CHROMATIN STRUCTURE IN REGULATION OF MAMMALIAN GENE EXPRESSION

我们感兴趣的是如何时间和组织特异性表达的一个 基因是在发育过程中产生的。有序的激活和 抑制人类珠蛋白基因家族的个别成员是一种 这一现象的突出例子。我们正在研究 这些基因被激活时所经历的染色质结构。我们 使用DNA酶I足迹法和电泳迁移率变动分析 在体外检测红细胞核蛋白之间的相互作用， 和非红细胞系细胞以及来自调控区的DNA序列， 和3'端的人ε-珠蛋白基因。至少有一种互动 我们观察到的是红细胞特异性的，对应于 EryF 1/NF-E1/GF-1启动子区的两个位点。这种蛋白质还具有 强结合位点位于基因的3'区。其他互动包括 在分析过程中。为了研究功能性 K562中这些蛋白质-DNA相互作用瞬时测定的意义 细胞和HeLa细胞，其中来自细胞的转录被使用。 通过氯霉素监测ε-珠蛋白启动子 乙酰转移酶报告基因。一个附加型载体系统正在被 为了研究这些蛋白质DNA在体内的作用， 对高级染色质结构和转录的相互作用 ε-珠蛋白基因。初步研究表明， 载体可以作为扩增的附加体在K562细胞中维持。明显 将ε-珠蛋白基因插入载体中， 将被研究。

We are interested in how the temporal and tissue specific expression of a gene is brought about during development. The orderly activation and repression of individual members of the human globin gene family is a prominent example of this phenomenon. We are studying the changes in chromatin structure that these genes undergo when they are activated. We have used DNAse I footprinting and the electrophoretic mobility shift assay to detect, in vitro, interactions between nuclear proteins from erythroid and non-erythroid cells and DNA sequences from regulatory regions both 5' and 3' to the human epsilon-globin gene. At least one of the interactions we observed was erythroid specific and corresponded to binding of EryF1/NF-E1/GF-1 to two sites in the promoter region. This protein also has strong binding sites in the region 3' to the gene. Other interactions are in the process of being analyzed. To investigate the functional significance of these protein-DNA interactions transient assays in K562 cells and HeLa cells are being employed in which transcription from the epsilon-globin promoter is monitored via a chloramphenicol acetyltransferase reporter gene. An episomal vector system is being developed in order to study in vivo the effect of these protein DNA interactions on higher order chromatin structure and transcription of the epsilon-globin gene. Preliminary studies suggest that an EB virus-based vector can be maintained as an amplified episome in K562 cells. A marked epsilon-globin gene is being inserted into the vector and its regulation will be studied.