Summary
The two-hybrid system is a genetic method to search for and to identify direct interaction partners of a protein of interest. This method is instrumental to elucidate the transformation mechanism of several oncogenes that play a role in childhood leukaemia. With respect to mixed lineage leukaemia gene (MLL) fusions, two-hybrid screening was applied to discover proteins that bind to various MLL fusion partners. Here we describe a streamlined protocol that enables any average molecular biology laboratory to conduct and evaluate a standard two-hybrid screen. Starting with a general explanation of the biological background of the two-hybrid method, this chapter covers the construction of bait vectors and two comprehensive protocols for screening either by yeast mating or yeast transformation. In addition, it also gives guidelines for the evaluation of two-hybrid results.
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References
Fields S, Song O. (1989). A novel genetic system to detect protein–protein interactions. Nature;340:245–246.
Legrain P, Selig L. (2000). Genome-wide protein interaction maps using two-hybrid systems. FEBS Lett;480:32–36.
Zeisig DT, Bittner CB, Zeisig BB, Garcia-Cuellar MP, Hess JL, Slany RK. (2005). The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin. Oncogene;24:5525–5532.
Erfurth F, Hemenway CS, de Erkenez AC, Domer PH. (2004). MLL fusion partners AF4 and AF9 interact at subnuclear foci. Leukemia; 18:92–102.
Bursen A, Moritz S, Gaussmann A, Moritz S, Dingermann T, Marschalek R. (2004). Interaction of AF4 wild-type and AF4.MLL fusion protein with SIAH proteins: indication for t(4;11) pathobiology? Oncogene;23:6237–6249.
Srinivasan RS, de Erkenez AC, Hemenway CS. (2003). The mixed lineage leukemia fusion partner AF9 binds specific isoforms of the BCL-6 corepressor. Oncogene;22:3395–3406.
Begay-Muller V, (2002). Ansieau S, Leutz A. The LIM domain protein Lmo2 binds to AF6, a translocation partner of the MLL oncogene. FEBS Lett;521:36–38.
Hemenway CS, de Erkenez AC, Gould GC. (2001). The polycomb protein MPc3 interacts with AF9, an MLL fusion partner in t(9;11)(p22;q23) acute leukemias. Oncogene;20:3798–3805.
Garcia-Cuellar MP, Zilles O, Schreiner SA, Birke M, Winkler TH, Slany RK. (2001). The ENL moiety of the childhood leukemia-associated MLL-ENL oncoprotein recruits human Polycomb 3. Oncogene;20:411–419.
Fuchs U, Rehkamp G, Haas OA, et al. (2001). The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia. Proc Natl Acad Sci U S A;98:8756–8761.
So CW, So CK, Cheung N, Chew SL, Sham MH, Chan LC. (2000). The interaction between EEN and Abi-1, two MLL fusion partners, and synaptojanin and dynamin: implications for leukaemogenesis. Leukemia;14:594–601.
Garcia-Cuellar MP, Schreiner SA, Birke M, Hamacher M, Fey GH, Slany RK. (2000). ENL, the MLL fusion partner in t(11;19), binds to the c-Abl interactor protein 1 (ABI1) that is fused to MLL in t(10;11)+. Oncogene;19:1744–1751.
Shinobu N, Maeda T, Aso T, et al. (1999). Physical interaction and functional antagonism between the RNA polymerase II elongation factor ELL and p53. J Biol Chem;274:17003–17010.
Yam JW, Jin DY, So CW, Chan LC. (2004). Identification and characterization of EBP, a novel EEN binding protein that inhibits Ras signaling and is recruited into the nucleus by the MLL-EEN fusion protein. Blood;103:1445–1453.
Ma J, Ptashne M. (1987). Deletion analysis of GAL4 defines two transcriptional activating segments. Cell;48:847–853.
Guthrie C FGR. (2002). Methods in Enzymology, Guide to Yeast Genetics and Molecular and Cellular Biology, Part B., Vol. 350. New York: Academic.
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Work in the authors’ laboratory is funded by DFG, Deutsche Krebshilfe, and the EU.
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Garcia-Cuellar, MP., Mederer, D., Slany, R. (2009). Identification of Protein Interaction Partners by the Yeast Two-Hybrid System. In: Eric So, C.W. (eds) Leukemia. Methods in Molecular Biology™, vol 538. Humana Press. https://doi.org/10.1007/978-1-59745-418-6_18
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DOI: https://doi.org/10.1007/978-1-59745-418-6_18
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