To analyze the formation and the recruitment on DNA of the considered Dnmt1-including complexes, U251 cells were blocked into the different cell cycle phases by using serum deprivation and the thymidine, nocodazole or taxol treatments. As expected, cell cycle phase analysis indicated that serum deprivation mainly blocked cells in G0/G1 phases, thymidine treatment mainly blocked cells in S phase nocodazole treatment mainly blocked cells in G2/M phase, while taxol treatment mainly blocked cells in G2/M phase and more specifically in M phase since taxol blocks metaphasis/anaphasis transition of mitosis (Table 1) 12.

As already used to analyze the formation of the Dnmt1/PCNA-including complexes and its recruitment on DNA in glioma cells, we used Proximity Ligation In Situ Assay (P-LISA) and ApoTome technology to dissect the dynamic of formation of the Dnmt1/PCNA-including complex and its recruitment on DNA during the different phases of cell cycle 4. Thus, we noted that the Dnmt1/PCNA-including and Dnmt1/UHRF1-including complexes were mainly formed and recruited on DNA during the S-phase of cell cycle (Figures 1A and 1B).

Among the transcription factors already described as been potential partners of interactions of Dnmt1, we focused our analysis on the dynamic of formation and recruitment on DNA of 6 Dnmt1/transcription factors-including complexes. P-LISA experiments indicated that the Dnmt1/p53-including and Dnmt1/YY1-including complexes were mainly formed and recruited on DNA during the G2/M phases of cell cycle (Figure 2A). The Dnmt1/Sp1-including and Dnmt1/PPARγ-including complexes were mainly formed and recruited on DNA during the G0/G1 and G2/M phases of cell cycle. The Dnmt1/Ets1-including complexes were mainly formed and recruited on DNA during the G0/G1 phase of cell cycle, while the Dnmt1/E2F3-including complexes were mainly formed and recruited on DNA during the S and G2/M phases of cell cycle. Interestingly, all these data indicated that the formation and the recruitment on DNA of the complex(es) including the Dnmt1, PCNA and/or UHRF1 proteins mainly occur during the S phase of the cell cycle, while the formation and the recruitment on DNA of the Dnmt1/transcription factor-including complexes are not specific of one phase of the cell cycle (Figure 2B).

Next, we hypothesized that the kinetics of the interactions existing between Dnmt1 and its considered partners could be due to the putative changes in the expression level and/or of subcellular localization of these proteins. To validate these points, we performed western blots analyses using nuclear and cytosolic extracts from cells blocked in the different cell cycle phases. Actin and Lamin-B1 were used as cytosol and nuclear internal controls, respectively. No change in the expression and subcellular localization was observed for Dnmt1, UHRF1, Sp1 and E2F3 (Figures 3A and 3B). YY1 expression was higher in G2/M phase supporting the idea that the Dnmt1/YY1 interactions mainly occur in G2/M phase. Ets1 expression was higher in G0/G1 phase supporting the fact that Dnmt1/Ets1 interaction mainly occurs in G0/G1 phase. PCNA expression was higher in S phase supporting the fact that Dnmt1/PCNA interaction mainly occurs in S phase. With regards to p53, we noted that the p53 expression increased during the cell cycle progression. This observation can explain why Dnmt1/p53 interaction was higher in G2/M phase. Western blot analyses also revealed that the nuclear expression of PPARγ decreased during the cell cycle progression. This point supports the fact that the number of the Dnmt1/PPARγ dots seen in G0/G1 phase was superior to that seen in S phase, but not the observation that the number of the Dnmt1/PPARγ dots seen in S phase was superior to that seen in G2/M phase. Without explaining all variations of Dnmt1/partner-X interactions, the variations in the expression level of the partners of Dnmt1 appears as a rational explanation for the dynamic of the Dnmt1/partner-X interactions existing during the different cell cycle phases.

