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

Transcription of the B-myb gene is regulated at the G1/S boundary of t he cell cycle. To begin to examine the mechanism controlling expressio n of this gene during the cell-cycle, a mouse B-myb 5' flanking sequen ce 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 re leased 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 negativel y in G0. Mutagenesis of an E2F consensus binding site within this regi on was sufficient to relieve transcription repression in G0, resulting in a promoter with constitutively high activity. Specific G0 and S ph ase E2F complexes binding to this B-myb element were detected in NIH3T 3 cell extracts by mobility shift assays. These studies demonstrate fo r the first time a direct role for E2F in regulation of cell cycle gen e expression by repression of transcription in G0/early G1.