Abstract
The human COMPASS complexes regulate gene expression during development and cell differentiation. Three distinct subunits, KMT2C, KMT2D, and KDM6A (also known as UTX), are frequently mutated in urothelial carcinoma, possibly disrupting the formation of functional COMPASS complexes. Here, we describe methods to evaluate the formation of these large native protein complexes in urothelial carcinoma (UC) cell lines harboring different mutations in KMT2C/D. To this end COMPASS complexes were purified from nuclear extracts by size exclusion chromatography (SEC) using a Sepharose 6 column. SEC fractions were then separated by 3–8% Tris–acetate gradient polyacrylamide gel and the COMPASS complex subunits KMT2C, UTX, WDR5, and RBBP5 were detected by immunoblotting. In this fashion, the formation of a COMPASS complex could be observed in UC cells with wild-type but not in cells with mutant KMT2C and KMTD.
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Acknowledgments
This work was supported by the Wilhelm Sander Foundation (grant 2016.038.2). PW was supported by the DPST scholarship, Thailand.
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Peter, C., Schulz, W.A., Whongsiri, P. (2023). Characterization of Native COMPASS Complex in Urothelial Carcinoma Cells by Size Exclusion Chromatography. In: Hoffmann, M.J., Gaisa, N.T., Nawroth, R., Ecke, T.H. (eds) Urothelial Carcinoma. Methods in Molecular Biology, vol 2684. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3291-8_5
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DOI: https://doi.org/10.1007/978-1-0716-3291-8_5
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