Transcription Factor NF-IL6 (C/EBP β ) Activates the Expression of the Mouse MHC Class I H2-K b Gene in Response to TNF- α via the Intragenic Downstream Regulatory Element

Abstract

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-α(TNF-α) treatment. The only defined regulatory region in this construct is the intragenic downstream regulatory element (H2DRE). Computational inspection uncovered two potential NF-IL6 (C/EBPβ) binding motifs within the H2DRE. Chroramphenicol 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-α-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/EBPβ) in the course of the cellular response to TNF-α, and we discuss some consequences of this conclusion in a general framework of inducible expression of the H2-K^b gene.