Lam E W, Watson R J
Ludwig Institute for Cancer Research, St Mary's Hospital Medical School, London, UK.
EMBO J. 1993 Jul;12(7):2705-13. doi: 10.1002/j.1460-2075.1993.tb05932.x.
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.
B-myb基因的转录在细胞周期的G1/S边界受到调控。为了开始研究细胞周期中控制该基因表达的机制，从小鼠黏粒文库中分离出小鼠B-myb 5'侧翼序列，并将其转染到NIH3T3成纤维细胞中时，发现它能有效地促进荧光素酶报告基因的转录。进一步研究表明，在通过重新添加血清使细胞从G0期释放的转染细胞中，当细胞进入S期时，由B-myb启动子指导的荧光素酶活性显著诱导，这与内源性B-myb的调控情况相似。对B-myb启动子的分析确定了一个区域，该区域似乎没有内在的启动子活性，但在G0期却起到负向调节转录的作用。该区域内E2F共有结合位点的诱变足以解除G0期的转录抑制，从而产生一个具有组成性高活性的启动子。通过迁移率变动分析在NIH3T3细胞提取物中检测到与该B-myb元件结合的特定G0期和S期E2F复合物。这些研究首次证明了E2F在通过抑制G0/早期G1期转录来调控细胞周期基因表达中的直接作用。
