methylating agent - (Jul/09/2009 )

Hi,

My gene of interest shows reduced expression in my normal cell line but overexpression in my cancer cell lines. have heard of de-methylating agents. Are there any methylating agents out there? I would like to test if teh overexpression of my gene is due to demethylation? How can I approach this?

Thanks

SF_HK on Jul 9 2009, 08:34 PM said:

Well, first I would suggest you to check the methylation status of your promoter of interest in both normal and cancer cells. There is a lot of possibilities for overexpression of a gene, epigenetics is just one of them. And before you waste your time and money, better check first... Bisulfite sequencing seems to be quick and rather inexpensive. You can also check many CpG islands at once.
I never heard of methylating agents... The only ones I know of are DNMT1 and 3

Cheers,
Minna

Minna on Jul 10 2009, 12:48 PM said:

Hi,
Thank you for your response. I agree with you that there could be several reasons for overexpression. What is the accepted way to check teh methylation status of the promoter? Is it treatment with 5'azacitidine? or bisulphite sequencing? If I use 5'azacitidine, I should expect expression in cell lines don't have reduced expression and no changes in expression in cell lines that show overexpression?

Sorry if any comments appear silly. I'm new to this area of research.

5-azacytidine and bisulfite sequencing are two completely different things, as you might know. To check the methylation status of your promoter, you usually use bisulfite sequencing (or other methods, like methylation-chip or this restriction enzyme, the name I just forgot...). If you find different methylation levels, you can try treatment with 5-aza (DNMT inhibitor) and see if that changes the expression of your gene. But better start with bisulfite.
Cheers,
Minna

Minna on Jul 11 2009, 12:27 AM said:

5-azacytidine is a demethylating agent and thus wouldn't it better to do a quick experiment by treating my cell lines with 5-azacytidine to check is they are methylated or not? I mean there would be a change in expression if they are methylated and no change in expression if they are not methylated? Isn't this faster than bisulphite treatment? And then this experiment could be followed by bisulfite sequencing?

Well, 5-aza is a Dnmt inhibitor, and does not induce demethylation per se. But that's nitpickery...
Sure you can treat your cells with 5-aza and check if there's a change in expression. I guess that is really quicker than doing bisulphite. If there is a change in expression if a promoter is methylated or not, is actually under scientific discussion. Most people agree that methylation inhibits gene expression, but there seem to be rare cases where methylation induces expression of certain genes.
I would start with bisulphite, but you seem to be very dedicated to do 5-aza, so go for it! But make sure you get reproducible data...
Best,
Minna