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

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