E2F AND REGULATION OF DHFR GENE EXPRESSION

描述E2 F调节dhfr的机制 转录，扩增的CH 0 C 400细胞dhfr基因将被 用作E2 F活性的内源报告基因。 CH0C 400 条件性表达E2 F、DP或RB家族成员的细胞系 蛋白质将被用来与特定的 E2 F/DP/pRB家族蛋白复合物：1）dhfr基因 通过核连续测定测量的转录，2）dhfr mRNA 水平，3）dhfr启动子的基因组足迹，和4） 通过细胞周期的进展。基因组足迹 同步小区还将用于获得关于 转录因子在dhfr启动子上的组装顺序 DNA复制后 体外DNA结合研究， 重组E2 F/DP复合物和确定的DNA底物将 可以用来研究重叠的保守结构， dhfr启动子处的反向E2 F位点。 的序列改变 将测试体外E2 F/DP DNA结合影响 体内转录调控。 启动子重建和 突变E2 F、SP和pRB家族成员蛋白的表达 将用于测试全细胞中dhfr基因表达的模型。 在相关的工作中，已经表明DP-1是泛素化的， 在CH 0和人细胞中高水平表达。 DP-1地区 所需的泛素化将被映射，和作用的 将检查DP-1功能中的泛素化。 这些研究 将提供有关机制的新信息， E2 F、DP和pRB家族成员一致行动以调节 在细胞周期中内源性细胞基因的转录。

To describe the mechanism by which E2F regulates dhfr transcription, the amplified dhfr gene of CH0C 400 cells will be used as endogenous reporter genes for E2F activity. CH0C 400 cell lines that conditionally express E2F, DP, or RB family member proteins will be used to relate the formation of specific E2F/DP/pRB family protein complexes to: 1) dhfr gene transcription as measured by nuclear run-on assays, 2) dhfr mRNA levels, 3) the genomic footprint of the dhfr promoter, and 4) progression through the cell cycle. Genomic footprinting in synchronized cells also will be used to gain information regarding the order of assembly of transcription factors on the dhfr promoter after DNA replication. In vitro DNA binding studies with recombinant E2F/DP complexes and defined DNA substrates will be used to study the conserved architecture of the overlapping, inverted E2F sites at the dhfr promoter. Sequence alterations that influence E2F/DP DNA binding in vitro will be tested for transcriptional regulation in vivo. Promoter reconstruction and expression of mutant E2F, SP and pRB family member proteins will be used to test models for dhfr gene expression in whole cells. In related work, it has been shown that DP-1 is ubiquitinated when expressed at high levels in CH0 and human cells. Regions of DP-1 required for ubiquitination will be mapped, and the role of ubiquitination in DP-1 function will be examined. These studies will provide new information concerning the mechanism by which E2F, DP and pRB family members act in concert to regulate transcription of an endogenous cellular gene during the cell cycle.