Identification Of HPYGO2 Interacting Protein By Yeast Two-Hybrid

Identification of Pygopus in Drosophila as a dedicated component of the Wg(fly homolog of mammalian Wnt) signaling cascade initiated many inquiries into the mechanism of its function.Surprisingly,the nearly exclusive role for Pygopus in Wg signal transduction in flies has dual functions in mice,where Pygopus appears to have both Wnt-related and Wnt-independent functions.In this dissertation addresses the canonical Wg/Wnt signaling pathway and the recent studies of Pygopus function.We further analyze the roles of the two conserved domains of Pygopus proteins in transcription.To further understand the function of hPYGO2^△NHD in the Wg/Wnt signaling pathway, hPYGO2^△NHD was utilized the bait to screen human mammary gland cDNA library using the yeast two-hybrid system.Seventy yeast positive clones were obtained.The positive clones were analyzed with BLAST after sequenced.Four candidate proteins were chosen for further characterization:CDC73(cell division cycle 73), BCL6(B-cell CLL/lymphoma 6),CDC34(cell division cycle 34 homolog), EEF1G(eukaryotic translation elongation factor 1 gamma).The recombinant plasmids of p3×Flag-CMV-hpygopus^△NHD,pcDNA3.1-Myc-cdc73, pCMV5-Myc-bcl6 were constructed successfully.The expressions of these proteins have been confirmed in 293T cells.However,only pcDNA3.1-Myc-cdc73 can interact with p3×Flag-CMV-hpygopus^△NHD by immunoprecipitate and Luciferase assay,other interactions still need to be validated.Therefore,I make a conclusion that these four proteins are able to interact with hPYGO2^△NHD in the yeast strain.Firstly,Parafibomin,the human ortholog of CDC73 and the component of the Polymerase-Associated Factor1(PAF1) complex,are required for nuclear transduction of the Wg/Wnt signal.Secondly,BCL6 functions as a transcriptional repressor which can suppress the expression of the P53 gene.Thirdly, CDC34 is a member of the ubiquitin-conjugating enzyme family.And it is a part of the large multiprotein complex,which is required for ubiquitin-mediated degradation of cell cycle G1 regulators,and for the initiation of DNA replication.Fourthly, EEF1G is a eukaryotic translation elongation factor,and is probably related to the elongation since that Pygopus has the transactivation function.My research contributes to further understand the function of hPYGO2^△NHD which is significant for elucidating the mechanism of Wg/Wnt signal pathway.