Replacement of 5-methylcytosine by cytosine: a possible mechanism for transient DNA demethylation during differentiation.

Abstract

In an earlier study it was discovered that when Friend erythroleukemia cells (FELC) were exposed to a variety of chemical agents capable of inducing differentiation, their DNA underwent genome-wide transient demethylation.In an attempt to elucidate the biochemical mechanism responsible for this phenomenon we have induced FELC with 5 mM hexamethylenebisacetamide and labeled the DNA in vivo with a density label, 5-bromodeoxyuridine, and a radioactive label, deoxy [5-3H]cytidine. Newly replicated DNA (heavy-light) was separated from parental DNA (light-ight) by isopycnic centrifugation. Incorporation of deoxy[5-3H]cytidine into lightlight duplex DNA has been observed only in induced cells concomitantly with the demethylation of the DNA, whereas, in parallel experiments, deoxy[G-3Hladenosine was not incorporated into light-light DNA. It was also found that the labeling of light-light DNA with deoxy[5-3H]cytidine is transient since the H label was removed from the DNA during the period of de novo DNA methylation that follows the demethylation. These results, taken together, strongly suggest that the demethylation of the DNA during differentiation is achieved by an enzymatic mechanism whereby 5-methylcytosine is replaced by cytosine.In a previous study, while analyzing changes in DNA methylation during terminal differentiation of Friend erythroleukemia cells (FELC), some of us have observed that genome-wide DNA hypomethylation is induced by a variety of agents that are known to cause differentiation in these cells (1). Genome-wide hypomethylation has also been observed in developing mouse (2) and rabbit (3) early embryos and in teratocarcinoma mouse cells in response to inducers of differentiation (2,4,5). It is noteworthy that the hypomethylation observed in FELC was found to be transient: demethylation takes place relatively rapidly (within several hours) and is followed by de novo methylation (1). The kinetics of this demethylation process is not consistent with a passive mechanism of hypomethylation-namely, loss of methyl groups by replication of the DNA due to the absence or inhibition of maintenance methylation. In addition to this observation, two other instances have been reported recently that suggest an active mechanism for DNA demethylation.One Hpa II site upstream to the chicken vitelogenin II gene undergoes demethylation in response to estrogen treatment in the absence of replication (6), and a region in the EpsteinBarr virus DNA undergoes demethylation in response to induction by phorbol 12-myristate 13-acetate and butyrate before viral DNA amplification takes place (7). In an attempt to elucidate the mechanism by which active demethylation takes place, we have considered three possible mechanisms: (i) removal of the methyl group from 5-methylcytosine by direct demethylation; (ii) removal of5-methylcytosine and its replacement with cytosine through an enzymatic mechanism not previously described; (iii) removal of a stretch of DNA that would include the 5-methylcytosine moiety by the conventional ...

DNA glycosylases in the base excision repair of DNA

“…This implies that methylation and demethylation of cytosine must be regulated by complex mechanisms. 5-MeC is apparently removed and replaced by cytosine by an enzymic mechanism resembling the BER process [171]. While one report suggested that 5-meC is removed by an endonucleolytic process [172], others have demonstrated release of the free base (5-meC, which is subsequently deaminated to thymine) by HeLa nuclear extracts [173].…”

Polymerase chain reaction-aided genomic sequencing of an X chromosome-linked CpG island: methylation patterns suggest clonal inheritance, CpG site autonomy, and an explanation of activity state stability.

“…They showed that an equilibrium methylation level will be reached that will depend on the ratio of the efficiency of maintenance methylation (Em) to the efficiency ofde novo methylation (Ed). Much ofthe reasoning is directly applicable to our system, but because of recent evidence that methylation can be lost quickly and without DNA replication (38,39), we present here an alternative treatment that does not specify how, or from which strand, methylcytosines are lost.…”

Enzymic removal of 5-methylcytosine from poly(dG-5-methyl-dC) by HeLa cell nuclear extracts is not by a DNA glycosylase

“…A mammalian DNA methyltransferase, specific for the CpG dinucleotide sequences that are the major targets for cytosine methylation in vivo, has been characterized in considerable detail and shown to be critical for mouse embryogenesis by gene knockout (2). The mechanism for de-methylation of 5-methylcytosine (5-MeC) residues in DNA is less clear, but it appears to involve removal of either the modified base or nucleotide and subsequent replacement through incorporation of an unmethylated cytidine nucleotide (3). An excision/repair activity has been described in nuclear extracts ofchicken embryos and differentiating mouse myoblasts that appears to work through a 5-methylcytidine-specific, EDTA-resistant endonuclease activity (4,5).…”