CHROMATIN STRUCTURE IN REGULATION OF MAMMALIAN GENE EXPRESSION

哺乳动物基因表达调节中的染色质结构

• 批准号: 3839591
• 负责人: A DEAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3839591
• 关键词: chromatin; developmental genetics; gene expression; genetic enhancer element; genetic mapping; genetic promoter element; genetic regulation; genetic transcription; globin; human genetic material tag; nucleic acid sequence; nucleoproteins; regulatory gene; reporter genes; transcription factor

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.

ε-珠蛋白基因是第一个β-样珠蛋白基因， 在人类发展过程中。 最大ε-珠蛋白合成 发生在胚胎卵黄囊的大的有核红细胞中。 基因的转录在第6周和第10周之间逐渐停止 红细胞生成的部位转移到胎儿的肝脏。 到 为了研究这个基因的调控，我们在体外绘制了 红细胞核蛋白与非红细胞核蛋白相互作用 细胞和ε-珠蛋白启动子中的DNA序列。 我们确定了一个 红细胞系因子加塔-1的位点，位于α-葡聚糖的位置-165处， 珠蛋白启动子 需要在该位点结合加塔-1来介导 人β-珠蛋白LCR HS II增强子的作用。 但在 增强子加塔-1的缺失不参与转录， 这个发起人。 增强子中的加塔-1位点不能取代 它们不需要启动子位点，也不与启动子位点相互作用。 的 相反，增强子依赖于AP-1/NF-E2位点，以影响 从这个启动子的增强。 因此，生产性启动子-增强子 增加ε-珠蛋白基因转录的相互作用可以 需要少至两个蛋白质通过两个调控位点相互作用， DNA β-球蛋白LCR表现出至少两种性质： 对染色质结构以及经典增强子的范围效应 活动 我们设计了一个小染色体载体， ε-珠蛋白基因，以研究LCR序列对 染色质中ε-珠蛋白基因的结构。 微型染色体是 作为稳定的附加体元件携带，组装成染色质，在红细胞中 和非红细胞人类细胞。 在没有LCR的情况下，我们发现， 微型染色体上的ε-珠蛋白基因不被转录。 的 基因可能需要其自身的增强子来表达，即使在红细胞中也是如此。 环境，这表明红细胞转录的可用性 这些因素不足以表达。 微型染色体系统可以 提供了一种研究LCR对染色质结构影响的方法， 以及其增强剂活性。