Next step after DNA methylation - What is the next step? (Apr/10/2007 )
What is the next logical step after you have shown your gene to be aberrantly methylated? Histone methylation, acetylation analysis? And which is the best technique?
I would go for histone modifications, best technique is Chip and there is this protocol by KPDE, that makes it very fast...
What about expression of the protein? Is that not a logical step?
Actually making sure there is an impact on mRNA expression would be the next step...
Sure, I thought, expression was already done ... (why should one look at the methylation of a gene that is normally expressed?)
Good point krumelmonster! I guess I was assuming it was detected in some sort of genome wide array etc...
also a good point beccaf22 ... interesting, how we tend to assume that everybody is doing the same thing (in the same order) as we do, isn't it?
OK, to clarify it a bit more:
Suppose the microarray has been done to look at RNA expression, validated by QPCR, the methylation patterns of specific genes have been verified in cell lines, tissues and expression has been correlated with DNA methylation.
So after all that,
Do we start looking at histone acetylation and methylation? Do we add ubiquitinilation and phosphorylation? Which lysine, arginine (proline) residues shall we start with? Do we look at methyl binding proteins? Do we just use the normal CHIP method or adjusted protocols? Is it worth doing luciferase constructs to look at the effect of DNA meth on the DNA fragment of interest? Do we need to do a western and northern blot if we have the microarray and QPCR data?
What are your opinions?
Okay, man - a lot of work already done!
A first experiment in the cell culture could be the use of Trichostatin A - if this doesn't change the gene's expression, it is not likely that histone acetylation is involved.
The histone modification most often looked at (in my opinion) is lysine 9 acetylation of H3 or H4. After that, there are a lot more modifications to look at (there is this nice poster from Upstate, you can pin every new discovered modification at the proper residue), or maybe there is a transcription factor interesting for your disease? It may be worth to have a look at the promoter for TF binding sites (e.g. mathinspector - www.genomatix.de). Maybe Chiping interesting TFs for your gene is also interesting?
If nothing is known about the regulation of your gene, the use of luciferase may be worth it - but I know several coleagues who curse the day they started it
Finally mRNA is not equivalent to protein - so western may bring up different results.
Many options, I don't know if there is a superb logical way of going through it? There is this interesting paper by Weaver et al from Michael Meaneys group (Nature Neurosci 2004) - they performed qPCR, BSP, H3 & H4 K9 acetylation Chip and afterwards TF-Chip for NGFI-A (neuronal TF). Maybe a good idea for the next steps?
Sorry, I don#t think I am of too much help...