internal assay controls for histone ChIP - (May/02/2006 )

Hi,

our lab recently has seen instability issues with various transfected cell lines. Preliminary experiments suggest that this might be due to histone modifications and / or DNA methylation.

I'm trying to develop a ChIP assay that would allow me to see which histone modification may be present (I will tackle methylation assays later). Specifically I'm thinking about looking at H3 K9 methylation (conducive to transcriptional downregulation) and H3 K9 acetylation (transcriptionally active).

My question: what kind of internal controls could I be using to see whether these 2 Abs are working properly? I was thinking housekeeping genes, but I don't have any promoter sequence available for any of these (which I would need in order to design primers, correct?)

Also, the pulldown efficiencies for these 2 Abs might be quite different which would mean that I would not be able to directly compare the results (such as in a ratio)......

Any suggestions?

selection of loci to assay is very important.

I would suggest you have a look at cancer associated gene promoters which are known to be silenced and repressed or activated depending on the cell line you are using. Another place to look is if your line is female and mammalian, then you could probably target genes on the X-chromosome.

I take it the instability in your cell lines is the instability of the vector you introduced? I was wondering if you have ruled out all the selections (antibiotics etc) that may be off?

Nick

Great suggestion (I spent the better part of my morning looking for developmentally regulated i.e silenced genes. I may have actually found some. Getting the respective sequences is going to be the next step).

Yes, we are fairly certain at this point that the expression instabilities are NOT due to inadequate / non-existent selective pressure, and / or rearrangements.

Thanks so much!

You'll probably see more "cry for help" kind of posts from me in the near future since ChIP is essentially a new technique in our lab.

Hey,

I think that it is also a good idea to use the promoters of housekeeping genes as you have mentioned previously.

There are some pretty good PCR primer sequences at the following link:

http://ops.abcam.com/index.html?pageconfig...ocols&mode=prot

There are also primers for liver, muscle specific genes that you can reply on being transcriptionaly inert and depleted for tri-methylated histone H3 K4.

Take it easy

Hey spotty,

great suggestion and link - unfortunately I'm working in a hamster system, and genomic sequences are hard to come by. I'll keep looking. I think I might have to resort to non-promoter sequences (not ideal, but it should work - correct?)

Thanks to all of you for the great input I've gotten so far!

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