Consistent with our last results, we then hypothesized that the complexes including the Dnmt1/Ets1, Dnmt1/UHRF1 and Dnmt1/YY1 proteins were mainly recruited on DNA during the G0/G1, S or G2/M phases, respectively. To cure these points, we analyzed by sequential chromatin immunoprecipitation (reChIP) experimentations the co-recruitment of Dnmt1/Ets1, Dnmt1/UHRF1 and Dnmt1/YY1 on specific genes during the different phases of the cell cycle. Thus, after a blockade of cells in the different cell cycle phases, chromatin was immunoprecipitated with an antibody directed against Dnmt1 in the first time and with an antibody directed against Ets1, UHRF1 or YY1 in a second time, an antibody directed against GFP was used as control. For the targeted genes, we focused our analyses on genes coding for effectors or regulators of apoptosis since our group is involved in the analysis of mechanisms modulating apoptosis. As such, we analyzed the co-recruitment of Dnmt1/Ets1, Dnmt1/UHRF1 and Dnmt1/YY1 on caspase-1 gene (a target of Ets1), caspase-4 gene and DR5 gene (a target of YY1) 1314. As shown in Figure 4, we observed that the Dnmt1/UHRF1 complex was mainly recruited on the caspase1 gene in G0/G1 phase and that the recruitment of Dnmt1/UHRF1 and Dnmt1/YY1 on the caspase4 and DR5 gene mainly occurred during the S and G2/M phases, respectively. Associated with our previous data, these last results strongly support the idea that the Dnmt1/UHRF1, Dnmt1/YY1 and Dnmt1/Ets1 complexes were mainly formed and recruited on DNA during the S, G2/M and G0/G1 phases of the cell cycle, respectively.

The fact that the formation and the recruitment on DNA of the Dnmt1/Partner-X occurs in different phases suggests that the inheritance of DNA methylation on its targeted genes by this type of complex can occur after the cellular division or that DNA can exist under a hemi-methylated status in one or several cell cycle phases. Thus, we compared, the methylation status of caspase1, caspase4 and DR5 genes during the different cell cycle phases. Methylated DNA Collected (MeDCol) and hemi-Methylated DNA Isolated (hemi-MeDIs) were used to determine the status of methylation or hemi-methylation of the considered genes since DNA was purified using recombinant proteins having the capacity to specifically bind methylated (MBD2b/MBD3L1 protein complex, according to Active Motif kit) or hemi-methylated DNA (UHRF1). The hemi-MeDIs and MeDCol indicated that caspase1 gene was mainly hemi-methylated in S and G2/M phases and was mainly methylated in G0/G1 phase since maximum of amplification of caspase1 gene was observed when PCR were realized from DNA isolated by hemiMeDIs and MeDCol issue to cells blocked in S and G2/M, and G0/G1 phases, respectively (Figure 5). Similar experiments indicated that caspase4 gene was hemi-methylated in S phase and methylated in G0/G1 and G2/M phases, and DR5 was hemi-methylated in S phase and methylated in G0/G1 phase (Figure 5).

Consistently with results obtained from reChIP experiments, our data indicated that the maintenance of methylation of capsase1 gene occurs during G0/G1 phase and implicates Dnmt1/Ets1, the maintenance of methylation of capsase4 gene occurs during S phase and implicates Dnmt1/UHRF1, and that the maintenance of methylation of DR5 gene occurs during G2/M phase and implicates Dnmt1/YY1.

Cells were fixed with 4% paraformaldehyde in PBS pH7.4 for 15 min at room temperature. Permeabilization is performed with PBS containing 0.5% Triton 100 × 4 for 20 min at room temperature and staining were realized according to manufacturer's instructions (Olink Bioscience) 29. Fluorescence was visualized by using the Axiovert 200 M microscopy system (Zeiss, Le Pecq, France) with ApoTome module (X63 and numerial aperture 1.4). Pictures acquisition was realized in structured illumination microscopy 30. Thus, the lateral resolution (rl) is, according to the Rayleigt criteria: rl = 0.61λ/NA (λ: wavelength; NA: numerical aperture of the objective), and the axial resolution, in ApoTome, is defined by the full-width half maximum (FWHMz)

F W H M ( z ) = 3 . 83 16 π ⋅ λ × 10 - 3 n ⋅ sin 2 α 2 v ̄ 1 - v ̄ 2

In this equation, λ is the emission wavelength, n is the index of medium refraction, ν is the frequency and α is the angle of opening of the objective as previously described 31.

