Regulation of pluripotency and lineage decisions by histone methylation

胚胎干细胞（ESCs）提供了一个无与伦比的场所来研究定义多能性和谱系规范的基本过程，以及利用这些知识进行再生医学新兴疗法的潜力。ESCs的最新进展确定了多能性窗口期的三个阶段；即Nanog阳性ESCs (Nanog on)， Nanog阴性ESCs （Nanog off）和外胚层干细胞（EpiSCs）。在第一个资助期内，申请人的工作已经确定了这些ESC转变与赖氨酸4 （H3K4me3）和27 （H3K27me3）的组蛋白3三甲基化之间的联系，这也是基于trithorax-Group （trx-G）的基因激活/维持和Polycomb-Group （PcG）的沉默的相反调控系统的标志。出乎意料的是，我们最近发现谱系特异性基因的H3K27三甲基化是在从Nanog打开状态到Nanog关闭状态的转变过程中获得的。此外，许多外胚层谱系特异性基因启动子似乎受到trx-G因子Mll2的特异性调节。在拟议的研究中，我们将系统地检查和比较选定的trx-G、PcG和某些其他蛋白质在条件诱变后的三个多能性阶段的蛋白质相互作用、基因组结合位点和表达谱。这些分析已经开始，主要集中在已知参与H3K4和H3K27甲基化的两种组蛋白甲基转移酶（分别为Mll2和Ezh2）上。利用强大的蛋白质标记，基因靶向和条件诱变策略，以及质谱和深度测序方法，我们的目标是确定多能性调控层次的关键方面。

Embryonic stem cells (ESCs) provide an unrivalled venue to study the basic processes defining pluripotency and lineage specification, as well as the potential to harness this knowledge for emerging therapies in regenerative medicine. Recent progress with ESCs has identified three stages within the pluripotent window; namely Nanog positive ESCs (Nanog on), Nanog negative (Nanog off) and Epiblast stem cells (EpiSCs). Work by the applicants, supported during the first funding period, has identified linkages between these ESC transitions and histone 3 trimethylation at lysines 4 (H3K4me3) and 27 (H3K27me3), which are also hallmarks of the opposing regulatory systems based on gene activation/maintenance by the trithorax-Group (trx-G) and silencing by the Polycomb-Group (PcG) respectively. Unexpectedly, we recently found that H3K27 trimethylation of lineage specific genes is acquired during transition from Nanog on to Nanog off states. Furthermore many of the ectodermal lineage specific gene promoters appear to be specifically regulated by the trx-G factor, Mll2. In the proposed research, we will systematically examine and compare selected trx-G, PcG and certain other proteins for protein-protein interactions, genome binding sites and expression profiles after conditional mutagenesis, in the three pluripotent stages. These analyses, which have already begun, are focused on the two histone methyltransferases already known to be involved in H3K4 and H3K27 methylation (Mll2 and Ezh2 respectively). Using robust protein tagging, gene targeting and conditional mutagenesis strategies, together with mass spectrometry and deep sequencing methods, we aim to identify key aspects of the regulatory hierarchy of pluripotency.