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Papers In Press, published online ahead of print November 22, 2004
J. Biol. Chem, 10.1074/jbc.M410015200
Submitted on August 31, 2004
Revised on November 17, 2004
Accepted on November 20, 2004

Oct-3/4 and Sox2 regulate Oct3/4 gene in ES cells

Sayaka Okumura-Nakanishi, Motoki Saito, Hitoshi Niwa, and Fuyuki Ishikawa

Graduate School of Biostudies, Kyoto University, Kyoto 606-8502

Corresponding Author: fishikaw{at}lif.kyoto-u.ac.jp

Oct-3/4 is a key transcriptional factor whose expression level governs the fate of primitive inner cell mass (ICM) and embryonic stem (ES) cells. Previously, an upstream 3.3-kb distal enhancer (DE) fragment was identified to be responsible for the specific expression of mouse Oct-3/4 in ICM and ES cells. However, little is known about the cis-elements and trans-factors required for the DE activity. In this study, we identified a novel cis-element, called Site 2B here, located approximately 30-bp downstream of Site 2A that was previously revealed in DE by an in vivo chemical modification experiment. Using the luciferase reporter assay, we demonstrated that both Site 2A and Site 2B are necessary and sufficient for activating DE in the contexts of both the native Oct-3/4 promoter and the heterologous thymidine kinase minimal promoter. In the electrophoretic mobility shift assay (EMSA), we showed that Site 2B specifically binds to Oct-3/4 and Sox2 when ES-derived cell extracts were used, whereas Site 2A binds to a factor(s) present in both ES and NIH 3T3 cells. Furthermore, we showed that the physiological level of Oct-3/4 in ES cells is required for the Site 2B-mediated DE activity using the inducible knockout system of Oct-3/4 in ES cells. These results indicate that Oct-3/4 is a member of the gene family regulated by Oct-3/4 and Sox2, as reported before for the FGF4, UTF1, Sox2 and Fbx15 genes. Thus, Oct-3/4 and Sox2 comprise a regulatory complex that controls the expression of genes important for the maintenance of the primitive state, including themselves. This auto-regulatory circuit of the Sox2-Oct-3/4 complex may contribute to robustly maintaining the precise expression level of Oct-3/4 in primitive cells.


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