Regulation of the transforming growth factor-beta 2 gene promoter in embryonal carcinoma cells and their differentiated cells: differential utilization of transcription factors.
Previous studies demonstrated that differentiation of embryonal carcinoma (EC) cells increases the expression of the TGF-beta 2 gene and identified a CRE/ATF-like motif in the TGF-beta 2 promoter that is necessary for its activity. This suggested that differentiation may increase the transcription of this gene by differential binding of transcription factors to the CRE/ATF-like motif. To test this possibility, we performed gel mobility shift analysis using double-stranded oligodeoxynucleotides containing the TGF-beta 2 CRE/ATF-like motif and nuclear extracts prepared from F9 EC cells and F9-differentiated cells. We determined that the DNA/protein complexes formed by the EC nuclear extracts, but not the complexes formed by differentiated cell nuclear extracts, are recognized and supershifted by an ATF-1 specific antibody. This observation is consistent with our Western immunoblot analysis that detects AFT-1 in the EC cells, but not in their differentiated counterparts. In addition, we provide evidence that protein phosphorylation influences the formation of complexes between F9 nuclear proteins and the CRE/ATF-like motif. Together, our studies identify a likely role for the CRE/ATF-like motif in the regulation of TGF-beta 2 and suggest that this site binds one set of nuclear proteins in EC cells, where the gene is not expressed, and a different set of nuclear proteins in the differentiated cells, where the gene is expressed.
Kelly
D
D
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805, USA.
Scholtz
B
B
Orten
D J
DJ
Hinrichs
S H
SH
Rizzino
A
A
D051697:Activating Transcription Factor 1; D000818:Animals; D001483:Base Sequence; D018236:Carcinoma, Embryonal; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D015342:DNA Probes; D004268:DNA-Binding Proteins; D015972:Gene Expression Regulation, Neoplastic; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000235:genetics*; D014407:Tumor Cells, Cultured
D051697:Activating Transcription Factor 1; C497100:Atf1 protein, mouse; D015342:DNA Probes; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D016212:Transforming Growth Factor beta