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

[kopi-o]

It's definitely used for that sort of thing.

http://scholar.google.se/scholar?q=C...+modifications

[Melissa]

I think there must be many similar experiments in NCBI SRA unless you're using different cancer cells. If you want to look for histone modifications and methylation patterns, you will need to do Chip-Seq and BS-seq separately or Chip-BS-Seq. You will need to do gene expression study to get a list of differentially expressed genes for your particular cancer/cells. The goal to identify a few genes is unrealistic.

[Daveybaby32]

I think there must be many similar experiments in NCBI SRA unless you're using different cancer cells. If you want to look for histone modifications and methylation patterns, you will need to do Chip-Seq and BS-seq separately or Chip-BS-Seq. You will need to do gene expression study to get a list of differentially expressed genes for your particular cancer/cells. The goal to identify a few genes is unrealistic.

Yes, the identification of numerous cancer causing genes is a bit of a stretch, my actual hope is to do the ChIP sequencing of the cancer and normal cells and compare the results. Based on those results I imagine there there would be a few discrepancies in methylation patterns on the histones of certain genes. The next step would be to insert interfering RNA strands that would knock down the expression levels of those genes from the first part of the study and then observe the cell cultures to see if any sorts of tumours develop as a result of the gene knockdown. I imagine that at this point there would only be one gene for which the methylation of histones was different in the cancer cells as compared to the tumor cells and showed the presence of cancer once that gene was knocked down in normal cells.