Wells J, Held P, Illenye S, Heintz N H
Program in Cell and Molecular Biology, University of Vermont College of Medicine, Burlington 05405, USA.
Mol Cell Biol. 1996 Feb;16(2):634-47. doi: 10.1128/MCB.16.2.634.
In mammals, two TATA-less bidirectional promoters regulate expression of the divergently transcribed dihydrofolate reductase (dhfr) and rep3 genes. In CHOC 400 cells, dhfr mRNA levels increase about fourfold during the G1-to-S phase transition of the cell cycle, whereas the levels of rep3 transcripts vary less than twofold during this time. To assess the role of DNA-binding proteins in transcriptional regulation of the dhfr and rep3 genes, the major and minor dhfr-rep3 promoter regions were analyzed by high-resolution genomic footprinting during the cell cycle. At the major dhfr promoter, prominent DNase I footprints over four upstream Sp1 binding sites did not vary throughout G1 and entry into the S phase. Genomic footprinting revealed that a protein is constitutively bound to the overlapping E2F sites throughout the G1-to-S phase transition, an interaction that is most evident on the transcribed template strand. On the nontranscribed strand, multiple changes in the DNase I cleavage pattern are observed during transit through G1 and entry into the S phase. By using gel mobility shift assays and a series of sequence-specific probes, two different species of E2F were shown to interact with the dhfr promoter during the cell cycle. The DNA binding activity of one E2F species, which preferentially recognizes the sequence TTTGGCGC, did not vary significantly during the cell cycle. The DNA binding activity of the second E2F species, which preferentially recognizes the sequence TTTCGCGC, increased during the G1-to-S phase transition. Together, these results indicate that Sp1 and the species of E2F that binds TTTGGCGC participate in the formation of a basal transcription complex, while the species of E2F that binds TTTCGCGC regulates dhfr gene expression during the G1-to-S phase transition. At the minor promoter, DNase I footprints at a consensus c-Myc binding site and three Sp1 binding sites showed little variation during the G1-to-S phase transition. In addition to protein binding at sequences known to be involved in the regulation of transcription, genomic footprinting of the entire promoter region also showed that a protein factor is constitutively bound to the first intron of the rep3 gene.
在哺乳动物中，两个无TATA盒的双向启动子调控着反向转录的二氢叶酸还原酶（dhfr）基因和rep3基因的表达。在CHOC 400细胞中，dhfr mRNA水平在细胞周期的G1期到S期转变过程中增加约四倍，而在此期间rep3转录本水平的变化小于两倍。为了评估DNA结合蛋白在dhfr和rep3基因转录调控中的作用，在细胞周期中通过高分辨率基因组足迹分析了主要和次要的dhfr-rep3启动子区域。在主要的dhfr启动子处，四个上游Sp1结合位点上显著的DNase I足迹在整个G1期及进入S期的过程中没有变化。基因组足迹分析显示，在整个G1期到S期转变过程中，一种蛋白质持续结合在重叠的E2F位点上，这种相互作用在转录模板链上最为明显。在非转录链上，在通过G1期及进入S期的过程中观察到DNase I切割模式有多种变化。通过凝胶迁移率变动分析和一系列序列特异性探针，发现两种不同的E2F在细胞周期中与dhfr启动子相互作用。其中一种E2F优先识别序列TTTGGCGC，其DNA结合活性在细胞周期中没有显著变化。第二种E2F优先识别序列TTTCGCGC，其DNA结合活性在G1期到S期转变过程中增加。这些结果共同表明，Sp1和结合TTTGGCGC的E2F参与了基础转录复合物的形成，而结合TTTCGCGC的E2F在G1期到S期转变过程中调控dhfr基因的表达。在次要启动子处，一个共有c-Myc结合位点和三个Sp1结合位点上的DNase I足迹在G1期到S期转变过程中变化很小。除了在已知参与转录调控的序列上有蛋白质结合外，整个启动子区域的基因组足迹分析还显示，一种蛋白质因子持续结合在rep3基因的第一个内含子上。
