Synergy of SF1 and RAR in Activation of Oct-3/4Promoter

Efrat Barnea,Yehudit Bergman

doi:10.1074/jbc.275.9.6608

Efrat Barnea, Yehudit Bergman

Open Access

https://doi.org/10.1074/jbc.275.9.6608

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Abstract

The Oct-3/4 transcription factor is expressed in the earliest stages of embryogenesis, and is thus likely to play an important role in regulation of initial decisions in development. For the first time, we have shown that SF1 and Oct-3/4 are co-expressed in embryonal carcinoma (EC) P19 cells, and their expression is down-regulated with very similar kinetics following retinoic acid (RA) induced differentiation of these cells, suggesting a functional relationship between the two. Previously, we have shown that the Oct-3/4 promoter harbors an RA-responsive element, RAREoct, which functions in EC cells as a binding site for positive regulators of transcription, such as RAR and RXR. In this study we have identified in the Oct-3/4 promoter two novel SF1-binding sites: SF1(a) and SF1(b). The proximal site, SF1(a), is located within the RAREoct, and the distal site, SF1(b), is located between nucleotide -193 and -209 of the Oct-3/4 promoter. Both sites contribute to activation of Oct-3/4 promoter in EC cells, with SF1(a) playing a more crucial role. SF1, and its isoforms ELP2 and ELP3 bind to both SF1 sites and activate the Oct-3/4 promoter. This activation depends on the presence of SF1 DNA-binding domain. Thus, Oct-3/4 is the first EC-specific gene reported that is regulated by SF1. Interestingly, SF1 and RAR form a novel complex on the RAREoct sequence that synergistically activate the Oct-3/4 promoter. Both RARE and SF1 cis regulatory elements, as well as the SF1 DNA-binding domain, are needed for this synergism. SF1 and Oct-3/4 transcription factors play a role in the same developmental regulatory cascade.

Highlights

Transcription factors play a critical role in embryonic development and cellular differentiation
steroidogenic factor-1 (SF1) Binds the RAREoct Site—We have previously reported the characterization of the Oct-3/4 promoter
To find whether SF1 plays a role in regulation of the Oct-3/4 gene, the proteins that bind to the RAREoct sequence were investigated by electrophoretic mobility shift assay using nuclear extracts prepared from undifferentiated P19 cells (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Cells—Murine P19 EC cells, L cells, and COS-1 cells were maintained in Dulbecco’s medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units of penicillin, and 100 ␮g of streptomycin/ml. The p0.4R* construct (designated in our previous articles as p0.4octR*-CAT), is identical to the above described plasmid except for four point mutations inserted in the RAREoct site (the mutated sequence is GGGCCcGtcGTgAAGGCTAGA, with the lowercase letters denoting mutated nucleotides). The p0.4-SF1(a)* construct is identical to the p0.4 plasmid except for four point mutations inserted in the SF1 site, present in the RAREoct motif (the mutated sequence is GGGCCAGAGGTCgctcCTAGA). COS-1 cells were transfected by the DEAE-dextran method [37] with 10 ␮g of plasmid containing the expression vector of the indicated nuclear receptor. One to 10 ␮g of WCE were incubated in a 20-␮l reaction mixture containing 0.3 ng of end-labeled oligonucleotide (30,000 cpm), in the presence of 10 mM Tris-HCl (pH 7.8), 14% glycerol, 1 mM dithiothreitol, and 2 ␮g of poly(dI-dC). Filters were washed at 65 °C in 2 ϫ SSC (1 ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1% SDS and autoradiographed with an intensifying screen at Ϫ70 °C

RESULTS

TABLE I Oligonucleotides and plasmids

DISCUSSION