Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Sci Signal. 2011 Mar 15;4(164):rs3. doi: 10.1126/scisignal.2001570.
To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those accompanying their differentiation. From these data, we identified a core hESC phosphoproteome of sites with similar robust changes in response to the two distinct treatments. These sites exhibited distinct dynamic phosphorylation patterns, which were linked to known or predicted kinases on the basis of the matching sequence motif. In addition to identifying previously unknown phosphorylation sites on factors associated with differentiation, such as kinases and transcription factors, we observed dynamic phosphorylation of DNA methyltransferases (DNMTs). We found a specific interaction of DNMTs during early differentiation with the PAF1 (polymerase-associated factor 1) transcriptional elongation complex, which binds to promoters of the pluripotency and known DNMT target genes encoding OCT4 and NANOG, thereby providing a possible molecular link for the silencing of these genes during differentiation.
为了阐明人类胚胎干细胞（hESC）多能性的细胞事件，我们对通过二酰基甘油类似物或转移到未被饲养细胞条件化的培养基中启动的分化过程中的 hESC 进行了平行的定量蛋白质组学和磷酸化蛋白质组学分析。我们对 6521 种蛋白质和 23522 个磷酸化位点进行了分析，其中近 50%的磷酸化状态在 24 小时的分化过程中发生了动态变化。这些数据是研究与 hESC 多能性维持相关的事件以及伴随其分化的事件的资源。从这些数据中，我们确定了一个 hESC 磷酸化蛋白质组的核心，其中有许多位点对两种不同的处理都有相似的强变化。这些位点表现出不同的动态磷酸化模式，根据匹配的序列基序与已知或预测的激酶相关。除了鉴定与分化相关的因素（如激酶和转录因子）上以前未知的磷酸化位点外，我们还观察到 DNA 甲基转移酶（DNMTs）的动态磷酸化。我们发现 DNMTs 在早期分化过程中与 PAF1（多聚酶相关因子 1）转录延伸复合物的特定相互作用，该复合物与多能性和已知的 DNMT 靶基因的启动子结合，这些基因编码 OCT4 和 NANOG，从而为这些基因在分化过程中的沉默提供了一个可能的分子联系。
