Abstract
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.
Highlights
Pluripotent embryonic stem cells (ESCs) are highly proliferative cells that can expand indefinitely
The isolate proteins on nascent DNA (iPOND) technique utilizes the rapid incorporation of the thymidine analogue 5-ethynyl-2 -deoxyuridine during deoxyribonucleic acid (DNA) replication, covalent cross-linking between DNA and proteins, the addition of a biotin moiety to the incorporated ethynyl deoxyuridine (EdU) using mild conditions and streptavidin-biotin affinity to capture sheared EdU-labelled chromatin
The other major repressive histone mark, the methylated lysine 27 at histone H3 and its acetylated form was not significantly affected. These results show that valproic acid (VPA) has pronounced effects on the deposition of epigenetic marks during DNA replication, and suggests that in ESCs, HDAC1 could act at nascent DNA by regulating the rapid deacetylation of H3K9 in ESCs, which is necessary for their subsequent methylation during replication
Summary
Pluripotent embryonic stem cells (ESCs) are highly proliferative cells that can expand indefinitely This unlimited expansion is sustained by their self-renewal capacity, which relies on a high fidelity of the genome and the epigenome transmission during deoxyribonucleic acid (DNA) replication [1,2]. The network is centred by the pluripotent transcription factors OCT4, NANOG and SOX2, which act in a coordinated manner with chromatin modifying complexes [1,3]. These complexes include Polycomb repressor complexes (PRC) 1 and 2, BRG1 associated factors (esBAF) complex and the nucleosomal remodelling and deacetylase (NuRD) complex [1,4]. This question is especially relevant for ESCs, which display a rapid cell cycle with a shortened G1 phase and a dominant DNA replication phase [2,8]
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