Pluripotency of embryonic stem cells (ESCs) is maintained by transcriptional activities

Pluripotency of embryonic stem cells (ESCs) is maintained by transcriptional activities and chromatin modifying complexes highly organized within the chromatin. we show that the chromatin remodeller HDAC1CNuRD organic is usually enriched at nascent DNA. Oddly enough, an acute stop of HDAC1 in ESCs prospects 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 explained 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 quick deacetylation-coupled methylation mechanism during the replication of DNA in ESCs that may participate in the preservation of pluripotency of ESCs during replication. INTRODUCTION Pluripotent embryonic stem cells (ESCs) are highly proliferative cells that can expand indefinitely. This unlimited growth is usually 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 self-renewal capacity and the plasticity to differentiate into all the cell types of an adult organism are orchestrated and balanced by a unique protein conversation network. The network is usually centred by the pluripotent transcription factors OCT4, NANOG and SOX2, which take action in a coordinated manner with chromatin changing 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). With the aim to elucidate the functionality of these complexes, rigorous efforts have been undertaken to understand precisely where these epigenetic complexes are situated within the genome in ESCs using chromatin immunoprecipitation combined with massive parallel sequencing (CHIP-seq) (5C7). However, less is usually known on the mechanics of these interactions, in particular during cell cycle progression. This question is usually especially relevant for ESCs, which display a quick cell cycle with a shortened G1 phase and a dominating DNA replication phase (2,8). Very recently, a novel technique was developed to isolate proteins on nascent DNA (iPOND). Using highly proliferative transformed cells, the iPOND technology has enabled the isolation of proteins already known to be associated with the replication fork (9), as well as, in combination with mass spectrometry, the recognition of new replication associated factors in HEK293T cells (10,11). Although core replication protein were consistently recognized, such transformed cell lines are predicted to display abnormalities determining the tumour 19210-12-9 cell state, including modifications of chromatin regulatory protein. Therefore, results from these studies lend 19210-12-9 limited insights of replication associated proteins in ESCs which display unique characteristics compared to other cell types such as a high fidelity during replication (12C14), a unique transcriptional protein network within chromatin and a specific epigenetic composition (1,2). In this study, we undertook the recognition of proteins associated with nascent DNA during the DNA replication phase of ESCs. For this purpose, we used an adapted iPOND technology in combination with high-resolution mass spectrometry. Beside the recognition of proteins associated to the replisome and found in recent iPOND screens, our data in ESCs showed a designated enrichment of protein complexes involved in chromatin remodelling and changes. Among these protein complexes, HDAC1-made up of 19210-12-9 complexes, such as NuRD, 19210-12-9 19210-12-9 emerged as central in the protein conversation network that participates in the maintenance of the epigenome in the replication fork of ESCs. MATERIALS AND METHODS Cell culture R1 mouse ESCs (from A. Nagy, Toronto, Canada) were cultured as explained previously (15,16). For the experiments, ESCs were produced for 48 h on tissue culture dishes coated with 0.1% gelatin (Sigma) and cultured in serum-free medium (Knockout Dulbecco’s modified Eagle’s medium [DMEM], 15% Knockout Serum Replacement, 1 non-essential amino acids, 2 mM glutamine, 5 mM HEPES, 0.4 mM 2-mercaptoethanol [Gibco]) supplemented with 1000 U/ml ESGRO Leukemia Inhibitory Factor (Calbiochem). NIH-3T3 cells (Sigma) were cultured in DMEM supplemented with 10% foetal calf serum (Gibco). Rabbit Polyclonal to EDNRA The cells were transfected (Lipofectamine 2000; Invitrogen) with 30 nM of siRNA directed against mouse HDAC1 or CHD4 (ON-TARGETplus SMARTpool siRNA; Dharmacon) or with a control siRNA (Non-targeting siRNA; Dharmacon) for 48 h. When indicated, valproic acid (1 mM, Sigma), cycloheximide (10 g/ml, Sigma) and aphidicolin (0.1C1 g/ml, Sigma) were used. For differentiation, ESCs cells were produced in N2W27 media (DMEM/F12:Neurobasal 1:1, 0.5 N-2 complement, 1 B-27 serum-free complement, 3.2 mM 2-mercaptoethanol; all from Gibco) on monolayer for 5 days (17). Western blotting The samples were analysed by western blot as explained previously (18), and the data was quantified using Image Lab software (v4.0.1; Bio-Rad). The antibodies are outlined in Supplementary Table H3. Isolation of protein on nascent DNA (iPOND) The cells were pulsed for 10 min with 100 M of the thymidine analogue, ethynyl deoxyuridine (EdU). For the.