Wnt/β-catenin signaling pathway safeguards epigenetic stability and homeostasis of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) are pluripotent and can differentiate into cells belonging to the three germ layers of the embryo. However, mESC pluripotency and genome stability can be compromised in prolonged in vitro culture conditions. Several factors control mESC pluripotency, including Wnt/β-catenin signaling pathway, which is essential for mESC differentiation and proliferation. Here we show that the activity of the Wnt/β-catenin signaling pathway safeguards normal DNA methylation of mESCs. The activity of the pathway is progressively silenced during passages in culture and this results into a loss of the DNA methylation at many imprinting control regions (ICRs), loss of recruitment of chromatin repressors, and activation of retrotransposons, resulting into impaired mESC differentiation. Accordingly, sustained Wnt/β-catenin signaling maintains normal ICR methylation and mESC homeostasis and is a key regulator of genome stability.


Immunofluorescence staining
For immunofluorescence staining, cells were fixed with 4% paraformaldehyde for 20 min at room temperature, and then washed twice with PBS. Fixed cells were then incubated in blocking solution containing 10% goat serum (Sigma) and 0.1% Triton X-100 (Sigma) for 1 h at room temperature. The cells were then left overnight at 4 °C in blocking solution containing the primary antibody. The primary antibodies used for immunofluorescence (β-catenin (BD, 610153), NANOG (Calbiochem, #SC1000), , Nestin (Abcam ab6142), Tuj1 (Millipore mab1637), and their working dilutions are listed in Table S6. The next day, the cells were washed three times with PBS and then incubated with the secondary antibody for 1 h at room temperature. Goat anti-mouse IgG, goat anti-rabbit IgG, (1:1000, Life Technologies) conjugated to Alexa Fluor-488 or Alexa Fluor-594 were used as secondary antibodies.

Virus preparation and cell infection
For mouse embryonic stem cell (mESC) infection, lentiviral particles were produced following the RNA interference Consortium (TRC) instructions for lentiviral particle production and infection in 6-well plates (http://www.broadinstitute.org/rnai/public/).
Briefly 5 ×10 5 HEK293T cells/well were seeded in 6-well plates in DMEM, supplied with 10% FBS, 10 u/ml penicillin, streptomycin 10µg/ml, 2 mM glutamine, 1mM sodium pyruvate and 100X non-essential amino acids. The day after plating, the cells were co-transfected with 1 µg pLKO-shCtrl, pLKO-shBcat #1, #2, #3, 750 µg pCMV-dR8.9, and 250 µg pCMV-VSV-G, using Polyfect reagent (Qiagen). The day after transfection, the HEK293T culture medium was substituted with the mESC culture medium. Then 5×10 4 mESCs/well were plated onto gelatin-coated 6-well plates the day before transduction. The lentiviral-containing medium was harvested from HEK293T cells at 48, 72 and 96 h after transfection, filtered, and added to the mESC plates. The day after transduction, these mESCs were washed twice in PBS and Hygromycin B selection (50 µg/ml) was applied. Not infected cells were used in parallel as a control to check the efficacy of Hygromycin B selection.

Flow cytometry analysis
The cells were washed once in PBS with 5% FBS. Next , the cells were incubated for 20 min at 4°C with either E-cadherin (0,5 µg/10 6 cells, Biolegend 147308) or SSEA-1 (0,5 µg/10 6 cells, Biolegend 125607) antibody in PBS with 5% FBS and DAPI (SIGMA 09542). E-cadherin or SSEA-1 expression was quantified by BD LSR Fortessa flow cytometer and analyzed by Flowjo software. Unstained cells were used as negative control and DAPI (SIGMA 09542) staining was performed to exclude dead cells from the analysis.
For cell cycle analysis cells were detached with trypsin (0.025% trypsin and 0.04% EDTA (SIGMA 25300-054)) and collected by centrifugation at 300rcf for 5 min. The cell pellet was resuspended and fixed overnight at -20°C in 3ml cold 70% ethanol. After fixation, the cells were centrifuged at 300rcf for 10 min at room temperature. The pellet was washed twice with 1ml PBS. During each wash, the cells were pelleted at 300rcf for 5 min at room temperature. Then cells were resuspended in DAPI solution (SIGMA 09542)(5µg/ml/10 6 cells in PBS) and incubated for 30 min on ice. Samples were analysed by BD LSR II and by using the commercially available Flowjo software.

RNA extraction and quantitative PCR detection of mRNA
Cells were washed with PBS, detached with trypsin (0.025% trypsin and 0.04% EDTA) and pelleted for RNA isolation. RNA was extracted and purified using RNeasy kits  Table S7. The oligos specific for IAP and MusD and the corresponding GenBank Entry were previously described by Guallar and colleagues 6 .

Chromatin immunoprecipitation assay
For ChIP experiments against H3K9me3, H3 and ZFP57, mESCs were trypsinized and crosslinked in 1% formaldehyde for 10 min at room temperature. Crosslinking was   Table S8, which were already used by Quenneville and colleagues 36 .

COBRA and pyrosequencing
Combined bisulfite and restriction analysis 2 were performed using sodium bisulfite treated genomic DNA samples and purified using EpiTect ® Bisulfite Kit (Qiagen).

Statistical Analysis.
Averages from three independent experiments were calculated for most of the shown experiments and Student's unpaired two-tailed t-test were performed for statistical analysis. p <0.05 defined statistical significance.          Table S9. List of primers used for nested BS-PCR, COBRA and pyro-sequencing. OUT and IN primers used for nested PCR. BIO indicates the biotinylated primer used for each region.

IG-DMR Fw -GGTATAAGTTAAGTGTGT Airn
Fw -AGGGTTTTATAGGAGGGAAG Grb10 Rv -TAAATTTAATCCTAAAATTCCT Peg10 Fw -TTTATAAGATTTAGAAATATAA Rasfgr1 Rv -AATAAATATAAAAACAACAAC Table S10. List of primers used for pyrosequencing. Primers used for sequencing are in the opposite strand with the respect to the biotinylated oligos.