title: The interferon regulatory factors 1 and 2 bind to a segment of the human c-myb first intron: possible role in the regulation of c-myb expression.
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.
authors.affiliation: Department of Microbiology and Immunology and Kimmel Cancer Institute, Thomas Jefferson University, Bluemle Life Sciences Building, 233 South 10th Street, Philadelphia, Pennsylvania 19107, USA.
authors.forename: L
authors.initials: L
authors.lastname: Manzella
authors.forename: R
authors.initials: R
authors.lastname: Gualdi
authors.forename: D
authors.initials: D
authors.lastname: Perrotti
authors.forename: N C
authors.initials: NC
authors.lastname: Nicolaides
authors.forename: G
authors.initials: G
authors.lastname: Girlando
authors.forename: M A
authors.initials: MA
authors.lastname: Giuffrida
authors.forename: A
authors.initials: A
authors.lastname: Messina
authors.forename: B
authors.initials: B
authors.lastname: Calabretta
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D020570:Genes, myb; D015698:Genomic Library; D018922:HL-60 Cells; D006801:Humans; D050856:Interferon Regulatory Factor-1; D050836:Interferon Regulatory Factor-2; D007438:Introns; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; C494229:IRF2 protein, human; D050856:Interferon Regulatory Factor-1; D050836:Interferon Regulatory Factor-2; C494258:Irf1 protein, mouse; C494230:Irf2 protein, mouse; D009687:Nuclear Proteins; D010750:Phosphoproteins; D020598:Proto-Oncogene Proteins c-myb; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate