Regulation of the cfos serum response element by C/EBPbeta.
Serum response element binding protein (SRE BP) is a novel binding factor present in nuclear extracts of avian and NIH 3T3 fibroblasts which specifically binds to the cfos SRE within a region overlapping and immediately 3' to the CArG box. Site-directed mutagenesis combined with transfection experiments in NIH 3T3 cells showed that binding of both serum response factor (SRF) and SRE BP is necessary for maximal serum induction of the SRE. In this study, we have combined size fractionation of the SRE BP DNA binding activity with C/EBPbeta antibodies to demonstrate that homodimers and heterodimers of p35C/EBPbeta (a transactivator) and p20C/EBPbeta (a repressor) contribute to the SRE BP complex in NIH 3T3 cells. Transactivation of the SRE by p35C/EBPbeta is dependent on SRF binding but not ternary complex factor (TCF) formation. Both p35C/EBPbeta and p20C/EBPbeta bind to SRF in vitro via a carboxy-terminal domain that probably does not include the leucine zipper. Moreover, SRE mutants which retain responsiveness to the TCF-independent signaling pathway bind SRE BP in vitro with affinities that are nearly identical to that of the wild-type SRE, whereas mutant SRE.M, which is not responsive to the TCF-independent pathway, has a nearly 10-fold lower affinity for SRE BP. We propose that C/EBPbeta may play a role in conjunction with SRF in the TCF-independent signaling pathway for SRE activation.
Sealy
L
L
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Malone
D
D
Pawlak
M
M
D016475:3T3 Cells; D000818:Animals; D001769:Blood; D022762:CCAAT-Enhancer-Binding Proteins; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D016762:Genes, fos / Q000235:genetics*; D016350:Leucine Zippers; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D026362:Serum Response Factor; D015398:Signal Transduction / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology*
D022762:CCAAT-Enhancer-Binding Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D026362:Serum Response Factor; D004247:DNA