ABSTRACT
The preferential expression of the protooncogene c-myb in hematopoietic cells is in part regulated by a mechanism of transcriptional block in the first intron. By electrophoresis mobility shift assays using probes corresponding to different segments of the putative human c-myb intron 1 transcription pause region and nuclear extracts from myeloid leukemia HL 60 and fibroblast WI 38 cells, we detected a HL-60-specific DNA-protein complex with a 123-bp fragment containing binding sites for the interferon regulatory factors (IRFs) nuclear proteins. Formation of the DNA-protein complex was abrogated by competition with an oligomer containing the wild-type, but not the mutated, IRF binding site and the complex was specifically supershifted by the anti-IRF-1 or the anti-IRF-2 antibody. Moreover, in vitro translated IRF-1 or IRF-2 protein did interact with the 123-bp c-myb intron 1 fragment. Upon TPA-induced differentiation, c-myb expression was readily down-modulated in parental HL 60 cells, but not in cells transfected with an antisense IRF-1 plasmid. Moreover, chloramphenicol acetyltransferase activity driven by a c-myb promoter containing the entire intron 1 was suppressed upon IRF-1, but not IRF-2 expression. Together, these results are consistent with the existence of a functional relationship between IRF-1 and c-myb in which IRF-1 negatively regulates c-myb expression at the transcriptional level by a mechanism that may depend on the interaction of IRF-1 with a segment of the c-myb gene implicated in transcription pausing.
Subject(s)
DNA-Binding Proteins/metabolism , Genes, myb , Introns , Phosphoproteins/metabolism , Repressor Proteins , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , Chloramphenicol O-Acetyltransferase/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation/drug effects , Genomic Library , HL-60 Cells , Humans , Interferon Regulatory Factor-1 , Interferon Regulatory Factor-2 , Mice , Nuclear Proteins/metabolism , Phosphoproteins/genetics , Proto-Oncogene Proteins c-myb/genetics , Recombinant Proteins/metabolism , Tetradecanoylphorbol Acetate/pharmacology , TransfectionABSTRACT
In this report we discuss the role of interferon regulatory factor 1 (IRF-1) in the regulation of ornithine decarboxylase (ODC) transcription during IFN gamma human macrophage activation. We show that a binding sequence for the transcription factor IRF-1 is contained in the first intron of the human ODC gene (from nt +2711 to nt +2722) and we demonstrate that the level of expression of IRF-1 increases in human macrophages and in the human promonocytic cell line, U937, previously differentiated in monocytes/macrophages by phorbol myristate acetate (PMA), after 2 h of IFN gamma stimulation. We also show that the hamster tk-ts13 cell line, stably transfected with the IRF-1 cDNA, over-expresses ODC. In addition, a specific complex was detected, by gel-shift assay after incubating a 20 bp double-stranded oligomer containing the binding sequence for IRF-1 with nuclear proteins extracted from human macrophages and from (PMA-differentiated) U937 cells stimulated with IFN gamma for 2 h.
