I am doing ChIP-seq for an activator of a transcription factor, that does not directly bind to DNA. I have initially used 25 min fixation with DSG at 2 mM, followed by 10 min of 1% formaldehyde fixation. However, we get so many peaks (~80.000 for both the activator and the transcription factor), that it is difficult to make sense of any biology. If I only fix with formaldehyde, I get about 5000 peaks for the activator and about 20.000 peaks for the transcription factor. If I look for the property of the peaks, then those are the "strongest peaks" (in terms of p- value, fold change and # of reads). I assume that "the truth" lies somewhere in the middle, but I was wondering if somebody has identified any artifacts due to DSG fixation in ChIP-seq experiments or how I would best test for this.
Thanks, Sylvia
Basically, both your datasets are "true", they just reflect two different situations. The longer you fixate your samples, the more you are susceptible to catch low affinity interactions between your TF and DNA. So with your short fixation, you only catch the strongest interactions compared to the longer one, which is what you observe. TF fixation on DNA is dynamic and probably very elusive, but fixation doesn't mean that your TF is functional at this site, nor that it regulates any close-by gene / interact with an expressed gene promoter. Having performed ChIP-seq myself, it is not always the strongest peaks that explain gene regulation, it could also be dependent on cofactors, proximity, time... The only way to assess a functional interaction between a TF binding site and a promoter / gene locus is to perform chromosome conformation capture experiments of some kind.
Hi Fabrice,
thank you for your answer. That seems to be the consensus (talking to other people, too). On the other hand, in the literature, people have taken along an unrelated protein, like GFP, and see chromatin capture with prolonged fixation. I will look into the HI-C, 3C experiments to see if it would be applicable to our experimental setup. Thank you.
Sylvia
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