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Purification and Identification of Interactive Proteins with PYGO2 and Primary Study of Their Functions

Author: LiJie
Tutor: LiBoAn
School: Xiamen University
Course: Cell Biology
Keywords: PYGO2 tandem affinity purification MED12
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 117
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Abstract


Pygopus（Pygo） is a new component of Wnt signaling pathway.In mammals, pygo gene has two homologs:pygol and pygo2.Pygo2 is widely expressed in various tissues.PYGO2 protein locates in the nucleus.It has two domains,which are PHD domain in the C-terminal and NHD domain in the N-terminal.PYGO2 functions downstream ofβ-catenin.It binds with BCL9 through its PHD domain,then forms a complex withβ-catenin,and the three of them coactivate transcriptional factor TCF to activate Wnt target genes.So far,the function of NHD domain is still unknown,but some researchers consider it of transactive activity.Recent studies show that Pygo2 may mediate Wnt-independent signal transductions in mammals.Tandem affinity purification（TAP） technology adds two specific protein tags to the C- or N-terminal of the target protein.After two-step specific affinity purifications,proteins which interact with the target protein under physiological conditions could be obtained and identified by mass spectrometry. By integrating classical affinity purification with co-immunoprecipitation, the TAP technology acquires high concentration and purity of interactive protein complexes via simple and rapid processes.We purify two new interactive proteins from a DLD-1 colon cancer cell line which stably expressing ProtA-CBP-PYGO2 fusion protein,using tandem affinity purification and mass spectrometry technologies.The two candidates are MED12 （Mediator of RNA polymeraseⅡtranscription subunit 12 ） and PIK3C2A （Phosphatidylinositol-4-phosphate 3-kinase C2 domain-containing alpha polypeptide）.Afterward we construct eukaryotic expression plasmids of PYGO2NHD and MED12,then transfect them to 293T cell line,and we find that exogenous PYGO2 could bind with exogenous MED12 through its NHD domain.Subsequent luciferase assays show that,both overexpression of MED12 protein and siRNA induced silencing of Med12 gene have little influence on the transacitive activity of Pygo2 NHD domain,while silencing of Med12 gene promotes the transacitive activity of Pygo2 PHD domain,suggesting MED12 could inhibit the transacitive activity of the PHD domain,which may have a relationship with chromatin remodeling activities during transcriptional processes.

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