Tài liệu Báo cáo Y học: Dnmt3a and Dnmt1 functionally cooperate during de novo methylation of DNA

Dnmt3a and Dnmt1 functionally cooperate during de novo

methylation of DNA

Mehrnaz Fatemi*, Andrea Hermann*, Humaira Gowher and Albert Jeltsch

Institut fu¨r Biochemie, Justus-Liebig-Universita¨t, Gießen, Germany

Dnmt3a is a de novo DNA methyltransferase that modifies

unmethylated DNA. In contrast Dnmt1 shows high pre￾ference for hemimethylated DNA. However, Dnmt1 can be

activated for the methylation of unmodified DNA. We show

here that the Dnmt3a and Dnmt1 DNA methyltransferases

functionally cooperate in de novo methylation of DNA,

because a fivefold stimulation of methylation activity is

observed if both enzymes are present. Stimulation is obser￾ved if Dnmt3a is used before Dnmt1, but not if incubation

with Dnmt1 precedes Dnmt3a, demonstrating that methy￾lation of the DNA by Dnmt3a stimulates Dnmt1 and that

no physical interaction of Dnmt1 and Dnmt3a is required. If

Dnmt1 and Dnmt3a were incubated together a slightly increa￾sed stimulation is observed that could be due to a direct

interaction of these enzymes. In addition, we show that

Dnmt1 is stimulated for methylation of unmodified DNA if

the DNA already carries some methyl groups. We conclude

that after initiation of de novo methylation of DNA by

Dnmt3a, Dnmt1 becomes activated by the pre-existing

methyl groups and further methylates the DNA. Our data

suggest that Dnmt1 also has a role in de novo methylation of

DNA. This model agrees with the biochemical properties of

these enzymes and provides a mechanistic basis for the

functional cooperation of different DNA MTases in de novo

methylation of DNA that has also been observed in vivo.

Keywords: DNA methylation; enzyme mechanism; DNA

methyltransferase; Dnmt1; Dnmt3a.

Cytosine residues are methylated at the 5-position for

70–80% of all CG sequences in mammalian DNA. The

pattern of DNA methylation serves as an epigenetic mark

in general leading to a repression of gene expression

[reviews 1,2,3]. It is used to memorize developmental

decisions of the cell and to control monoallelic expression

of genes during imprinting [review 4] and X-chromosome

inactivation [5]. Work with knockout mice has shown that

DNA methylation is an absolutely essential process in

mammals during late embryogenesis [6,7]. The methylation

pattern is created by de novo methylation and demethyla￾tion of the DNA, and maintained during mitosis by

maintenance methylation [reviews 8,9]. De novo methyla￾tion of DNA is most prevalent during embryogenesis,

where the methylation is restored after an almost complete

demethylation of the genome that takes place during the

first cleavage divisions [review 10]. In addition, de novo

methylation can also occur later in development and even

in adult cells to silence acquired proviral DNA or to alter

the developmental program of the cell. Aberrant de novo

methylation may lead to hypermethylation of promotor

regions of tumor suppressor genes in cancer cells and is an

important mechanism for cancer progression [11–13]. The

mechanism of de novo methylation of DNA is still poorly

understood and requires further investigation as this

process (together with specific demethylation events) cre￾ates the pattern of DNA and therefore transfers the

epigenetic information to the DNA.

DNA methylation is introduced by DNA methyltrans￾ferases (MTases) which use S-adenosylmethionine as donor

for an activated methyl group [reviews 3,14,15,16]. Four

candidate DNA MTases have been identified in mammals

so far: Dnmt1, Dnmt2, Dnmt3a and Dnmt3b. Results

obtained with Dnmt1 knock-out mice have implicated this

enzyme in maintenance methylation [6], a role that is in

agreement to its pronounced preference for methylation of

hemimethylated DNA in vitro [17,18]. However, Dnmt1

also shows capabilities for de novo methylation of DNA

[15]. Interestingly, the de novo activity of Dnmt1 is

stimulated by binding of methylated DNA to an allosteric

site located in the N-terminal domain of the enzyme

[18,19]. However, de novo methylation activity is also

associated to the Dnmt3a and Dnmt3benzymes, which

do not show a preference for methylation of hemimeth￾ylated CG sites [20–23]. It has been shown that one target

for the Dnmt3benzyme are satellite sequences [7,24,25],

whereas specific targets for the Dnmt3a enzyme are not

yet known. In biochemical studies, it has been shown that

Dnmt3a methylates DNA in a distributive mechanism

[21]. This was a surprising observation, because it makes

the enzyme badly adapted for a fast methylation of one

domain of the DNA. However, the intriguing possibility

appeared that Dnmt3a and Dnmt1 might functionally

cooperate during de novo methylation of DNA. This

model assumes that Dnmt3a might initiate de novo

methylation by transferring methyl groups to one region

of DNA. This would recruit and stimulate Dnmt1, which

could then methylate the whole domain of the DNA

[3,21].

Correspondence to A. Jeltsch, Institut fu¨r Biochemie, FB 8,

Justus-Liebig-Universita¨t, Heinrich-Buff-Ring, 58 35392 Gießen,

Germany. Fax: + 49 641 99 35409, Tel.: + 49 641 99 35410,

E-mail: [email protected]

Abbreviations: Mtases, methyltransferases.

Note: *These authors contributed equally to the work.

(Received 21 June 2002, revised 8 August 2002,

accepted 21 August 2002)

Eur. J. Biochem. 269, 4981–4984 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03198.x