Recombination signal sequence binding protein Jkappa is constitutively bound to the NF-kappaB site of the interleukin-6 promoter and acts as a negative regulatory factor.
Analysis by electrophoretic mobility shift assays (EMSA) of the different proteins associated with the kappaB sequence of the interleukin-6 (IL-6) promoter (IL6-kappaB) allowed us to detect a specific complex formed with the recombination signal sequence binding protein Jkappa (RBP-Jkappa). Single-base exchanges within the oligonucleotide sequence defined the critical base pairs involved in the interaction between RBP-Jkappa and the IL6-kappaB motif. Binding analysis suggests that the amount of RBP-Jkappa protein present in the nucleus is severalfold higher than the total amount of inducible NF-kappaB complexes but that the latter bind DNA with a 10-fold-higher affinity. A reporter gene study was performed to determine the functional implication of this binding; we found that the constitutive occupancy of the IL6-kappaB site by the RBP-Jkappa protein was responsible for the low basal levels of IL-6 promoter activity in L929sA fibrosarcoma cells and that RBP-Jkappa partially blocked access of NF-kappaB complexes to the IL-6 promoter. We propose that such a mechanism could be involved in the constitutive repression of the IL-6 gene under normal physiological conditions.
Plaisance
S
S
Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Belgium.Stephen@lmb1.rug.ac .be
Vanden Berghe
W
W
Boone
E
E
Fiers
W
W
Haegeman
G
G
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D015850:Interleukin-6 / Q000235:genetics*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000502:physiology*; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D015850:Interleukin-6; D016328:NF-kappa B; D009687:Nuclear Proteins; C497571:Rbpj protein, mouse; D012097:Repressor Proteins