It is known that c-Jun and fos dimerize to form AP1 factor that binds to a sequence on DNA containing PyPuGACGTCNNNNGAGGTCPyPU. In esophageal cancer cell lines there is no expression of the fos gene. However we still see expression of genes regulated by AP1 factor. One of the possibility is another isoform of fos is expressed in these cells (e.g. fra1).
- How will you check that fra1 is expressed or not in these cells.
- How will you check that the heterodimer bound to the given DNA contains fra-1 along with c-jun.
My thoughts on the above questions
For first question we can do immunofluorescence assay or we can do DNA affinity assay in which we chemically synthesize DNA sequence and bind it on agarose gel column pass through it protein extract of oesophageal cancer cells and our fra-1 protein if being expressed in cell line will bind to DNA. later we can elute protein DNA complex and by anti-fra1 antibody we can check if it is being expressed. But the drawback of both these technique is we should have knowledge of epitope of fra-1.
For second question - we can perform ChIP using antibody tagged Against c-jun and then eluting out DNA bound to c-jun. Then we can simultaneously label the complex with anti fra-1 in one sample and anti fos in another sample. We can check for results. If fluorescence is obtained in case of fra-1 sample that means both c-jun and fra are bound to probe and not fos.