Chromatin Structure In Regulation Of Mammalian Gene Expr

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。人类-珠蛋白基因被一个距离基因本身6 - 60kb的增强子/基因座控制区激活。远端LCR的强增强子活性如何在这些基因的启动子上表现出来尚不清楚，但它包括改变染色质结构。为了研究LCR如何改变染色质，我们正在研究人红细胞K562细胞病毒发作的一个模型5kb珠蛋白位点。我们将LCR的强HS2增强子成分与epsilon珠蛋白基因联系起来，该基因从这些细胞的内源性位点积极转录。有两种类型的复合物可以通过染色质修饰来缓解核小体的抑制：改变核小体稳定性或位置的SWI/SNF复合物，以及使组蛋白n端尾部乙酰化的组蛋白乙酰转移酶（HAT）复合物。为了探索LCR基因激活的事件顺序，我们分析了与野生型HS2相关的活性epsilon珠蛋白基因的核小体动员和超乙酰化，并将它们与无增强基因或与失活HS2相关的基因进行了比较。我们确定HS2和TATA盒子是在epsilon珠蛋白启动子上SWI/SNF动员和HAT超乙酰化所必需的。BAF 155是SWI/SNF的组成部分之一。我们发现SWI/SNF首先作用于这个启动子，在超乙酰化发生之前。我们对转录因子和pol II与增强子和启动子的关联的进一步研究支持了这些调控元件在转录活跃时彼此接近的观点。我们将继续研究HS2增强子在整个模型位点诱导的染色质变化，以及染色质绝缘体如何中断这一过程。

We study how enhancers activate transcription in the chromatin environment of eukaryotic cells. The human beta-globin genes are activated by an enhancer/locus control region that lies between 6 and 60 Kb distant from the genes themselves. How the strong enhancer activity of the distant LCR is manifest at the promoters of these genes is not well understood, but it encompasses altering chromatin structure. To investigate how the LCR changes chromatin, we are studying a model 5 Kb globin locus on viral episomes in human erythroid K562 cells. We linked the strong HS2 enhancer component of the LCR to the epsilon globin gene, which is actively transcribed from the endogenous locus in these cells. Two types of complexes have been described that carry out chromatin modifications to relieve nucleosomal repression: SWI/SNF complexes that alter nucleosome stability or position, and histone acetyltransferase (HAT) complexes that acetylate histone N-terminal tails. To explore the order of events underlying gene activation by the LCR, we assayed nucleosome mobilization and hyperacetylation at active epsilon globin genes linked to wild type HS2, and compared them to an enhancerless gene, or one linked to an inactivated HS2. We determined that HS2 and the TATA box are required for SWI/SNF mobilization and HAT hyperacetylation at the epsilon globin promoter. BAF 155 is one of the components of SWI/SNF that is involved. We found that SWI/SNF acts first at this promoter, before hyperacetylation occurs. Our additional studies on association of transcription factors and pol II with the enhancer and promoter support the idea that these regulatory elements are in close proximity to each other when transcription is active. We continue to investigate chromatin changes induced throughout our model locus by the HS2 enhancer, and how a chromatin insulator might interrupt this process.