Felsenfeld G, Boyes J, Chung J, Clark D, Studitsky V
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0540, USA.
Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9384-8. doi: 10.1073/pnas.93.18.9384.
It is now well understood that chromatin structure is perturbed in the neighborhood of expressed genes. This is most obvious in the neighborhood of promoters and enhancers, where hypersensitivity to nucleases marks sites that no longer carry canonical nucleosomes, and to which transcription factors bind. To study the relationship between transcription factor binding and the generation of these hypersensitive regions, we mutated individual cis-acting regulatory elements within the enhancer that lies between the chicken beta- and epsilon-globin genes. Constructions carrying the mutant enhancer were introduced by stable transformation into an avian erythroid cell line. We observed that weakening the enhancer resulted in creation of two classes of site: those still completely accessible to nuclease attack and those that were completely blocked. This all-or-none behavior suggests a mechanism by which chromatin structure can act to sharpen the response of developmental systems to changing concentrations of regulatory factors. Another problem raised by chromatin structure concerns the establishment of boundaries between active and inactive chromatin domains. We have identified a DNA element at the 5' end of the chicken beta-globin locus, near such a boundary, that has the properties of an insulator; in test constructions, it blocks the action of an enhancer on a promoter when it is placed between them. We describe the properties and partial dissection of this sequence. A third problem is posed by the continued presence of nucleosomes on transcribed genes, which might prevent the passage of RNA polymerase. We show, however, that a prokaryotic polymerase can transcribe through a histone octamer on a simple chromatin template. The analysis of this process reveals that an octamer is capable of transferring from a position in front of the polymerase to one behind, without ever losing its attachment to the DNA.
现在已经清楚地了解到，在表达基因的附近，染色质结构会受到干扰。这在启动子和增强子附近最为明显，在这些区域，对核酸酶的超敏感性标记了不再携带典型核小体的位点，转录因子也会结合到这些位点上。为了研究转录因子结合与这些超敏感区域产生之间的关系，我们对鸡β-珠蛋白基因和ε-珠蛋白基因之间的增强子内的单个顺式作用调节元件进行了突变。携带突变增强子的构建体通过稳定转化被引入到一种鸟类红细胞系中。我们观察到，削弱增强子会导致产生两类位点：一类仍然完全可被核酸酶攻击，另一类则完全被阻断。这种全或无的行为表明了一种机制，通过该机制染色质结构可以增强发育系统对调节因子浓度变化的反应。染色质结构引发的另一个问题涉及活性和非活性染色质结构域之间边界的建立。我们已经在鸡β-珠蛋白基因座的5'端附近，靠近这样一个边界处，鉴定出一个具有绝缘子特性的DNA元件；在测试构建体中，当它置于增强子和启动子之间时，会阻断增强子对启动子的作用。我们描述了该序列以及对其部分剖析的特性。第三个问题是由转录基因上持续存在的核小体提出的，这可能会阻止RNA聚合酶通过。然而，我们表明，原核聚合酶可以在一个简单的染色质模板上转录通过一个组蛋白八聚体。对这个过程的分析表明，一个八聚体能够从聚合酶前方的位置转移到后方的位置，而不会失去其与DNA的附着。
