In Vivo Activity of Murine De Novo Methyltransferases, Dnmt3a and Dnmt3b

TLDR

Dnmt3a and Dnmt3b are de novo DNA methyltransferases that were previously shown to have weak activity on the 3′ LTR of Moloney murine leukemia virus in vitro. The authors evaluated in vivo methylation by Dnmt3a and Dnmt3b using a stable episomal plasmid system in human cells. Overexpressing murine Dnmt3a or Dnmt3b in human cells induced nonrandom de novo methylation of specific CpG sites on episomal plasmids, a process dependent on the catalytic cysteine, whereas human DNMT1 or murine Dnmt3b did not, indicating distinct target preferences and confirming in vivo de novo activity of Dnmt3a and Dnmt3b.

Abstract

The putative de novo methyltransferases, Dnmt3a and Dnmt3b, were reported to have weak methyltransferase activity in methylating the 3′ long terminal repeat of Moloney murine leukemia virus in vitro. The activity of these enzymes was evaluated in vivo, using a stable episomal system that employs plasmids as targets for DNA methylation in human cells. De novo methylation of a subset of the CpG sites on the stable episomes is detected in human cells overexpressing the murine Dnmt3a or Dnmt3b1 protein. This de novo methylation activity is abolished when the cysteine in the P-C motif, which is the catalytic site of cytosine methyltransferases, is replaced by a serine. The pattern of methylation on the episome is nonrandom, and different regions of the episome are methylated to different extents. Furthermore, Dnmt3a also methylates the sequence methylated by Dnmt3a on the stable episome in the corresponding chromosomal target. Overexpression of human DNMT1 or murine Dnmt3b does not lead to the same pattern or degree of de novo methylation on the episome as overexpression of murine Dnmt3a. This finding suggests that these three enzymes may have different targets or requirements, despite the fact that weak de novo methyltransferase activity has been demonstrated in vitro for all three enzymes. It is also noteworthy that both Dnmt3a and Dnmt3b proteins coat the metaphase chromosomes while displaying a more uniform pattern in the nucleus. This is the first evidence that Dnmt3a and Dnmt3b have de novo methyltransferase function in vivo and the first indication that the Dnmt3a and Dnmt3b proteins may have preferred target sites.