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，我们继续扩展我们早期的研究，表明CTCF分子与辅助因子相互作用。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。我们最近的研究结果已经确定SA2是直接与CTCF相互作用的内聚蛋白亚基，并且还确定了与CTCF相互作用的结构域。

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 continued to extend our earlier studies showing that CTCF molecules interact with co-factors. 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. Our recent results have identified SA2 as the cohesin subunit that directly interacts with CTCF, and also identified the domain of CTCF with which it interacts. We have also used mass spec analysis to detect other co-factors that appear to be important for CTCF insulator function. Using these techniques, we have 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. We also have evidence that at least one role of p68/SRA is to help stabilize the CTCF/cohesin interaction.