Wells J M, Illenye S, Magae J, Wu C L, Heintz N H
Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
J Biol Chem. 1997 Feb 14;272(7):4483-92. doi: 10.1074/jbc.272.7.4483.
Previously genomic DNase I footprinting showed changes in protein binding to two overlapping E2F sites correlates with activation of dhfr gene expression at the G1/S boundary of the Chinese hamster cell cycle (Wells, J., Held, P., Illenye, S., and Heintz, N. H. (1996) Mol. Cell. Biol. 16, 634-647). Here gel mobility and antibody supershift assays were used to relate changes in the components of E2F DNA binding complexes in cell extracts to repression and induction of dhfr gene expression. In extracts from log phase cells, E2F complexes contained predominantly E2F-4 and E2F-2 in association with DP-1, and DNA binding assays showed complexes containing E2F-2 preferentially interact with only one of the two overlapping E2F sites. In serum starvation-stimulation experiments, arrest in G1 by low serum was accompanied by decreased levels of dhfr mRNA and the appearance of an E2F-4.DP-1.p130 complex. After serum stimulation, induction of dhfr gene expression was preceded by loss of the p130 complex in mid G1 and coincided with marked increases in two free E2F.DP-1 complexes in late G1, one of which contained E2F-4 and a second which contained an unidentified E2F. We suggest activation of dhfr gene expression after serum stimulation requires at least two temporally distinct processes, relief of p130-mediated repression and subsequent activation of transcription by free E2F.
此前，基因组DNA酶I足迹分析表明，与两个重叠的E2F位点结合的蛋白质变化与中国仓鼠细胞周期G1/S边界处二氢叶酸还原酶（dhfr）基因表达的激活相关（Wells, J., Held, P., Illenye, S., and Heintz, N. H. (1996) Mol. Cell. Biol. 16, 634 - 647）。在此，凝胶迁移率和抗体超迁移分析被用于将细胞提取物中E2F DNA结合复合物成分的变化与dhfr基因表达的抑制和诱导联系起来。在对数期细胞的提取物中，E2F复合物主要包含与DP-1结合的E2F-4和E2F-2，并且DNA结合分析表明，含有E2F-2的复合物仅优先与两个重叠的E2F位点之一相互作用。在血清饥饿-刺激实验中，低血清使细胞停滞于G1期，同时伴随着dhfr mRNA水平的降低以及E2F-4.DP-1.p130复合物的出现。血清刺激后，dhfr基因表达的诱导在G1中期伴随着p130复合物的消失，并且与G1晚期两种游离E2F.DP-1复合物的显著增加同时发生，其中一种含有E2F-4，另一种含有未鉴定的E2F。我们认为血清刺激后dhfr基因表达的激活至少需要两个时间上不同的过程，即p130介导的抑制的解除以及随后游离E2F对转录的激活。