After decovolving (3.5 Huygens Essential software (SVI)), 3D view was obtained by using Amira.4.1.1 program. Finally, the images were analyzed by using the freeware "BlobFinder available for download from http://www.cb.uu.se/~amin/BlobFinder. Thus, we obtained either number of signals per nuclei since nuclei can be automatically identified. In other terms, the use of this program participates to the normalization, standardization, reproducibility and to the definition of the cut off signal to accept/quantify or not a dot.

Briefly, chromatin was purified from cells after cross-linking with 1% formaldehyde for 10 min at room temperature. ChIP and Re-ChIP assays were performed with the ChIP-IT™ and Re-ChIP-IT™ kit (ActiveMotif, France) with indicated antibodies and primers. In ChIP and reChIP assays, quantitative PCRs (MX4000 system and the Brilliant SYBR Green QPCR Core Reagent Kit) were performed on 2 μl of input, ChIP or reChIP sample DNA. The relative levels of the fragments of interest in the immunoprecipitated DNA were determined from the threshold cycle (CT) for each PCR. To ensure the reliability of our ChIP and reChIP data, two control samples specific for the ChIP and reChIP experiments have been included: the input sample (indicative of the presence and amount of chromatin used in the ChIP reaction) and the control antibody (GFP antibody) sample (indicative of the amount of background signal generated by the chromatin preparations and ChIP procedure). The calculations of the relative enrichment values were as described below. (i) We normalized the quantitative PCR signals obtained from the immunoprecipitated ChIP sample to the input sample, i.e., CT input - CT ChIP. The PCR efficiency, corresponding to the different sets of primers used in our quantitative PCR, was then raised to the power of this CT difference, i.e., (primer PCR efficiency)(CT input - CT ChIP). (ii) The enrichment (n-fold) of the immunoprecipitated sequence of interest was obtained by normalizing the values to the ChIP background (relative to IP GFP antibody). (iii) To ensure that the observed binding of the tested proteins reflect specific binding to the considered promoter, we also amplified an unrelated control region in a quantitative PCR. The relative enrichment values were calculated by dividing the enrichment (n-fold) derived from the sequence of interest by the signal derived from this control locus (unrelated control region).

DNA was extracted by using the QiaAmp DNA mini Kit (Qiagen, France). DNA was sonicated by using a Bioruptor Sonicator (Diagenode, France). Methylated DNA Collected (MeDCol) was realized by using the MethylCollector™ Ultra kit (Active Motif, France). HemiMethylated DNA Isolated (hemiMeDIs) was realized by using his-tagged UHRF1 protein to isolated hemimethylated DNA. Briefly, 2 μg of His-UHRF1 were loading on Handee™ Spin Column containing 50 μl of Immobilized Cobalt Chelate resin (ProFound™ Pull-Down PolyHis Protein:Protein Interaction Kit Pierce, Thermo Scientific, France). Next, 100 ng of sonicated genomic DNA was incubated on previous tube/column, on a rotisserie shaker for 1 hour at 4°C in binding buffer issue to the MethylCollector™ Ultra kit (Active Motif, France). Washes, recovery of methylated DNA fragments, DNA clean-up steps were performed such as described in MethylCollector™ Ultra kit (Active Motif, France). Finally, methylated and hemi-methylated DNA was analyzed by qPCR. To evaluate the relative enrichment of target sequences, we normalized (for each amplicon tested) the C_t of the MeDCOl/HemiMeDIs fraction to the C_t of the input (ΔC_t). Subsequently we normalized the ΔC_t of each target sequence to the ΔC_t of an unmethylated control sequence (ΔΔC_t). Finally, we calculated the relative enrichment E = 2^ΔΔCt.

In brief, proteins were size fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose or PVDF membrane. Saturation and blotting were realized by using SNAP i.d™ Protein Detection System (Millipore, France). The detection of proteins was performed using ECL™(Amersham Biosciences) and/or SuperSignal west femto Maximum Sensitivity (Pierce) chemilumenscence reagents.

Antibodies and primers are listed in additional file 2 and 3.