Disruption of Dnmt1/PCNA/UHRF1 Interactions Promotes Tumorigenesis from Human and Mice Glial Cells
Figure 6
The disruption of the Dnmt1/PCNA/UHRF1 interactions promotes the global DNA hypomethylation in astrocytes (Astro#40) and in glial precusor cells (Ntv-a).
(A) Monitoring by immunoprecipitation of Dnmt1 and ELISA of the effect of the expression of the UP protein” (a chimera protein composed by the 163–171 amino-acids of PCNA and the 526–614 amino-acids of UHRF1) on the Dnmt1/PCNA/UHRF1 interactions and on the level of 5-methylcytosine (5 mC). (B) Use of proximity ligation in situ assay (P-LISA) to monitor the “UP”-induced disruption of the Dnmt1-PCNA interactions. Nucleus/DNA are in blue and Dnmt1-PCNA interaction in red. Quantification was performed from the analysis of 100 cells of three independent experiments. (C) Impact of the “UP”-induced disruption of the Dnmt1-PCNA-UHRF1 interactions on the co-recruitment of Dnmt1, PCNA and UHRF1 on Alu, a DNA repeat element. Chromatin Immunoprecipitation (ChIP) was performed by using the EZ-ChIP (Millipore, France). For each point, the relative quantity of immunoprecipitated DNA is obtained by using input as reference. (D) Impact of the “UP”-induced disruption of the Dnmt1-PCNA-UHRF1 interactions on the methylation status of Alu by coupling the Methylated DNA COllection and PCR amplification (MeDCO) via the use of the MethylCollector Ultra kit (Active Motif, France). (I:input: M:Methylated and collected DNA).