Debate on the existence of DNA demethylase - (Aug/27/2005 )
I am interested in DNA demethylation, and there are active DNA demethylation in two stages in mammalian development: one is the PGC stage, and the other is after fertilization. Several papers have proven this phenomenon, but no one find protein associated.
Then what is your opinion?
I believe the results, as reveiwed by Dean and Santos 2005, who ever characterses the protein responsible will have a guarunteed nature or science paper under their belt.
There has been some conjecture of MBD2 being responsible because it's a glycosylase. But it's great seeing the two bigshots of DNA methylation disagree with each other, Bird and Syzf. Bird can't replicate Syzf's findings and therefore thinks it is untrue, but seeing these guys in action at a conference really blows me away.
I believe it's ture. But we must prove it, the best way is to find out a protein or a complex associated with DNA demethylation. Because of the meterials and manipulation, progress is hard to make. Maybe we have to change our diretion? I think it's a challenge for a epigenetist.
and to find the active protein at the timepoints where we see active demethylation, where there is so little material (cells) to start with is rather challenging.
global approaches could be used but it would be a rather expensive fishing expedition!!
hello, nick, could you tell me what opinion Szyf and Bird hold?What is their difference? Just in brief
essentially Szyf performed experiments whereby overexpression of MBD2 causes active demethylation of methylated reporter promoters....implicating MBD2 as the demethylase, because such experiments were attempted in the Bird lab and could not be replicated, Bird thinks that MBD2 is not the demethylase as first reported.....that was the jist of things at the conference.
Which conference was that Nick?
Papers from Szyf's group claiming the existence of demethylase (MBD2)
1: Detich N, Theberge J, Szyf M.
Promoter-specific activation and demethylation by MBD2/demethylase.
J Biol Chem. 2002 Sep 27;277(39):35791-4. Epub 2002 Aug 12.
PMID: 12177048 [PubMed - indexed for MEDLINE]
2: Szyf M, Bhattacharya SK.
Extracting DNA demethylase activity from mammalian cells.
Methods Mol Biol. 2002;200:163-76. No abstract available.
PMID: 11951651 [PubMed - indexed for MEDLINE]
3: Szyf M, Bhattacharya SK.
Measuring DNA demethylase activity in vitro.
Methods Mol Biol. 2002;200:155-61. No abstract available.
PMID: 11951650 [PubMed - indexed for MEDLINE]
4: Cervoni N, Szyf M.
Demethylase activity is directed by histone acetylation.
J Biol Chem. 2001 Nov 2;276(44):40778-87. Epub 2001 Aug 27.
PMID: 11524416 [PubMed - indexed for MEDLINE]
5: Ramchandani S, Bhattacharya SK, Cervoni N, Szyf M.
DNA methylation is a reversible biological signal.
Proc Natl Acad Sci U S A. 1999 May 25;96(11):6107-12.
PMID: 10339549 [PubMed - indexed for MEDLINE]
6: Cervoni N, Bhattacharya S, Szyf M.
DNA demethylase is a processive enzyme.
J Biol Chem. 1999 Mar 26;274(13):8363-6.
PMID: 10085064 [PubMed - indexed for MEDLINE]
7: Bhattacharya SK, Ramchandani S, Cervoni N, Szyf M.
A mammalian protein with specific demethylase activity for mCpG DNA.
Nature. 1999 Feb 18;397(6720):579-83.
Papers against demethylase
1. Ng, H. H., Zhang, Y., Hendrich, B., Johnson, C. A., Turner, B. M., Erdjument-Bromage, H., Tempst, P., Reinberg, D., and Bird, A. (1999) Nat. Genet. 23, 58-61
2. Boeke, J., Ammerpohl, O., Kegel, S., Moehren, U., and Renkawitz, R. (2000) J. Biol. Chem. 275, 34963-34967
3. Wade, P. A., Gegonne, A., Jones, P. L., Ballestar, E., Aubry, F., and Wolffe, A. P. (1999) Nat. Genet. 23, 62-66
Explanation given in a JBC Paper from Szyf's group why others couldn't reproduce their results:
Although it is accepted in recent publications that MBD2 (the demethylase) acts as a transcriptional repressor (11, 12, 24, 25), the data presented here do not necessarily contradict previously published data once the experimental conditions stated in these reports are carefully considered.
First, in all the studies demonstrating repression by MBD2/dMTase, transcriptional assays were performed anywhere from 24 to 48 h post-transfection. Since we do not see activation of the SV40 promoter until 96 h post-transfection, it is possible that some of the promoters in these reports could be activated by MBD2/dMTase provided that the transfection time is extended. The fact that a longer time is necessary for activation by MBD2/demethylase may be explained by previous data showing that demethylation of ectopically methylated DNA in living cells is a slow process (3).
Second, previous studies (11, 25) used the Gal4 DNA binding domain to bind MBD2/GAL4 chimeras to their promoters rather that looking at the effect of cognate MBD2 on methylated DNA as done in this study, which may also account for some of the discrepancies.
Third, not all promoters are repressed by MBD2, and not all concentrations of transfected MBD2 bring about repression. For example, Boeke et al. (12) demonstrated that although the TK promoter was repressed by methylation, transfection of MBD2 had no effect; and in a study by Sekimata et al. (25), certain concentrations of transfected Gal4-MBD2 alone did not repress their reporter construct. The data presented above are consistent with our findings that MBD2 had no effect on the p19-ARF promoter (Fig. 1C) and that the effect on transcription we observed is dose-dependent, where the dose causing activation varies with the promoter type.
Last, although MBD2 has been found to associate with the NuRD repressor complex (24), it was not originally purified as part of this complex (26), and there is evidence that the NuRD complex may associate with different DNA-binding proteins, not only MBD2, depending on the physiological state of the cell (24). Thus, it is possible that under certain cellular conditions and within certain promoters MBD2/dMTase may act as a transcriptional repressor by recruiting the NuRD complex. However, it is equally possible that in a different cell environment and within different promoters MBD2 may act independently of NuRD as a demethylase and activator. In support of the latter, expression of dMTase is correlated with demethylation within the promoter of c-erbB-2 and survivin genes (23). In addition, a recent report has demonstrated that the Drosophila homolog of MBD2, dMBD2/3, formed foci that associated with DNA at the cellular blastoderm stage, concurrent with the activation of the embryonic genome, and also associated with the active Y chromosome
Their conclusion: it is feasible that MBD2 is a protein with dual functions. It is possible that both repressor and demethylase functions reside in one protein to coordinate a program of gene expression that requires suppression of some methylated genes and activation of others.
I don't know if others have repeated their experiments under the exact same conditions such as the same promoter, same transfection period, and everything. If not I tend to believe their story. Sometimes, things may work in one system but not another.
couldn't have summerised that myself, good job pcrman