Figure 1.
Principle of targeted DNA methylation and gene silencing using Zinc fingers (ZF) fused to the catalytic domain of the DNA methyltransferase Dnmt3a (Dnmt3aCD).
The blue bar represents the ZF binding site, unfilled lollipops represent unmethylated CpGs and filled lollipops represent methylated CpGs.
Figure 2.
Genome context of the EpCAM gene (indicated by a blue bar) on chromosome 2 p21.
The gene is shown in blue, its CpG island in green and the amplicon in black. The amplicon sequence is given below, the ZF binding site sequence is shaded in red. This picture was generated using University of California Santa Cruz genome browser (http://genome.ucsc.edu/) [66].
Figure 3.
Examples of the results of the DNA methylation analysis of the EpCAM gene promoter in SKOV3 cells.
The following abbreviations were used: SKOV3 cells, untreated cells; ZF, SKOV3 cells transfected with Zinc finger construct, ZF-Dnmt3aCD, cells transfected with the Zinc finger Dnmt3a catalytic domain construct; VEC cntrl, cells transfected with empty vector; Dnmt3aCD, cells transfected with a Dnmt3aCD construct without Zinc finger; CD1, stable cell line expressing ZF-Dnmt3aCD 1; CD2, stable cell line expressing ZF-Dnmt3aCD 2. The horizontal rows indicate the CpGs in the amplicon analyzed and the vertical rows represent individual clones that were sequenced. The blue and red colors represent unmethylated CpG and methylated CpG, respectively, for white colored sites, the methylation state is unknown due to technical reasons.
Figure 4.
Absence of off-target methylation in SKOV3 cells analyzed by bisulfite sequencing.
Methylation of four non-target genes was analyzed (KIAA0179, DSCR3, Sumo3 and WRB). Data presentation is as in Fig. 3. The sequences of the regions analyzed here are given in the Supplementary Information S1.
Figure 5.
Analysis of EpCAM gene expression after targeted promoter methylation in stable cell lines.
A) Example of the RT qPCR analysis of EpCAM (left) and beta actin (right) mRNA amounts in SKOV3 cells and in two independent cell lines which stably express the ZF-Dnmt3a construct (CD1 and CD2). B) Quantification of the RT qPCR analysis of EpCAM expression as shown in panel A. We carried out two independent RNA preparations each analyzed in three technical repeats. The image shows the average of both results, the error bars indicate the standard error. C) Example of the Western blot analysis of EpCAM expression in SKOV3 cells and the CD1 and CD2 stable cell lines (upper panel). Beta actin was used as loading control (lower panel). The EpCAM and beta actin bands are marked with arrows. D) Quantification of the Western Blot analysis of EpCAM expression as shown in panel C. The figure shows an average of two independent experiments, the error bars indicate the standard deviation of the data.
Figure 6.
Down regulation of EpCAM expression inhibits the proliferation of SKOV3 cells.
A) Results of CCK8 cell proliferation assays conducted with the CD1 and CD2 stable cell lines and SKOV3 cells as reference. The results plotted are from four independent experiments and the error bars indicate the standard error of the mean. B) Viable cell counting performed by Trypan blue staining. The graph represents the data from two independent experiments and the error bars indicate the standard error of the mean.