title: 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.
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.
authors.affiliation: Biochemistry Laboratory, School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany.
authors.forename: Abu Nasar
authors.initials: AN
authors.lastname: Siddique
authors.forename: Suneetha
authors.initials: S
authors.lastname: Nunna
authors.forename: Arumugam
authors.initials: A
authors.lastname: Rajavelu
authors.forename: Yingying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Renata Z
authors.initials: RZ
authors.lastname: Jurkowska
authors.forename: Richard
authors.initials: R
authors.lastname: Reinhardt
authors.forename: Marianne G
authors.initials: MG
authors.lastname: Rots
authors.forename: Sergey
authors.initials: S
authors.lastname: Ragozin
authors.forename: Tomasz P
authors.initials: TP
authors.lastname: Jurkowski
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
mesh_terms: D002460:Cell Line; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis*
chemical_list: C000717967:DNMT3A protein, human; D011993:Recombinant Fusion Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C412484:DNMT3L protein, human; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A