Cyclic AMP response element-binding protein (CREB) and CAAT/enhancer-binding protein beta (C/EBPbeta) bind chimeric DNA sites with high affinity.
Basic region leucine zipper (bZIP) proteins are transcription factors that interact selectively with duplex DNA to regulate gene expression. Specifically, the cAMP response element-binding protein (CREB) interacts with the cAMP response element (CRE) DNA site with high affinity, while it binds the CAAT/enhancer-binding protein (CEBP) DNA site with low affinity. Despite the selectivity of CREB for the CRE site, CREB-dependent transcription is observed via chimeric DNA sites with similarities to both CRE and CEBP sites. Because CRE/CEBP and CEBP/CRE chimeric DNA are relevant for transcription regulation but have not been rigorously characterized, quantitative electrophoretic mobility shift assays were used to characterize the binding affinity and specificity of CREB to the sites. In addition to CREB, C/EBPbeta was tested because chimeric DNA was shown to stabilize CREB-C/EBPbeta heterodimerization. Despite previous work, no CREB-C/EBPbeta heterodimer was observed in the presence of chimeric DNA; only CREB and C/EBPbeta homodimers were seen. The CREB homodimer bound to the chimeric sites with high affinity, demonstrating that the presence of one CRE half-site is sufficient for high-affinity interaction. A comparison of CREB and C/EBPbeta homodimers indicated that they bind the chimeric sites with similar, high affinity. Whereas the CRE and CEBP sites preferentially interact with CREB and C/EBPbeta, respectively, the chimeric sites bind CREB and C/EBPbeta competitively. Because DNA binding correlates with transcription regulation, the results suggest that gene expression from chimeric sites can be altered by small changes in relative bZIP concentrations or bZIP accessory factors.
Flammer
Jamie R
JR
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Popova
Katerina N
KN
Pflum
Mary Kay H
MK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000737:chemistry* / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006801:Humans; D051381:Rats; D014158:Transcription, Genetic
D022782:CCAAT-Enhancer-Binding Protein-beta; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D004247:DNA