Functional dissection of the ETS transcription factor MEF

We previously indicated that myeloid elf-1-like factor (MEF) but not elf-1, specifically activated lysozyme gene expression in epithelial cells. MEF is highly homologous at the nucleotide and amino acid level, with elf-1 especially in the ETS domain. Here, we report the functional analysis of the nuclear localization and transactivation properties of MEF. To investigate the intracellular localization of MEF, we transiently transfected MEF-green fluorescence protein (GFP) fusion protein expression vector into HeLa cells. A region spanning residues 177-291 is required for nuclear localization. We produced deletion mutants of MEF to determine the transactivation domain. The data showed that the N-terminal region, encompassing amino acids 1-52 is a potent transactivation domain. The C-terminal region spanning residues 477-663 can also mediate transactivation but not as strongly as the N-terminal region. The activity of the amino acid residues 1-52 was confirmed by experiments with fused constructs of MEF to the DNA binding-domain of the yeast GAL4 protein. These results, which determined the localization of the functional domains of MEF, will provide us with new clues to its transactivation mechanisms to regulate lysozyme gene expression in epithelial cells.     

Identification of direct genomic targets downstream of the nuclear factor-kappaB transcription factor mediating tumor necrosis factor signaling

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-kappaB (NF-kappaB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kappaB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kappaB inhibitor to systematically identify NF-kappaB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kappaB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F)<0.001) and demonstrated a change in signal intensity of+/-3-fold relative to control. Of these, 28 were NF-kappaB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-kappaB-dependent genes and five genes not previously known to be NF-kappaB-dependent (Gro-beta and-gamma, IkappaBepsilon, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-kappaB regulated. Expression of constitutively active enhanced green fluorescent.NF-kappaB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-kappaB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IkappaBalpha/epsilon, Gro-beta/gamma, and Naf-1 promoters directly bound NF-kappaB/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-kappaB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.