UHRF1 is an oncoprotein that is overexpressed in various types of cancers. The genomic DNA of cancer cells is marked by a global DNA hypomethylation that could activate previously silenced retroviruses or transposons and significantly destabilize the genome. UHRF1 contains a C-terminal E3 ligase domain and can polyubiquitinate selected substrate proteins, such as the <i>de novo</i> DNA methyltransferase DNMT3A1, for proteasome degradation. The UHRF1-induced degradation of DNMT3A1 is partially responsible for the global DNA demethylation in cancer. Our hypothesis is that the inhibitors of UHRF1-DNMT3A1 interaction can potentially increase DNMT3A1 level and thereby reverse global DNA hypomethylation in cancer cells. In this study, we developed a NanoBRET assay that allow us to test chemical compound effects on UHRF1-DNMT3A1 interactions in live cancer cell. In the NanoBRET assay, the Nano-luciferase (NanoLuc) is used as blue-shifted light donor while a fluorophore-labeled HaloTag ligand is the acceptor. When the NanoLuc fusion protein interacts with the HaloTag fusion protein, an increase of NanoBRET (460nm/618nm) ratio can be observed; alternatively, the NanoBRET ratio will decrease after adding protein-protein interaction (PPI) inhibitors. The NanoBRET assay has the advantages of evaluating PPI in its native cellular environments – the proteins are properly folded and likely to form normal protein complexes (with other necessary cellular components). In our study, we used GoldenBraid cloning to generate 8 vectors that express different pairs of NanoLuc or HaloTag-fused UHRF1 and DNMT3A1 (also test the effects of N- or C-terminal labeling). During NanoBRET optimization, the 8 vectors were transfected into HEK293 and the best donor/acceptor combinations were selected. We expect this assay will be a useful primary and secondary method to identify and characterize PPI inhibitors of UHRF1-DNMT3A1 interaction.

Develop NanoBRET assay to examine small molecule effect on DNMT3A1-UHRF1 interaction in cancer cells

2019 Fall National Meeting

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