Cancer development is not only the result of genetic mutations but also stems from modifications in the epigenetic code leading to an aberrant expression of genes relevant for cancer. The most studied epigenetic mark is DNA methylation of cytosines in the promoters of genes, which is associated with repressed gene expression. Hallmarks of cancer are aberrant silencing of tumor suppressor genes and the activation oncogenes by epigenetic means. We reasoned that the induction of targeted DNA methylation at endogenous loci will lead to the transcriptional repression of the target genes. Therefore, we used zinc finger based DNA-binding domains designed to target specifically an 18 bps DNA sequence. The DNA-binding domain was then fused to the catalytic active proportion of the DNA methyltransferase DNMT3A to induce targeted DNA methylation at the gene of interest. We show that targeted DNA methylation lead to silencing of gene expression of both, an oncogene and a tumor suppressor gene. Repression of a tumor suppressor gene lead to an increase of tumor cell growth; while silencing of the oncogenic transcription factor SOX2, lead to a decrease in cancer cell proliferation in vitro and to the inhibition of tumor growth in xenografts. The induced DNA methylation was maintained over cell generations and detectable up to 100 days post inoculation, which was associated with the repression of target gene expression and decreased expression of the proliferation marker Ki67. Overall our findings show that targeted DNA methylation can be used to specifically and stably silence gene expression.
|Qualification||Doctor of Philosophy|
|Place of Publication||[S.l.]|
|Publication status||Published - 2014|