I have been trying to study DNA binding of a yeast transcription factor under different growth conditions using ChIP. Positive control shows enrichment under a specific growth condition and that is expected. However, even negative control loci (incl. ones that do not have response element) are also getting enriched when cells are grown under same condition. The response element sequence is known and mutating this sequence results in lose of transcriptional regulation. Fixation time, Antibodies and temperature of fixation were changed to see if it would reduce non-specific enrichment. Enrichment is qualitatively observed using endpoint PCR. Is there any other explanation other than my protein of interest promiscuously associating with non specific sequences without exerting its transcriptional regulatory activity? Thank you.
Hi! Stevin,
Did you include a no-ab control? Or even better, negative control antibody (goat/rabbit/mouse serum, depending on the other antibodies you use)? It could simply be background binding to the beads, and these controls are the best way to rule that out.
best,
vegard
Yes, There was no background binding issue with untagged protein control. Thank you
Hi Stevin,
Are you over expressing the transcription factor on a 2u plasmid or a CEN plasmid? You could be causing promiscuous binding if you are using an inducible (Gal) or high expression promoter (Adh1). How many negative control loci are you looking at? Are they all promoter regions or have you selected ORFs and 3' regions as well? It could be that the loci you are looking at could have binding as well but the regulatory role is not identifiable under these conditions.
Hi. Thank you for the response. the protein was expressed under a weak constitutive promoter and integrated into genome. Several negative control loci were looked including sites without consensus binding sequence. I looked only in the promoter regions.
Hi Stevin!
It is possible that your transcription factor of interest is indirectly associating with the DNA through another transcription factor. If you know of any protein-interaction partners for your TF of interest, it is worth investigating whether it's interaction partner has DNA element(s) at the locus you're assaying for.
Also, as Paul mentioned, I recommend designing negative control primers. Since you did this, I'm curious to know whether you also see amplification at these negative control loci. If not, you may have discovered a new and uncharacterized DNA binding element for your TF! Promoter-deletion analyses will help you answer that :)
Good luck :)
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