3 replies to this topic

[ky_kev]

Hi there,

I'm sure some of the experts in this forum will have a simple answer for a newbie to epigenetics. I've treated my cells over 5-days with 5-aza-dC and seen a nice dose-dependent induction of my gene of interest. I've since sodium bisulfite sequenced the the promoter region which is of low-density containing only 9 CpGs spanning 800bp and found that there is no demethylation occuring. I've digested the same gDNA samples with McrBC and there is a clear global demethylation taking place so I know the treatment has worked. So this might indicate that it is an upstream factor being demethylated to activate transcription or I'm not focussing on the correct CpG sites? But why aren't all CpGs demethylated following 5-aza-dC treatment? Is there a preference of 5-aza-dC action towards large islands rather than low-density regions? Any explanation will go a long way to addressing some comments!

Much appreciated,
Kev

Edited by ky_kev, 06 September 2009 - 07:01 PM.

[methylnick]

A recent paper in Leukemia by Flotho et. al. showed differential demethylation with a range of demthylating agents including 5-aza-C demethylating certain genes and not others and I think is the first paper to show differential action of these drugs on the promoter.

Happy to email it to you if you pm me your address.

cheers

Nick

[ky_kev]

Much appreciated Nick, I was hoping you were going to come through with the goods!

Kev

[Davo]

Hi Kev,

This is something that I have seen before as well. I know the confiused feeling of seeing an increase in expression without a great change in CpG methylation. We are trying to publsih a paper on this at the moment, so fingers crossed it goes through!

Dave