I found that knockout of my gene of interest causes higher Wnt activity (as shown by TOP/FOP flash luciferase assay as well as RNA-seq) and hence, I am trying to figure out the mechanism that leads to this observation. However, when I examined the level of beta-catenin in my knockout, the protein level of beta-catenin is lesser than the wild type. Can anyone provide me some explanation why the beta-catenin is lower when I observe higher Wnt activity in the cells?
Sounds likely the mechanism is via Wnt non canonical pathway, which does not lead to beta catenin stabilization. If it was canonical Wnt signalling and Wnt activity was increased then yes you would expect beta catenin to be increased.
By working through non canonical route you are probably getting feedback that is downregulating the canonical route, so beta catenin is down.
Wnt pathway activation depends of various Wnt ligands, Wnt receptors, Wnt agonists & antagonists. you may have knockout one target but beta-catenin is regulated by multiple factors. So, upon knockdown, beta-catenin level came down but the Wnt pathway remained active may be due to Wnt ligand, receptors & agonist.
Have you checked the stability status of components of the death complex i.e., APC, GSK3b, AXIN?
Also, as Simon said, the Wnt pathway has two types of machinery, one canonical i.e., Wnt/beta-catenin pathway and another is Wnt non-canonical (PCP or calcium led), there is a chance of switchover taking place from canonical to the non-canonical pathway.
You may find some useful information from the attached paper.
Article The regulation of β-catenin activity and function in cancer:...
My protein of interest interacts with TCF3 and TLE, so I assume it works under the canonical pathway? Or am I wrong?
I also did not check on the stability status of the destruction complex, probably I should try to get these antibodies.
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