Conserved kappa B element located downstream of the tumor necrosis factor alpha gene: distinct NF-kappa B binding pattern and enhancer activity in LPS activated murine macrophages.
Transcriptional activation of various genes by lipopolysaccharide (LPS) is known to be mediated, at least in part, by the NF-kappa B/Rel family of transcription factors. We have identified a novel kappa B element located immediately downstream of the TNF-alpha gene that is conserved together with its flanking sequences across species lines and can act as an LPS-responsive enhancer for reporter gene constructs driven by the minimal TNF promoter. In extracts from activated murine macrophages and macrophage cell lines this element binds several non-canonical NF-kappa B/Rel complexes, in addition to p50 (NFKB1) homodimer and p50-p65 (NKFB1-RelA) heterodimer. Combination of high-resolution electrophoretic mobility shift assays (EMSA) with monospecific antibodies and u.v.-cross-linking indicates that the prominent slow migrating complex III contain p65 homodimer and c-Rel. The appearance of complex III in EMSA parallels the translocation of p65 and c-Rel into the nucleus and occurs shortly after LPS induction. Transfection experiments with reporter constructs driven by this kappa B element indicate strong inducibility by LPS and p65, moderate inducibility by c-Rel and repression by p50. Functional activity of sandwich TNF-CAT-TNF constructs further suggests that LPS-inducible transcriptional activation of the TNF gene in murine macrophages may be partly mediated by a downstream enhancer.
Kuprash
D V
DV
Biological Carcinogenesis and Development Program, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland, 21702-1201, USA.
Udalova
I A
IA
Turetskaya
R L
RL
Rice
N R
NR
Nedospasov
S A
SA
D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017124:Conserved Sequence; D004247:DNA; D004742:Enhancer Elements, Genetic; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D020569:Proto-Oncogene Proteins c-rel; D012045:Regulatory Sequences, Nucleic Acid; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D008070:Lipopolysaccharides; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase