E2F AND REGULATION OF DHFR GENE EXPRESSION

描述E2F调节DHFR的机制 转录，扩增的CH0C400细胞dhfr基因将被 作为E2F活性的内源报告基因。CH0C 400 有条件表达E2F、DP或RB家族成员的细胞系 蛋白质将被用来关联特定的 E2F/DP/PRB家族蛋白复合体与：1)DHFR基因 核连续法检测转录，2)dhfr基因 水平，3)dhfr启动子的基因组足迹，以及4) 在细胞周期中进行。基因组足迹研究 同步的单元格还将用于获取有关 转录因子在dhfr启动子上的组装顺序 在DNA复制之后。与DNA结合的体外研究 重组E2F/DP复合体和确定的DNA底物将 被用来研究重叠的保守建筑， DHFR启动子上的E2F位点倒置。序列的改变 将测试体外对E2F/DP DNA结合的影响 体内转录调控。启动子重建和 突变型E2F、SP和PRB家族成员蛋白的表达 将被用于测试dhfr基因在整个细胞中的表达模型。 在相关工作中，已经证明DP-1在以下情况下是泛素化的 在CH0和人类细胞中高水平表达。DP-1的区域 泛素化所需的将被映射，并且角色 将考察DP-1功能中的泛素化。这些研究 将提供有关机制的新信息 E2F、DP和PRB家族成员协调行动，以监管 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.