When doing ChIP, the antibody you use is the output you'd see when looking at a specific promoter correct?

11 November 2018 2 5K Report

So, I am reading a paper and I seem to be confused. In this paper they did a ChIP assay to observe the promoter region of p21 to determine if there was an interaction with p53. They claim their results prove a dependency of p53 on p21 expression. While this is not unheard of and I don't doubt the accuracy of this statement, I am a bit perplexed at some of the language used to describe this interaction and the explanation of the experiment used. From my understanding of ChIP, you use specific primers to target the promoter region of interest, then to determine the interaction that region may have with other genes or transcription factors you use a specific antibody. The output of data should tell you the level of expression of that particular antibody correct?

So they looked at the p21 promoter using specified primers then probed with the p53 antibody. In their description of results for the ChIP assay, they say they found increases in p21 expression as a result of p53. In the previous section they do report increases in p21 mRNA expression which makes me think it's a matter of semantics or I am understanding the experiment wrong. If the antibody was specific to p53, would they not have found increases in p53 expression at the promoter of p21 thereby showing a relationship with the two and possibly a dependency?

They do show increases in p53 protein expression along side p21 which supports their line of thinking, and I think it makes sense to associate elevated levels of p21 with elevated levels of p53 (wt) since p53 is interacting with p21 at its promoter. Elevated levels of p53 at the p21 promoter would make sense, and would not change the conclusions of this particular study.

I'm simply wanting to ensure I understand how the experiment works.

Pietro Pilo Boyl

Hi Kendra, since you don't mention the paper it is difficult to discuss its conclusions based on what you summarize... In science the exact wording makes the whole difference!

But, to clarify the experimental approach: ChIP starts with an antibody and the fragmented genomic material. Your antibody is directed against a gDNA binding protein (or a protein part of a gDNA binding complex). First thing you do is the immunoprecipitation, then from the IPed material you extract the DNA and finally using specific primers (and often a quantitative approach such as qPCR for a targeted study) you measure how much of the target gDNA you are interested in was immunoprecipitated by the antibody (and you normally calibrate it with your fragmented gDNA input material). In order to make quantitative statements you need a control and an experimental condition.

So, I could imagine that in the paper they overexpressed p53 and after ChIP with an anti-p53 antibody they could measure increased amount of the p21 promoter region DNA compared to a mock transfected condition. If p53 association to the p21 promoter is functional to the expression of p21, you should in the same conditions measure increased levels of p21 mRNA, which they did. Then the conclusion would be that p53 has a positive role on the expression of p21 by direct association with it's promoter region.

Keep in mind that if p53 would not specifically bind to the p21 promoter, overexpressing p53 would not increase the amount of p21 promoter region DNA found in the IP! In a standard WB you would see the increased levels of p53, of course, but that is not the principle of ChIP. ChIP is used to see the association of a protein to a genomic DNA region by measuring the amount of this genomic DNA that is co-immunoprecipitated with the protein you pull down with the Ab.

Hope I made myself clear...

Cheers

Kendra Royston

Thanks so much! As you explained is how I understood ChIP to work. Since there's so much literature in support of p21 and p53 regulating one another they are in a safe zone. I think in this case, their wording was just funny. They did not find an increase in p21 expression using ChIP as they write, they found an increase in p53 immunopercipitation of the p21 promoter thereby proving p53's positive role in p21 mRNA expression.

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