3/4 and Nanog Targets

描述（由申请人提供）：从鼠囊胚内细胞团中分离的胚胎干（ES）细胞是多能性的，能够进行无限对称细胞分裂，并可产生嵌合动物。最近分离的人ES细胞对于治疗各种退行性疾病（包括但不限于帕金森病和糖尿病）具有很大的希望。同源结构域转录因子Oct 3/4和Nanog被认为在维持ES细胞多能性中起关键作用。令人惊讶的是，Nanog的目标是完全未知的，很少有人提出Oct 3/4目标。因此，抑制ES细胞分化和促进自我更新的分子机制还不清楚。我们已经开发出一种新的方法来识别复杂的后生动物调节子称为SACO（染色质占据序列分析），它结合了染色质免疫沉淀与修改长SAGE。我们将使用SACO来鉴定小鼠ES细胞中Nanog和Oct 3/4基因组靶标的完整互补。这些知识不仅有助于表征管理干细胞的细胞命运定型的机制，而且还可以使现有的人类干细胞系无限繁殖。深入了解这些转录网络也可能导致从成人组织或细胞中产生多能干细胞。我们的研究旨在提供多能干细胞中Nanog和Oct 3/4靶点的完整定义。 通过表征相应转录物的调节，我们期望表征控制ES细胞自我更新和多能性的分子途径。代表新型Nanog和Oct 3/4驱动转录本的微阵列和基因组结合位点的目录将提供给干细胞研究界。代表新Nanog和Oct 3/4靶标的微阵列的创建将促进对它们在早期胚胎发生期间的表达及其通过调节分化和多能性的途径的调节的高通量分析。这组研究将代表迄今为止对任何后生动物系统中转录因子结合调节的最全面的分析。

DESCRIPTION (provided by applicant): Embryonic stem (ES) cells isolated from the inner cell mass of murine blostocysts are pluripotent, capable of indefinite symmetric cell division, and can generate chimeric animals. The recent isolation of human ES cells holds great promise for the treatment of a variety of degenerative disorders including, but not limited to, Parkinson's disease and diabetes. The homeodomain transcription factors, Oct3/4 and Nanog, are believed to play critical roles in sustaining ES cell pluripotency. Surprisingly, targets of Nanog are completely unknown and few Oct3/4 targets have been proposed. Thus, the molecular mechanisms that repress differentiation of ES cells and promote self-renewal are poorly defined. We have developed a novel approach for identifying complex metazoan regulons called SACO (Serial Analysis of Chromatin Occupancy), which combines chromatin immunoprecipitation with a modification of Long SAGE. We will use SACO to identify the entire complement of Nanog and Oct3/4 genomic targets in mouse ES cells. Such knowledge would not only aid efforts to characterize mechanisms that govern cell-fate commitment of stem cells, but could also enable the indefinite propagation of existing human stem cell lines. Insight into these transcriptional networks could also lead to the generation of pluripotent stem cells from adult tissues or cells. Our studies are designed to provide a complete definition of Nanog and Oct3/4 targets in pluripotent stem cells. By characterizing the regulation of the corresponding transcripts, we expect to characterize the molecular pathways that control self-renewal and pluripotency of ES cells. The microarrays representing novel Nanog and Oct3/4 driven transcripts and the catalog of genomic binding sites will be provided to the stem cell research community. The creation of microarrays representing novel Nanog and Oct3/4 targets will facilitate high-throughput analysis of their expression during early embryogenesis and their regulation by pathways that regulate differentiation and pluripotency. This set of studies will represent the most comprehensive analysis to date of the regulation of transcription factor binding in any metazoan system.