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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 Activity

Author:
Siddique, Abu Nasar, Nunna, Suneetha, Rajavelu, Arumugam, Zhang, Yingying, Jurkowska, Renata Z., Reinhardt, Richard, Rots, Marianne G., Ragozin, Sergey, Jurkowski, Tomasz P., Jeltsch, Albert
Source:
Journal of Molecular Biology 2013 v.425 pp. 479-491
ISSN:
0022-2836
Subject:
DNA, DNA methylation, amino acids, catalytic activity, cell growth, endothelial cells, enzymes, gene silencing, genes, humans, proteins, vascular endothelial growth factor A
Abstract:
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.
Agid:
1021741