Kinetics of DNA methylation inheritance by the Dnmt1-including complexes during the cell cycle

Abstract

Background: The clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during the cellular division is a crucial biological process controlled by the DNA methyltransferase Dnmt1, mainly. To investigate possible dynamic mechanisms of DNA methylation inheritance during the cell cycle, we used a Proximity Ligation In Situ Assay (P-LISA) to analyze the kinetic of formation and DNA recruitment of Dnmt1-including complexes.

Results: P-LISA, sequential chromatin immunoprecipitation and quantitative methylation specific PCR revealed that the Dnmt1/PCNA/UHRF1-including complexes are mainly formed and recruited on DNA during the S-phase of cell cycle, while the formation and the DNA recruitment of several Dnmt1/transcription factors-including complexes are not S-phase dependent but are G0/G1 and/or G2/M phases dependent.

Conclusion: Our data confirm that DNA methylation inheritance occurs in S-phase, and demonstrate that DNA methylation inheritance can also occur in G0/G1 and G2/M phases of the cell cycle.

Figure 1

Dynamic of the formation and of the recruitment on DNA of the Dnmt1/PCNA-including complexes during the cell cycle. (A) ApoTome view of the Dnmt1/PCNA dots in U251 cells during the different phases of the cell cycle. Red dots symbolize Dnmt1/PCNA dots. Nucleus/DNA are stained in blue via the use of DAPI. Graph (mean ± SEM) illustrating the number of Dnmt1/PCNA dots per nucleus in U251 cells during the different phases of the cell cycle. The number of Dnmt1/PCNA interactions is calculated from the analysis of, at least, 50 nucleus in three independent experimentations. (B) Graph (mean ± SEM) illustrating the number of Dnmt1/UHRF1 interactions per nucleus in U251 cells during the different phases of the cell cycle. The number of Dnmt1/UHRF1 interactions is calculated from the analysis of, at least, 50 nucleus in three independent experimentations.

Figure 2

Dynamic of the formation and of the recruitment on DNA of the Dnmt1/transcription factor-including complexes during the cell cycle. (A) Graph (mean ± SEM) illustrating the number of Dnmt1/transcription factor dots per nucleus in U251 cells during the different phases of the cell cycle. The number of Dnmt1/transcription factor interactions is calculated from the analysis of, at least, 50 nucleus in three independent experimentations. (B) Schematic representation of the dynamic of the Dnmt1-including complexes during the cell cycle.

Figure 3

Dynamic of expression and subcellular localization of the Dnmt1 and its partners of interaction during the cell cycle. (A) Pictures illustrate the results obtained after Western blot analyses. Nuclear (nuc.) and cytosolic (cyto.) were obtained by using the Nuclear Extract Kit (Active Motif, France). 30 μg and 50 μg of nuclear and cytosolic extracts were used for the western blot analysis concerning the Dnmt1, UHRF1, Sp1, E2F3, YY1, p53, Ets1, PCNA and PPARγ proteins. 20 μg of nuclear and cytosolic extracts were used for the western blot analysis concerning the actin and lamina-B1 proteins. (B) Graphs illustrate the relative expression of indicated proteins in nuclear fraction i.e. the results obtained from the densitometric analyses of western blot (Fusion Fx7 Imager, Fisher Scientific, France and ImageJ software). Lamina-B1 was used as internal control.

Figure 4

ReChiP experiments analysis the recruitment on the caspase1, caspase4, and DR5 genes of the Dnmt1/Ets1, Dnmt1/UHRF1, and complexes during the cell cycle. Sequential chromatin ImmunoPrecipitation (reChIP) are used to analyze the recruitment of Dnmt1, UHRF1 and transcription factor on the considered genes. Results are expressed as enrichment relative to input and corrected for GFP control levels. The results are averages of at least three independent experiments with error bars indicating standard deviation.

Figure 5

Methylation status of the caspase1, caspase4 and DR5 during the cell cycle. DNA: sonicated DNA (input), Ctrl: hemiMeDIs realized in absence of His-UHRF1 on column, hemiMeDIs: hemi-methylated DNA Isolated, MeDCol: Methylated DNA collected. The results are averages of at least three independent experiments with error bars indicating standard deviation.