Chromatin Structure In Regulation Of Gene Expression

我们研究增强剂如何激活真核细胞染色质环境中的转录。人类β-珠蛋白基因由一个增强子/位点控制区激活，该区域距离基因本身6至60kb。远距离LCR的强增强子活性如何在这些基因的启动子上变得明显还不是很清楚，但它包括改变染色质结构。已经描述了两类进行染色质修饰以缓解核小体抑制的复合体：改变核小体稳定性或位置(动员)的SWI/SNF复合体，以及乙酰化组蛋白N末端尾部的组蛋白乙酰转移酶(HAT)复合体。为了研究LCR如何改变染色质和激活转录，我们研究了人红系K562细胞中病毒表型上一个6kb的珠蛋白基因座。该基因座包含一个完整的人类表珠蛋白基因，该基因与强大的β-珠蛋白LCRHS2增强子相连。 我们已经将这种增强子依赖基因的结构和功能与非增强子基因或与失活的HS2连锁的基因进行了比较。利用染色质结构的探针和实时定量聚合酶链式反应，我们确定由于含有SWI/SNF复合体的BRG1导致的增强子依赖的核小体动员可能先于该启动子上的组蛋白超乙酰化。此外，这些研究提供了当转录活跃时增强子和基因启动子接近的证据，正如增强子作用的循环模型所预测的那样。然而，其他研究表明，组蛋白乙酰化和RNA PolII在增强子和启动子之间的整个序列中都被检测到，这表明组蛋白乙酰化可能从增强子通过基因座扩散，至少一些聚合酶分子可能通过基因座从增强子进入启动子，正如增强子作用的跟踪模型所预测的那样。在增强子和基因之间插入染色质绝缘体支持这一结论，因为我们看到增强子明显阻止了乙酰化和聚合酶的移动和聚合酶的积累。综上所述，这些结果与增强剂作用的简化跟踪模型最一致。这种观点认为，增强子招募激活剂、重塑复合体和聚合酶，然后移动到启动子，而不会失去与增强子的接触。这种运动最终会导致环路的形成。我们继续研究增强子激活的基因在我们的模型基因座和内源性人类染色体中的转录。

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 becomes manifest at the promoters of these genes is not well understood, but it encompasses altering chromatin structure. 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 (mobilization), and histone acetyltransferase (HAT) complexes that acetylate histone N-terminal tails. To investigate how the LCR changes chromatin and activates transcription, we have studied a model 6 Kb globin locus on viral episomes in human erythroid K562 cells. The locus contains a complete human epsilon-globin gene linked to the strong beta-globin LCR HS2 enhancer. We have compared aspects of structure and function of this enhancer dependent gene to an enhancerless gene, or one linked to an inactivated HS2. Using probes of chromatin structure and quantitative real time PCR, we determined that enhancer dependent nucleosome mobilization due to a Brg1 containing SWI/SNF complex likely preceeds histone hyperacetylation at this promoter. In addition, these studies provided evidence for proximity of the enhancer and gene promoter when transcription is active, as would be predicted by the looping model of enhancer action. However, other studies indicated that histone acetylation and RNA pol II were detected throughout sequences intervening between the enhancer and promoter, suggesting that histone acetylation may spread through the locus from the enhancer, and that at least some polymerase molecules may access the promoter from the enhancer by moving through the locus, as a tracking model of enhancer action would predict. Interposition of an chromatin insulator between the enhancer and gene supported this latter conclusion, as we saw an apparant block to acetylation and polymerase movement and accumulation of polymerase at the enhancer. Taken together, these results are most consistent with a facilitated tracking model of enhancer action. In this view, an enhancer recruits activators, remodeling complexes and polymerase which then move to the promoter without loosing contact with the enhancer. This movement eventually results in loop formation. We continue to investigate enhancer activated gene transcription in our model locus and in the endogenous human chromosome.