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

The preferential expression of the protooncogene c-myb in hematopoietic cel ls is in part regulated by a mechanism of transcriptional block in the firs t intron. By electrophoresis mobility shift assays using probes correspondi ng to different segments of the putative human c-myb intron 1 transcription pause region and nuclear extracts from myeloid leukemia HL 60 and fibrobla st 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 abrogate d by competition with an oligomer containing the wild-type, but not the mut ated, 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. Upo n 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 e-myb promoter containing the entire intron 1 mas suppressed upon IRF-1, but not IRF-2 expression. Together, these results are consistent with the e xistence of a functional relationship between TRF-1 and c-myb in which IRF- 1 negatively regulates c-myb expression at the transcriptional level by a m echanism that may depend on the interaction of IRF-1 with a segment of the c-myb gene implicated in transcription pausing. (C) 2000 Academic Press.