Chromatin Structure In Regulation Of 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。人类β-珠蛋白基因被增强子/基因座控制区激活，该区域距离基因本身6至60 Kb。远距离LCR的强增强子活性如何在这些基因的启动子处变得明显还不清楚，但它包括改变染色质结构。已经描述了两种类型的复合物，其进行染色质修饰以减轻核小体抑制：改变核小体稳定性或位置（移动）的SWI/SNF复合物，和乙酰化组蛋白N-末端尾部的组蛋白乙酰转移酶（HAT）复合物。为了研究LCR如何改变染色质并激活转录，我们研究了人红系K562细胞中病毒附加体上的模型6 Kb珠蛋白基因座。该基因座包含与强β-珠蛋白LCR HS 2增强子连接的完整的人ε-珠蛋白基因。 我们已经比较了这个增强子依赖基因的结构和功能方面的增强子无基因，或一个连接到一个失活的HS 2。使用染色质结构探针和定量真实的时间PCR，我们确定，增强子依赖的核小体动员由于Brg 1含有SWI/SNF复合物可能先于组蛋白在这个启动子的超乙酰化。此外，这些研究提供了证据的增强子和基因启动子的接近时，转录是活跃的，将预测的增强子作用的循环模型。然而，其他研究表明，组蛋白乙酰化和RNA pol II在增强子和启动子之间的整个序列中被检测到，这表明组蛋白乙酰化可以从增强子通过基因座传播，并且至少一些聚合酶分子可以通过移动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子支持了后一个结论，因为我们看到了乙酰化和聚合酶运动的明显阻断以及聚合酶在增强子处的积累。两者合计，这些结果是最一致的促进跟踪模型的增强子的行动。在这种观点中，增强子募集激活剂、重塑复合物和聚合酶，然后它们移动到启动子而不与增强子失去接触。这种运动最终导致形成环。我们继续研究增强子激活的基因转录在我们的模型基因座和内源性人类染色体。

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.