Transcription factor NF-IL6 (C/EBPbeta) activates the expression of the mouse MHC class I H2-Kb gene in response to TNF-alpha via the intragenic downstream regulatory element.
The 5'-enhancer-deleted genomic construct of the H2-K(b) gene, stably integrated into the genome of L(tk-) fibroblasts, retains full competence to be induced by tumor necrosis factor-alpha (TNF-alpha) treatment. The only defined regulatory region in this construct is the intragenic downstream regulatory element (H2DRE). Computational inspection uncovered two potential NF-IL6 (C/EBPbeta) binding motifs within the H2DRE. Chloramphenicol acetyltransferase (CAT) reporter gene assay revealed that NF-IL6 is able to elevate transcription from H2DRE. Moreover, transient transfection of an NF-IL6 expression vector increased both constitutive and TNF-alpha-induced mRNA levels of endogenous H2 class I genes, and transfection of an NF-IL6 dominant negative construct decreased the expression of endogenous H2 class I genes in a dose-dependent manner. Using the electrophoretic mobility shift assay (EMSA) and antibody supershift assay, we were able to qualify the two computationally identified NF-IL6 binding motifs as one high-affinity and one low-affinity binding site. We conclude that the H2-K(b) gene belongs to target genes of the NF-IL6 (C/EBPbeta) in the course of the cellular response to TNF-alpha, and we discuss some consequences of this conclusion in a general framework of inducible expression of the H2-K(b) gene.
Charles University, Medical Faculty in Pilsen, Institute of Biology, CZ-301 66 Plzen, Czech Republic. jiri.hatina@lfp.cuni.cz
Jirí
J
Hatina
Petr
P
Jansa
Josef
J
Reischig
D016475:3T3 Cells; D024506:5' Flanking Region / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D005805:Genes, MHC Class I; D005809:Genes, Regulator; D017930:Genes, Reporter; D006183:H-2 Antigens / Q000235:genetics*; D007249:Inflammation; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D022782:CCAAT-Enhancer-Binding Protein-beta; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha