Targeted methylation and gene silencing of VEGF-A in human cells by using a designed Dnmt3a–Dnmt3L single-chain fusion protein with increased DNA methylation …

AN Siddique, S Nunna, A Rajavelu, Y Zhang… - Journal of molecular …, 2013 - Elsevier
AN Siddique, S Nunna, A Rajavelu, Y Zhang, RZ Jurkowska, R Reinhardt, MG Rots
Journal of molecular biology, 2013Elsevier
The C-terminal domain of the Dnmt3a de novo DNA methyltransferase (Dnmt3a-C) forms a
complex with the C-terminal domain of Dnmt3L, which stimulates its catalytic activity. We
generated and characterized single-chain (sc) fusion proteins of both these domains with
linker lengths between 16 and 30 amino acid residues. The purified sc proteins showed
about 10-fold higher DNA methylation activities than Dnmt3a-C in vitro and were more active
in bacterial cells as well. After fusing the Dnmt3a-3L sc enzyme to an artificial zinc-finger …
The C-terminal domain of the Dnmt3a de novo DNA methyltransferase (Dnmt3a-C) forms a complex with the C-terminal domain of Dnmt3L, which stimulates its catalytic activity. We generated and characterized single-chain (sc) fusion proteins of both these domains with linker lengths between 16 and 30 amino acid residues. The purified sc proteins showed about 10-fold higher DNA methylation activities than Dnmt3a-C in vitro and were more active in bacterial cells as well. After fusing the Dnmt3a-3L sc enzyme to an artificial zinc-finger protein targeting the vascular endothelial cell growth factor A (VEGF-A) promoter, we demonstrate successful targeting of DNA methylation to the VEGF-A promoter in human cells and observed that almost complete methylation of 12 CpG sites in the gene promoter could be achieved. Targeted methylation by the Dnmt3a-3L sc enzymes was about twofold higher than that of Dnmt3a-C, indicating that Dnmt3a-3L sc variants are more efficient as catalytic modules in chimeric DNA methyltransfeases than Dnmt3a-C. Targeted methylation of the VEGF-A promoter with the Dnmt3a-3L sc variant led to a strong silencing of VEGF-A expression, indicating that the artificial DNA methylation of an endogenous promoter is a powerful strategy to achieve silencing of the corresponding gene in human cells.
Elsevier