Browsing by Author "Nunna, Suneetha"

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Open Access
Development of zinc finger methyltransferase fusion proteins for targeted DNA methylation and gene silencing in human cells
(2014) Nunna, Suneetha; Jeltsch, Albert (Prof. Dr.)
Epigenetic modifications such as DNA methylation and histone modifications play important roles in the regulation of gene expression. DNA methylation occurs at C5 position of cytosine residues mainly in CpG dinucleotides. In the human genome, about 70% of the CpGs are methylated. Most of the gene promoters are accompanied by CpG islands (regions rich in CpG dinucleotides) and methylation in these regions is inversely correlated with gene expression. Aberrant DNA methylation at the promoter region leads to variety of diseases including cancer. In the present study, we used catalytic domains of the Dnmt3a DNA methyltransferase and the GLP H3K9me3 lysine methyltransferase to silence two important oncogenes by targeted methylation. Zinc-finger proteins with predefined specificity were used as the targeting device. The first target gene was the vascular endothelial growth factor A (VEGF-A), which plays an important role in the vasculogenesis and angiogenesis. A zinc finger (VAZF) binding to the VEGF-A promoter was fused to either the catalytic domain of Dnmt3a (VAZF-Dnmt3aC) or to a fusion of Dnmt3a with its stimulator Dnmt3L (VAZF-Dnmt3a3Lsc). After transient transfection in ovarian cancer cells and magnetic activated cell sorting (MACS), we observed 25% methylation of the target region in the cells transfected with VAZF-Dnmt3aC and 49% in VAZF-Dnmt3a3Lsc transfected cells. VEGF-A expression was measured by quantitative -RTPCR and we observed a 36% reduction of VEGF-A expression in the cells that were transfected with VAZF-Dnmt3aC, and 56% in VAZF-Dnmt3a3Lsc transfected cells. However, transfection yields after MACS were only around 60-80% in these studies such that untransfected cells were still present. The second target gene of my study was the epithelial cell adhesion molecule (EpCAM), which is a transmembrane glycoprotein and is required for homophilic cell-cell adhesion. EpCAM is overexpressed in numerous cancers and its expression is inversely correlated with the promoter methylation status. In the present study, I used a zinc finger binding to the EpCAM promoter region, fused to the catalytic domain of the Dnmt3a DNA methyltransferase (EpZF-Dnmt3aC) for targeted methylation of the EpCAM promoter. After magnetic activated cell sorting, transfection yields of 60-80% were reached. With this approach 29% DNA methylation was achieved at the EpCAM promoter region in SKOV3 ovarian cancer cells. In stable cell lines expressing EpZF-Dnmt3aC, the methylation reached up to 48% which in turn led to 80% reduction of EpCAM protein expression. I also observed a reduction of cell proliferation in these stable cell lines which is a promising result suggesting that EpCAM repression might be an approach in cancer treatment. To improve the efficiency of gene delivery into the ovarian cancer cells, recombinant adenoviral vectors encoding the zinc-finger fused DNA methyltransferases and histone H3K9me3 methyltransferase catalytic domain were generated. Using adenovirus mediated gene delivery we achieved 53% methylation and 80% gene suppression at the VEGF-A promoter in SKOV3 ovarian cancer cells. Similarly, the zinc finger fused to GLP resulted in appearance of H3K9me3 at the promoter and 64% gene repression. At the EpCAM promoter, an EpCAM zinc finger fused to DNA methyltransferases led to 32% of methylation and 90% gene repression in the same ovarian cancer cells. Another important objective of the study was to measure the stability of DNA methylation and gene silencing of VEGF-A. After infection of SKOV3 ovarian cancer cells with adenovirus expressing VAZF-Dnmt3aC or VAZF-GLP, the methylation and gene expression was measured at different time points. In the cells that were infected with VAZF-Dnmt3aC constructs, establishment of DNA methylation was initiated 24 h after infection. The methylation reached to a maximum after five days, but surprisingly afterwards DNA methylation gradually declined to its basal level till day 15. VEGF-A supression correlated with the methylation levels, five days after infection 75% suppression of VEGF-A was observed which gradually declined to 8% after 15 days. In SKOV3 cells that were infected with recombinant adenoviral vectors encoding VAZF-GLP, H3K9 histone methylation peaked at day 5, but like the DNA methylation it gradually was lost afterwards. This result indicates that both silencing marks could not be introduced in a stable manner. As it was shown that DNA methylation and histone methylation acts synergistically, we measured DNA methylation after targeted introduction of histone H3K9 methylation and vice versa. However, in both the experiments we could not observe synergistic effects at the VEGF-A promoter. We started experiments to measure whether the decreased VEGF-A and EpCAM expression has an anti-tumor effect in a mouse model, which will explore the therapeutic potential of targeted methylation in cancer treatment.
Open Access
Targeted epigenome editing of an endogenouslocus with chromatin modifiers is not stably maintained
(2015) Kungulovski, Goran; Nunna, Suneetha; Thomas, Maria; Zanger, Ulrich M.; Reinhardt, Richard; Jeltsch, Albert
Background: DNA methylation and histone 3 lysine 9 (H3K9) methylation are considered as epigenetic marks that can be inherited through cell divisions. To explore the functional consequences and stability of these modifications, we employed targeted installment of DNA methylation and H3K9 methylation in the vascular endothelial growth factor A (VEGF-A) promoter using catalytic domains of DNA or H3K9 methyltransferases that are fused to a zinc finger protein which binds a site in the VEGF-A promoter. Results: Expression of the targeted DNA and H3K9 methyltransferases caused dense deposition of DNA methylation or H3K9 di- and trimethylation in the promoter of VEGF-A and downregulation of VEGF-A gene expression. We did not observe positive feedback between DNA methylation and H3K9 methylation. Upon loss of the targeted methyltransferases from the cells, the epigenetic marks, chromatin environment, and gene expression Levels returned to their original state, indicating that both methylation marks were not stably propagated after their installment. Conclusions: The clear anti-correlation between DNA or H3K9 methylation and gene expression suggests a direct role of these marks in transcriptional control. The lack of maintenance of the transiently induced silenced chromatin state suggests that the stability of epigenetic signaling is based on an epigenetic network consisting of several molecular marks. Therefore, for stable reprogramming, either multivalent deposition of functionally related epigenetic marks or longer-lasting trigger stimuli might be necessary.
Open Access
Targeted methylation of the epithelial cell adhesion molecule (EpCAM) promoter to silence its expression in ovarian cancer cells
(2014) Nunna, Suneetha; Reinhardt, Richard; Ragozin, Sergey; Jeltsch, Albert
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.