Insulator function and CTCF

我们将重点放在鸡β -珠蛋白位点5端1.2 kb的绝缘子DNA序列及其上游元件上。这种绝缘体既能阻断外部增强子的影响，又能防止可能关闭整个区域表达的浓缩染色质的侵入。我们之前已经证明增强子阻断活性与单个蛋白CTCF与增强子内某个位点的结合有关。我们已经证明这种蛋白在几个印迹位点上负责调控印迹基因的表达。为了了解CTCF的作用机制，我们确定了CTCF能与靶DNA位点完全结合的最小片段。我们还继续扩展我们早期的研究，表明CTCF分子之间相互作用。这为设计可以调节细胞核内CTCF功能的蛋白质提供了重要信息。目前正在研究的相互作用是CTCF与黏结蛋白复合物的相互作用，最近在Matthias Merkenschlager和其他人的实验室中显示，在细胞周期的某些阶段，黏结蛋白对DNA的募集至关重要。我们正在与Merkenschlager博士合作，剖析这种相互作用。我们最近的研究结果已经确定了直接与CTCF相互作用的内聚蛋白亚基，以及与CTCF相互作用的结构域。我们还使用质谱分析来确定对CTCF绝缘体功能似乎很重要的其他辅助因素。

We have focused attention on the 1.2 kb insulator DNA sequence at the 5 end of the chicken beta-globin locus, and elements upstream of it. This insulator is capable both of blocking the influence of outside enhancers and of preventing the encroachment of condensed chromatin that might shut down expression of the entire region. We have shown previously that enhancer blocking activity is associated with binding of a single protein, CTCF, to a site within the enhancer. We have shown that this protein is responsible for regulation of imprinted gene expression at several imprinted loci. In order to understand the mechanism of action of CTCF we have determined the minimum CTCF fragment that can bind to its target DNA site with full affinity. We have also continue to extend our earlier studies showing that CTCF molecules interact with one another. This provides important information for design of proteins that can modulate CTCF function within the nucleus. Among the interactions now under investigation is the interaction of CTCF with the cohesin protein complex, recently shown in the laboratories of Matthias Merkenschlager and others to be essential to the recruitment of cohesin to DNA at some stages of the cell cycle. We are collaborating with Dr. Merkenschlager in dissecting this interaction. Our recent results have identified both the cohesin subunit that directly interacts with CTCF, and the domain of CTCF with which it interacts. We have also used mass spec analysis to identify other co-factors that appear to be important for CTCF insulator function. Using these techniques, we have also identified a novel and important co-factor of CTCF, the regulatory protein p68. We have shown that this protein, together with its associated RNA co-factor,binds to CTCF, is present genome-wide at CTCF occupies sites on chromatin, and is essential for insulator activity.