Proximity ligation in situ assay for monitoring the global DNA methylation in cells

Abstract

Background: DNA methylation has a central role in the epigenetic control of mammalian gene expression, and is required for X inactivation, genomics imprinting and silencing of retrotransposons and repetitive sequences. Thus, several technologies have been developed to measure the degree of DNA methylation.

Results: We here present the development of the detection of protein-protein interactions via the adaptation of the proximity ligation in situ technology to evaluate the DNA methylation status in cells since the quantification of Dnmt1/PCNA interaction in cells reflects the degree of DNA methylation.

Conclusion: This method being directly realizable on cells, it appears that it could suggest a wide range of applications in basic research and drug development. More particularly, this method is specially adapted for the investigations realized from a weak quantity of biologic materiel such as stem cells or primary cultured tumor cells for examples.

Figure 1

Detection of endogenous Dnmt1/PCNA interactions using P-LISA. (A) Schematic representation of the Dnmt1/PCNA proximity ligation in situ assay (P-LISA). (B) Calibration of the microscopy (Axiovert 200 M Zeiss, Le Pecq, France) with ApoTome module. Left: picture of calibration performed with calibration balls (4 μm, Vue X-Y, Molecular Probes F36909). Right: picture of calibration performed with calibration balls (2.5 μm, Vue X-Z, Molecular Probes F36909). Bleu and red were merged in lateral and axial. (C) Decovolving of picture realized with calibration balls (140 nm). (D) Dnmt1/PCNA interactions were visualized in MCF10A cells. Red dots symbolize Dnmt1/PCNA interactions. Nucleus/DNA are stained in blue via the use of DAPI. Top left: ApoTome view, top right: orthogonal view, bottom: 3D views.

Figure 2

Decrease of Dnmt1/PCNA interaction is associated with the decrease of the DNA methylation. (A) Dnmt1/PCNA P-LISA in non-tumor cells (MCF10A) and in tumor cells (MDA-MD-231 and Cal-51). Red dots symbolize Dnmt1/PCNA interactions. Nucleus/DNA are stained in blue via the use of DAPI. The number of Dnmt1/PCNA interactions is calculated from the analysis of 100 nuclei. (B) Evaluation of the degree of DNA methylation in cells by using 5 mC-ELISA. (C) Correlation between the 5 mC (5-methylcyctosine) number and the number of Dnmt1/PCNA interactions.

Figure 3

siRNA-mediated down-expression of Dnmt1 promoted the decrease of Dnmt1/PCNA interactions and the decrease of 5 mC in U251 cells. (A) Impact of the siRNA-Dnmt1 treatments on the expression of Dnmt1 (ELISA method according to our previous report [17]), on the 5 mC (ELISA method) and on the number of Dnmt1/PCNA interactions (P-LISA method). siRNA-Dnmt1 (sc-35204, Tebu-Bio, France) (B) Correlation between the 5 mC (5-methylcyctosine) number and the number of Dnmt1/PCNA interactions.

Figure 4

Impact of the 5aza treatment on the DNA methylation status (ELISA method), the number of Dnmt1/PCNA interactions (P-LISA method) and on the Dnmt1 expression (ELISA method) in U251 cells.

Figure 5

Impact of conditions increasing the level of DNA methylation on the number of Dnmt1/PCNA interactions. (A) Characterization cell cycle of arrest. U251 cells were treated by serum deprivation (72 h) or by thymidine (2 mM, 24 h), previous to perform cell cycle analysis by using the NucleoCounter NC-3000™ Kit, Chemometec, France). (B) Dnmt1/PCNA P-LISA in U251 cells blocked in G0/G1 or S phases of the cell cycle. Red dots symbolize Dnmt1/PCNA interactions. Nucleus/DNA are stained in blue via the use of DAPI. (C) Analysis of the number of Dnmt1/PCNA interactions (P-LISA) and the 5 mC number (ELISA). The number of Dnmt1/PCNA interactions is calculated from the analysis of 100 nuclei. (D) Impact of the Dnmt3a overexpression in U87 cells on the level of methylation of the capase-8 gene (MeDIP), on DNA (5 mC ELISA) and on the number of Dnmt1/PCNA interactions. The number of Dnmt1/PCNA interactions is calculated from the analysis of 100 nuclei. U87-pORF-Dnmt3a and the U87-pORF (transfection control (-)) were obtained as previously described [16].