Aranda Sergi, Rutishauser Dorothea, Ernfors Patrik
Unit of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 17177 Stockholm, Sweden
Proteomics Karolinska, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Nucleic Acids Res. 2014 Jun;42(11):6972-86. doi: 10.1093/nar/gku374. Epub 2014 May 22.
Pluripotency of embryonic stem cells (ESCs) is maintained by transcriptional activities and chromatin modifying complexes highly organized within the chromatin. Although much effort has been focused on identifying genome-binding sites, little is known on their dynamic association with chromatin across cell divisions. Here, we used a modified version of the iPOND (isolation of proteins at nascent DNA) technology to identify a large protein network enriched at nascent DNA in ESCs. This comprehensive and unbiased proteomic characterization in ESCs reveals that, in addition to the core replication machinery, proteins relevant for pluripotency of ESCs are present at DNA replication sites. In particular, we show that the chromatin remodeller HDAC1-NuRD complex is enriched at nascent DNA. Interestingly, an acute block of HDAC1 in ESCs leads to increased acetylation of histone H3 lysine 9 at nascent DNA together with a concomitant loss of methylation. Consistently, in contrast to what has been described in tumour cell lines, these chromatin marks were found to be stable during cell cycle progression of ESCs. Our results are therefore compatible with a rapid deacetylation-coupled methylation mechanism during the replication of DNA in ESCs that may participate in the preservation of pluripotency of ESCs during replication.
胚胎干细胞（ESC）的多能性是由染色质内高度组织化的转录活性和染色质修饰复合物维持的。尽管人们在识别基因组结合位点方面付出了很多努力，但对于它们在细胞分裂过程中与染色质的动态关联却知之甚少。在这里，我们使用了改良版的iPOND（新生DNA上蛋白质的分离）技术来识别在胚胎干细胞新生DNA上富集的大型蛋白质网络。对胚胎干细胞进行的这种全面且无偏差的蛋白质组学表征表明，除了核心复制机制外，与胚胎干细胞多能性相关的蛋白质也存在于DNA复制位点。特别是，我们发现染色质重塑因子HDAC1-NuRD复合物在新生DNA上富集。有趣的是，胚胎干细胞中HDAC1的急性阻断会导致新生DNA上组蛋白H3赖氨酸9的乙酰化增加，同时甲基化丧失。一致地，与肿瘤细胞系中所描述的情况相反，这些染色质标记在胚胎干细胞的细胞周期进程中是稳定的。因此，我们的结果与胚胎干细胞DNA复制过程中一种可能参与维持胚胎干细胞复制过程中多能性的快速去乙酰化偶联甲基化机制相符。
