Chromatin Structure In Regulation Of Mammalian Gene Expr

我们研究增强剂如何激活真核细胞染色质环境中的转录。为了研究珠蛋白基因与β-珠蛋白基因位点控制区(LCR)元件的相互作用，我们在人红系K562细胞中使用了染色体显色的、稳定复制的表观染色体。双标记FISH证实每个细胞维持10-15个上染色体。不出所料，上染色体与细胞染色体结合在一起，提供了一种在细胞分裂时实现同等分离的手段。突变研究系统地消除了LCRHS2中的转录因子结合位点和微染色体中胚胎表型珠蛋白启动子中的转录因子结合部位，发现增强子和启动子相互影响染色质结构。对β-珠蛋白基因以及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沟通模型最为一致。为了进一步了解增强子依赖的启动子重构，我们研究了转录活性启动子和非活性启动子上的启动子重构和组蛋白乙酰化。CHIPS分析显示，在活性启动子的启动子近端，组蛋白H3和H4发生了显著的高乙酰化，而邻近的上游核小体没有发生差异乙酰化。启动子重塑和组蛋白超乙酰化都依赖于HS2增强子，有趣的是，还依赖于启动子中的TATA盒。我们继续探索由远距离增强子激活基因的潜在事件的顺序，以及染色质结构在体内对珠蛋白基因表达的调节作用。

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 in minichromosomes, 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. To further understand enhancer dependent promoter remodeling, we examined promoter remodeling and histone acetylation at transcriptionally active and inactive promoters. ChIPs assays revealed dramatic hyperacetylation of histone H3 and H4 specifically at the promoter proximal nucleosome in active promoters, while the adjacent upstream nucleosome was not differentially acetylated. Both promoter remodeling and histone hyperacetylation were dependent on the HS2 enhancer, and, interestingly, on the TATA box in the promoter. We continue to explore the order of events underlying gene activation by a distant enhancer and the regulatory role in vivo of chromatin structure in the expression of globin genes.