Regulation of the Pluripotency Marker Rex-1 by Nanog and Sox2

Wenjing Shi,Hui Wang,Guangjin Pan,Yijie Geng,Yunqian Guo,Duanqing Pei

doi:10.1074/jbc.m601811200

Abstract

Rex-1 (Zfp-42) is a known marker for undifferentiated embryonic stem cells and teratocarcinoma cells. However, the mechanism by which Rex-1 is regulated in pluripotent cells remains unresolved. Here we report that Nanog, an Nk-2 homeodomain protein known for its role in maintaining stem cell pluripotency, is a transcription activator for the Rex-1 promoter. Knockdown of Nanog in embryonic stem cells resulted in a reduction of Rex-1 expression, whereas forced expression of Nanog in P19 stimulated Rex-1 expression. Employing a Rex-1 reporter, we demonstrate that Nanog transactivates Rex-1 directly. Serial deletion studies mapped the Nanog-responsive element between -187 and -286 of the Rex-1 promoter. Although Oct-3/4 and Sox2 can both transactivate Rex-1 promoter, only Sox2 cooperates with Nanog in up-regulating Rex-1. Furthermore, we demonstrate that the C terminus of Nanog is responsible for transactivating the Rex-1 promoter, a function that can be substituted for by a viral transactivator Vp16 efficiently in NIH3T3 cells but less so in P19 cells. Taking these findings together, we conclude that Rex-1 is a direct target of Nanog, which is augmented by Sox2 and Oct-3/4.

Highlights

The Rex-1 gene is a developmentally regulated acidic zinc finger gene (Zfp-42) and a well recognized marker for the pluripotent state of both embryonic stem (ES)2 cells and embryonic carcinoma (EC) cells (1, 2)
We have recently demonstrated that Nanog functions as a transcription activator through binding to a consensus recognition motif determined by SELEX, despite the fact that Nanog was originally proposed as a transcription repressor to inhibit the expression of genes important for cell differentiation (9, 11, 12)
The complexes were eluted with chromatin preparations were divided into three portions. 500 ␮l of elution buffer (1% SDS, 0.1 M NaHCO3, 0.01 mg/ml One portion was precipitated with Nanog antibody (5 ␮l), herring sperm DNA)

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Plasmids—Cells (293T, NIH3T3, P19, and F9) were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal calf serum (Hyclone) and penicillin/streptomycin. First strand cDNAs were synthesized and analyzed by PCR to detect the expression of Nanog, Rex-1, and ␤-actin. For real-time PCR, the Nanog primers were: forward, 5Ј-ctcaagtcctgaggctgaca-3Ј; reverse, 5Ј-tgaaacctgtccttgagtgc3Ј. Transfection and Reporter Assay—All cells were seeded in 24 wells and transiently transfected with Rex-1 promoter reporters (0.25 ␮g/well) and effector plasmids (control empty vector, Nanog expression construct, and Nanog siRNA vector) with increasing doses (0.25– 0.75 ␮g) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. After centrifugation (2000 rpm, 2 min, 4 °C), the cells were lysed in 0.6 ml of ChIP sonication buffer (1% Triton X-100, 0.1% deoxycholate, 50 mM Tris, pH 8.1, 150 mM NaCl, 5 mM EDTA, and 1ϫ protease inhibitor) and sonicated to an average fragment length of 2000 bp.

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Findings

DISCUSSION