In colorectal cancer, some tumor suppressor genes are inactive. This is an important factor resulting in uncontrolled cell division. Two of the possible explanations for the inactive genes are: (1) a mutation in the coding region, resulting in an inactive protein, and (2) epigenetic silencing at the promoter of the gene, resulting in reduced transcription. How would you investigate each of these possibilities?

(1) a mutation in the coding region, resulting in an inactive protein

To check to see if there is a mutation, you could extract the DNA from the cancer cells and then perform PCR to amplify the gene of interest. You could then perform sanger sequencing and compare the sequence to the normal gene to see if a mutation is present. To test the effect of the mutation, you would want to see if an active protein has been formed.

To see if a normal sized protein has been formed, you could perform a western blot, comparing the protein band to the WT protein band. If the protein is absent or much smaller, it is likely not a functional protein.

(2) epigenetic silencing at the promoter of the gene, resulting in reduced transcription.

To check for changes in the epigenetic landscape of the promoter, you could perform chromatin immunoprecipitation by extracting the chromatin from the tumour cells and using antibodies for different chromatin marks to see what has changed between the normal cells and the tumor cells. E. g. H3K9me3, H3K27me3. You would perform a pull down with the antibody of interest and then PCR for your promoter to specifically look at changes at that gene compared to normal cells. To test DNA methylation, you could perform bisulfite sequencing.

To see how transcription is affected, you could extract RNA from the tumor and normal cells, and compare the levels of RNA between the two samples by qRT-PCR