Targeted methylation of the epithelial cell adhesion molecule (EpCAM) promoter to silence its expression in ovarian cancer cells

The Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in many cancers including ovarian cancer and EpCAM overexpression correlates with decreased survival of patients. It was the aim of this study to achieve a targeted methylation of the EpCAM promoter and silence EpCAM gene expression using an engineered zinc finger protein that specifically binds the EpCAM promoter fused to the catalytic domain of the Dnmt3a DNA methyltransferase. We show that transient transfection of this construct increased the methylation of the EpCAM promoter in SKOV3 cells from 4–8% in untreated cells to 30%. Up to 48% methylation was observed in stable cell lines which express the chimeric methyltransferase. Control experiments confirmed that the methylation was dependent on the fusion of the Zinc finger and the methyltransferase domains and specific for the target region. The stable cell lines with methylated EpCAM promoter showed a 60–80% reduction of EpCAM expression as determined at mRNA and protein level and exhibited a significantly reduced cell proliferation. Our data indicate that targeted methylation of the EpCAM promoter could be an approach in the therapy of EpCAM overexpressing cancers

The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism

The ubiquitin-like, containing PHD and RING finger domains protein 1 (UHRF1) is essential for maintenance DNA methylationby DNA methyltransferase 1 (DNMT1). UHRF1 has been shown to recruit DNMT1 to replicated DNA by the ability of its SET andRING-associated (SRA) domain to bind to hemimethylated DNA. Here, we demonstrate that UHRF1 also increases the activity ofDNMT1 by almost 5-fold. This stimulation is mediated by a direct interaction of both proteins through the SRA domain of UHRF1and the replication focus targeting sequence domain of DNMT1, and it does not require DNA binding by the SRA domain. Disruptionof the interaction between DNMT1 and UHRF1 by replacement of key residues in the replication focus targeting sequence domainled to a strong reduction of DNMT1 stimulation. Additionally, the interaction with UHRF1 increased the specificity of DNMT1for methylation of hemimethylated CpG sites. These findings show that apart from the targeting of DNMT1 to the replicatedDNA UHRF1 increases the activity and specificity of DNMT1, thus exerting a multifaceted influence on the maintenance of DNAmethylatio

DNA Methylation Analysis of Chromosome 21 Gene Promoters at Single Base Pair and Single Allele Resolution

Differential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read) Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicon's DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i) amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii) methylation levels of individual cells in one tissue are very similar, and iii) methylation patterns follow a relaxed site-specific distribution. Furthermore, iv) we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene regulation. Further, we illustrate genotype–epigenotype interactions by showing novel examples of allele-specific methylation

Examples of the results of the DNA methylation analysis of the EpCAM gene promoter in SKOV3 cells.

<p>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.</p

Absence of off-target methylation in SKOV3 cells analyzed by bisulfite sequencing.

<p>Methylation of four non-target genes was analyzed (KIAA0179, DSCR3, Sumo3 and WRB). Data presentation is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087703#pone-0087703-g003" target="_blank">Fig. 3</a>. The sequences of the regions analyzed here are given in the Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087703#pone.0087703.s001" target="_blank">Information S1</a>.</p

Specificity of Dnmt1 for Methylation of Hemimethylated CpG Sites Resides in Its Catalytic Domain

SummaryThe maintenance methylation of hemimethylated CpG sites by the DNA methyltransferase Dnmt1 is the molecular basis of the inheritance of DNA methylation patterns. Based on structural data and kinetics obtained with a truncated form of Dnmt1, an autoinhibition model for the specificity of Dnmt1 was proposed in which unmethylated DNA binds to Dnmt1's CXXC domain, which prevents its methylation. We have prepared CXXC domain variants that lost DNA binding. Corresponding full-length Dnmt1 variants did not display a reduction in specificity, indicating that the autoinhibition model does not apply in full-length Dnmt1. Furthermore, we show that the Dnmt1 M1235S variant, which carries an exchange in the catalytic domain of the enzyme, has a marked reduction in specificity, indicating that the recognition of the hemimethylated state of target sites resides within the catalytic domain

Genome context of the EpCAM gene (indicated by a blue bar) on chromosome 2 p21.

<p>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 (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087703#pone.0087703-Kent1" target="_blank">[66]</a>.</p

Analysis of EpCAM gene expression after targeted promoter methylation in stable cell lines.

<p>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.</p

Principle of targeted DNA methylation and gene silencing using Zinc fingers (ZF) fused to the catalytic domain of the DNA methyltransferase Dnmt3a (Dnmt3aCD).

<p>The blue bar represents the ZF binding site, unfilled lollipops represent unmethylated CpGs and filled lollipops represent methylated CpGs.</p

Down regulation of EpCAM expression inhibits the proliferation of SKOV3 cells.

<p>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.</p