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We have previously shown in mouse NIH3T3 fibroblasts that transcription of the B-myb gene, which encodes a transcription factor required for S phase entry, is repressed through a promoter E2F site in G0/early G1. Transcription repression at this stage of the cell cycle was correlated with binding of a specific p107/E2F complex to this site. We report here, however, that transfection of cells with the known components of this complex, p107, E2F-4 and DP-1, did not repress the B-myb promoter in cycling NIH3T3 cells, although p107 inhibited transcription transactivation by E2F-4/DP-1. To establish definitively the contribution of E2F to repression, the effects of further mutations within and surrounding the E2F site were examined. It was evident that E2F binding and repression were closely correlated, lending greater weight to the contention that E2F itself is implicated in this activity. These studies also identified a closely linked site, designated the downstream repression site (DRS), which was not required for E2F binding or transactivation but which was necessary for repression. These findings indicated that E2F-dependent repression and activation are independently regulated phenomena and suggest that repression involves additional interactions determined by the promoter context.

A minigene containing a cDNA encoding the normal mouse p80c-myb protein under strong promoter control was used to transfect Friend erythroleukaemia cells. Expression of the transfected gene was found to result in elevated levels of p80c-myb which were not subject to rapid down-modulation by inducers of Friend cell differentiation as was the product of the endogenous c-myb gene. Continued synthesis of p80c-myb was found to be associated with a marked decrease in differentiation of Friend cells and we concluded that normal down-regulation of c-myb expression may be a necessary event in differentiation of these cells.

The control of c-myb mRNA abundance was examined in three representative cell lines, (the erythroleukaemia F4-12B2, the myeloma MOPC-31C and the fibroblast NIH3T3), which display abundant, low and undetectable levels of this transcript, respectively. We observed a small difference in half-life between F4-12B2 and MOPC-31C c-myb mRNA (175 min and 105 min, respectively) insufficient to account for the approximately 20-fold lower levels of this transcript in myelomas. Using the run-on transcription assay we found that c-myb transcripts were initiated at similar rates in all three cell types and were elongated at this relatively high rate to a site approximately 2 kilobases into the first intron. NIH3T3 c-myb transcripts did not proceed detectably beyond this pause/attenuation site, while in F4-12B2 cells transcription of regions 3' of this site occurred at a rate approximately 12-fold greater than in MOPC-31C. We have concluded that this transcriptional arrest mechanism, together with small differences in RNA turnover, were sufficient to account for the spectrum of c-myb mRNA abundance observed. Despite evidence of transcript initiation, we were unable to detect c-myb mRNA in fibroblasts, even under conditions (e.g. serum stimulation) which induced high c-myc mRNA levels. However, a novel 3.0 kilobase transcript with homology to c-myb was detected in cycloheximide-treated NIH3T3 cells.