CHROMATIN STRUCTURE IN REGULATION OF MAMMALIAN GENE EXPRESSION

Epsilon-珠蛋白基因是第一个类似β的珠蛋白基因 在人类发展过程中表现出来的。最大表观珠蛋白合成量 发生在胚胎卵黄囊的大的有核红系细胞中。 基因的转录在第6至10周之间逐渐停止。 胎儿的生命，因为红细胞生成的部位转移到了胎儿的肝脏。至 研究这个基因的调控，我们已经在体外定位了 红系与非红系核蛋白相互作用的研究 细胞，以及epsilon-珠蛋白启动子的DNA序列。我们确定了一个 红系因子GATA-1在epsilon中-165位的位置- 珠蛋白启动子。GATA-1在这个位点上的结合需要介导 人β-珠蛋白LCR HS II增强子的作用然而，在 缺少增强子GATA-1不参与转录 这位推动者。增强子中的GATA-1位点不能取代 对启动子位点的要求，也不与启动子位点相互作用。这个 增强子依赖于AP-1/NF-E2位点来发挥作用 来自这个启动子的增强。因此，高效的启动子-增强子 增加epsilon-珠蛋白基因转录的相互作用可能 只需要两个蛋白质通过两个调控位点相互作用 DNA。 β-珠蛋白LCR至少表现出两种性质：它有很长的 染色质结构的范围效应，以及经典的增强剂 活动。我们设计了一个微小染色体载体，其中包含一个标记的 Epsilon-珠蛋白基因，以研究LCR序列对DNA合成的影响 染色质中表型珠蛋白基因的结构。微小染色体是 在红系中以稳定的上体成分的形式携带，组装成染色质 和非红系人类细胞。在没有LCR的情况下，我们发现 微染色体上的epsilon-珠蛋白基因没有转录。这个 基因可能需要其自身的增强子来表达，即使在红系中也是如此 环境，表明红系转录的可用性 因数不足以表达。微染色体系统可以 为研究LCR对染色质结构的影响提供了一种手段，如 以及它的增强剂活性。

The epsilon-globin gene is the first of the beta-like globin genes to be expressed during human development. Maximal epsilon-globin synthesis occurs in the large nucleated erythroid cells of the embryonic yolk sac. Transcription of the gene gradually ceases between the 6th and 10th weeks of fetal life, as the site of erythropoiesis shifts to the fetal liver. To investigate the regulation of this gene we have mapped, in vitro, the sites of interaction between nuclear proteins from erythroid and non-erythroid cells, and DNA sequences in the epsilon-globin promoter. We identified a site for the erythroid factor GATA-1 at position - 165 in the epsilon- globin promoter. GATA-1 binding at this site is required to mediate the effect of the human beta-globin LCR HS II enhancer. However, in the absence of the enhancer GATA-1 does not participate in transcription from this promoter. GATA-1 sites in the enhancer could not replace the requirement for, nor did they interact with, the promoter site. The enhancer depended instead upon AP-1/NF-E2 sites in order to effect enhancement from this promoter. Thus, productive promoter-enhancer interactions increasing transcription of the epsilon-globin gene may require as few as two proteins interacting through two regulatory sites in the DNA. The beta-globin LCR exhibits at least two kinds of properties: it has long range effects on chromatin structure, as well as classical enhancer activity. We have designed a minichromosomal vector containing a marked epsilon-globin gene, in order to study the effect of LCR sequences on the structure of the epsilon-globin gene in chromatin. The minichromosomes are carried as stable episomal elements, assembled into chromatin, in erythroid and non-erythroid human cells. In the absence of the LCR, we found that the epsilon-globin gene on the minichromosome was not transcribed. The gene may require its own enhancer to be expressed, even in an erythroid environment, suggesting that the availability of erythroid transcription factors is insufficient to allow expression. The minichromosome system may provide a means to study the effects of the LCR on chromatin structure, as well as its enhancer activity.