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

Siddique, Abu Nasar, Nunna, Suneetha, Rajavelu, Arumugam, Zhang, Yingying, Jurkowska, Renata Z. ORCID: https://orcid.org/0000-0002-4507-2222, Reinhardt, Richard, Rots, Marianne G., Ragozin, Sergey, Jurkowski, Tomasz P. ORCID: https://orcid.org/0000-0002-2012-0240 and Jeltsch, Albert 2012. 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. Journal of Molecular Biology 425 (3) , 479—491. 10.1016/j.jmb.2012.11.038

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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.

Item Type:	Article
Date Type:	Published Online
Status:	Published
Schools:	Schools > Biosciences
ISSN:	0022-2836
Date of Acceptance:	28 November 2012
Last Modified:	25 Oct 2022 13:09
URI:	https://orca.cardiff.ac.uk/id/eprint/119008

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