Can ChIP sequencing be used to make exploratory findings about histone methylation?

[Daveybaby32]

This is for a grad school course I'm taking about cancer biology. The assignment is to design any experiments of our choice and propose them via a grant proposal. The idea I have is to isolate DNA from tumor cells and from normal cells of the same cell type. Then I would shear the entire genome obtained from both cell types and use ChIP analyses to check the modifications made to the histone tails in the DNA.

I would then compare the results of the ChIP sequencing analyses to find out where in the genome his tones are being methylated/acetylation/phosphorylated differently in the cancer cells as compared to the normal cells. This would then point me to a few genes which have varied levels of expression in cancer cells.

I know that ChIP antibodies used in this sort of analyses are very specific to the histone so they bind to, for instance you can have antibodies that bind specifically only to a given modification on a given amino acid residue of a histone tail.

My question now is this: is this sort of ChIP sequencing something that is normally conducted or is this not really what it's used for. I'm a first year grad student and have never used ChIP sequencing myself but I'm familiar with the theoretical aspects behind it.

Can ChIP sequencing be used for this type of exploratory research (namely finding genes whose expression levels may be altered in cancer) or is ChIP typically used only with a specific short sequence of DNA in mind? My biggest worry is that the permutations of possible modifications to every different amino acid residue on the histone tails of all the different histones in the human genome are far too large. Am I correct in saying this? If so, do you have any suggestions as to how I may possibly achieve the same overarching goals with a more realistic lab technique?

Lastly, do you like my research proposal overall, or do you feel there are still places wherein I could strengthen it and make it more scientifically sound? Again, this is only an assignment in a course I'm in so all proposals are theoretical. None of this will be conducted in real life

[maciosz]

Sure, that's what ChIP-seq is for. What's more, there are many packages dedicated to comparing two (or more) sets of ChIP-seq data, obtained for example from different tissues or cells undergoing different treatment, so it definetely covers your idea. Some of these packages are better for transcription factors than histone modifications, but there are some that work pretty well with them too. My first suggestion would be DiffBind, if you're interested in details.

However, you must realise your experiment does sound a little bit unrealistic. First of all, you can't just say you're going to analyse all the possible modification - as you said, antibodies are very specific, so you should choose a very specific modifications (and when I say specific I don't mean being specific only about modification, but also a histone that undergoes the modification and even its specific aminoacid). Second, don't expect to get "a few genes", there will be a lot of genes with different modification.

You used expression "level of expression" - well, we could assume, that genes with different histone modification have different level of expression, but that's not for certain. So, if you want to messure expression level you should use something else, like RNA-seq, for example. Or microarrays.

[mlodato]

You might want to look up the ENCODE project, which is looking at histone marks, transcription factors, and other features of the regulation of the genome across many cell types, including cancer cells. This would give you a sense of how the field is thinking about these sorts of problems, but long story short they are doing something similar to what you propose on a massive scale.