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Can ChIP sequencing be used to make exploratory findings about histone methylati


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#1 Daveybaby32

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Posted 01 April 2014 - 01:46 PM

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

#2 pcrman

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Posted 08 April 2014 - 09:24 AM

So your question is to find genes differential expressed in cancer via epigenetic mechanisms and you want to approach this question by ChIP-seq. You don't need to go after all possible histone modifications, and can focus on a few such as H3K4m3 (active promoter), H3K36me3 (active transcription in gene body), H3k9me3 and H3K27me3 (repressive marks), To  get a sense of gene expression/transcription levels, you can also include RNAP II in ChIP-seq, alternative, you have to do a cDNA microarray or RNA-seq.

 

but all these ChIP-seq results just give a correlation between gene expression and epigenetic status, cannot really answer your question why some genes are deferentially expressed. Histone modification changes may just reflect the status of gene expression. If you really want to dive deep into this kind of questions, you have to take genetic factors, transcription factor binding and DNA methylation into consideration.    






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