Screening Of SOX4-interacting Protein By Yeast Two-hybrid Assasy And Functional Analysis Of The Protein Complex

SOX4 is an important member of SOX [SRY(Sex-related region Y)-box] transcription factor family, which plays key roles in regulating embryonic developent. SOX4 is specifically expressed in the ovary, testis, thymus and T, pre-B lymphocyte. Recently, there are increasing evidences that SOX4 is highly expressed in several tumors including breast cancer, colon cancer, lung cancer, meduloblastoma and hepatocellular carcinoma, suggesting that SOX4 may be involved in tumorigenesis. Additionally, SOX4 level is upgraded during prostaglandins induced apoptosis in hepatocellular carcinoma cells, suggesting that SOX4 may be involved in regulation of apoptosis. SOX4 participates in the regulation of many physiological and pathological processes, however, the accurate mechanism by which it plays its pivotal roles remains to be elucidated. In this study, we performed yeast two-hybrid screen to identify SOX4-interacting proteins and preliminary analysis on the biological functions of the interaction.We used two fragments of SOX4, SOX4(aal-133)and SOX4(aa 130-380), as baits respectively, to screen human mammary cDNA library. From 203 candidate clones, we finally identified 4 clones that interacted with SOX4(aal-133). Sequencing analysis showed that the four clones were all hUbc9, an essential E2 enzyme required for small ubiquitin-related modifier (SUMO) conjugation or sumoylation. Full-length cDNA for Ubc9 was then cloned from a mammary library. The interaction between SOX4 and Ubc9 was confirmed by in vitro GST pull-down assays and in vivo co-immunoprecitipation analysis. Fluorescence microscopy analyses also showed that the two proteins are co-localized in the nucleus of living cells. Furthermore, we found that the HMG domain of SOX4, which mediated DNA-binding of SOX proteins, was required for its interaction with Ubc9 by co-transfecting various truncated forms of SOX4 with wild type Ubc9 into yeast cells.To determine the significance of the interation between SOX4 and Ubc9, we established a luciterase reportor system in eukaryotic cells for analyzing transcriptional...