Advice on analysing Differentially Methylated Regions of a gene. - (Aug/08/2007 )
I hope someone can help me!
I'm working on a gene that i have shown to display monoallelic expression in a proportion of a breast and bladder cancer cohort. I would like to explain this phenomenon and want to look at differential methylation patterns between my samples. My gene doesn't have any CpG islands within the promoter, but i know that this does not exclude the fact that methylation may play a role in the monoallelic expression i have seen. I know this is a vague question, but if anyone had any ideas for a direction I could take i would be extremely grateful
Thank you very much, look forward to hearing from you.
is anything known about the regulation of your gene? I wouldn't start looking for methylation blindly in a promoter that does not contain CpG-islands. If it is possible, try to look in vitro using reporter assays.
there are many other regulatory mechanisms different than methylation, and just by the sound of it, i don't think your gene of interest is controlled by it. good luck on your research.
I know it is a long shot, but some genes can be regulated by sequences other than a promoter region - like imprinted Igf2 has DMR (and a CpG island) in its 5th or 6th exon (in rat). Have you looked within your gene sequence for CpG islands (but as you said before it doesn't have to be an island). However, even if you find something, you still have to prove that that particular region is involved in regulation of your gene. Good luck, I know how hard it is.
Many imprinting control regions that are differentially methylated are not situated at the promoter, but often many kb distant (eg Igf2/Dlk1 etc). You might want to scan within 50-100kb for other cpg islands and test these for differential methylation. These ICRs often contain repeat tandem repeat sequences, which might hep to narrow your search. In addition, it is possible that you have more than one promoter that differ in their imprinting status, have you mapped the 5' end of your imprinted transcript?
Thank you both for your replies, yes it is hard! but interesting, so that is what is motivating me at the moment! Yes, it's definitely the IGF2 story that i'm looking into with regard to differential methylation other than in the promoter region, so this is what i'm focusing on.
I haven't mapped the 5' end of my gene, so thank you for your suggestion. And as for proving that the differential methylation is regulation gene expression, well that's a whole other set of experiments!!!! The work is never done!! Cheers!