CHROMATIN STRUCTURE IN REGULATION OF MAMMALIAN GENE EXPRESSION

我们研究增强子如何在真核细胞的染色质环境中激活转录。为了研究球蛋白基因与β -球蛋白基因座控制区（LCR）元件的相互作用，我们在人红细胞K562细胞中使用了染色质化、稳定复制的片段。双标签FISH确认每个细胞维持10-15次发作。正如预期的那样，片段与细胞染色体相结合，在细胞分裂时提供了一种实现等效分离的手段。诱变研究系统地消除了LCR HS2和胚胎epsilon-globin启动子中的转录因子结合位点，揭示了增强子和启动子相互影响染色质结构。对-珠蛋白基因和LCR HS3和HS4的研究为这一概念提供了额外的支持。这些数据与增强剂作用的直接交流模型最为一致。目前体外纯化转录因子的研究解决了它们在这种交流中的作用。为了进一步了解增强子依赖性启动子重塑，我们检查了转录活性启动子和非转录活性启动子的核蛋白组成和组蛋白乙酰化。从激活转录的启动子/TATA核小体中恢复的核蛋白复合物与非激活的ε -珠蛋白基因相比显着减少，并且潜在的DNA为亚核小体大小。芯片分析显示，在活性启动子中，该核小体上的组蛋白H3和H4发生了剧烈的高乙酰化，而邻近的上游核小体没有发生差异乙酰化。在体内，曲古霉素A对组蛋白的全局乙酰化并没有模仿这种高度定向和特异性的核小体改变，这表明HS2的额外活性是必要的。我们将继续探索染色质结构在体内对珠蛋白基因表达的调控作用，以及-珠蛋白LCR的作用机制。

We study how enhancers activate transcription in the chromatin environment of eukaryotic cells. To study the interaction of globin genes and elements of the beta-globin locus control region (LCR), we have used chromatinized, stably replicating episomes in human erythroid K562 cells. Double label FISH confirms the maintenance of 10-15 episomes per cell. The episomes, as expected, associate with the cellular chromosomes, providing a means to achieve equivalent segregation at cell division. Mutagenesis studies which systematically eliminated transcription factor binding sites in LCR HS2 and in the embryonic epsilon-globin promoter, revealed that enhancer and promoter mutually affect each others chromatin structure. Studies with the beta-globin gene, and with LCR HS3 and HS4 provide additional support for this concept. These data are most consistent with a direct communication model of enhancer action. Current studies in vitro with purified transcription factors address their role in this communication. To further understand enhancer dependent promoter remodeling, we examined nucleoprotein composition and histone acetylation at transcriptionally active and inactive promoters. There was a marked decrease in nucleoprotein complexes recovered from the promoter/TATA nucleosome of actively transcribing versus inactive epsilon-globin genes, and the underlying DNA was of sub-nucleosomal size. ChIPs assays revealed dramatic hyperacetylation of histone H3 and H4 at this nucleosome in active promoters, while the adjacent upstream nucleosome was not differentially acetylated. Global acetylation of histones in vivo by Trichostatin A, did not mimic this highly directed and specific nucleosome alteration suggesting that additional activities of HS2 are necessary. We continue to explore the regulatory role in vivo of chromatin structure in the expression of globin genes, and the mechanism of action of the beta-globin LCR.