AN E2F-BINDING SITE MEDIATES CELL-CYCLE-REGULATED REPRESSION OF MOUSE B-MYB TRANSCRIPTION

Transcription of the B-myb gene is regulated at the G1/S boundary of the cell cycle. To begin to examine the mechanism controlling expression of this gene during the cell-cycle, a mouse B-myb 5' flanking sequence was isolated from a cosmid library and shown to promote efficiently the transcription of a luciferase reporter gene when transfected into NIH3T3 fibroblasts. It was further shown that in transfected cells released from G0 by readdition of serum, luciferase activity directed by the B-myb promoter was induced substantially as cells entered S phase, thus paralleling the regulation of endogenous B-myb. Analysis of the B-myb promoter identified a region that appeared to have no intrinsic promoter activity yet which acted to regulate transcription negatively in G0. Mutagenesis of an E2F consensus binding site within this region was sufficient to relieve transcription repression in G0, resulting in a promoter with constitutively high activity. Specific G0 and S phase E2F complexes binding to this B-myb element were detected in NIH3T3 cell extracts by mobility shift assays. These studies demonstrate for the first time a direct role for E2F in regulation of cell cycle gene expression by repression of transcription in G0/early G1.