I have a Zn finger protein that has allelic amino acid substitution in the zinc fingers. I have run some predictions on DNA binding specificity and the alleles are predicted to have differences in the DNA specificity. I would like to know if this might affect the functions of the gene net works that may be controlled by the Tf. I am working with Bovine genomes.
Hi Morris,
You could distinctly tag copies (i.e. Myc vs His) of the distinct ZnFngr protein variants and ectopically express them simultaneously, then do a comparative genome-wide ChIP analyses on their different binding affinities. This is assuming no lethal effect from the overexpression of these proteins. You will have to select the appropriate cell lines to use and compare - binding sites/affinities will undoubtedly vary from line to line.
If overexpression is lethal in bovine cells I would recommend either endogenously tagging your distinct alleles and diong as above or using an in vitro system to compare the protein variants such as SELEX-Seq or similar. The latter will require you express your protein variants separately in bacteria (or yeast) and harvest enough to test their relative affinities in vitro. Then locate your binding sites for each in the genome bioinformatically and use available packages (available in Bioconductor, etc) to isolate the sites with highest statistical confidence.
Best of luck with it,
Pegine
Thank you Pegine for the prompt response. I forgot to add that I am looking at in silico tools.
In that case, how familiar are you with Bioconductor's packages for R? Worth a look!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA