Insulator function and CTCF

我们将注意力集中在鸡β-珠蛋白基因座5端的1.2kb绝缘子DNA序列及其上游元件上，该绝缘子既能够阻断外部增强子的影响，又能够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质CTCF与增强子内位点的结合有关。我们已经表明，这种蛋白质是负责调节印迹基因的表达在几个印迹位点。为了了解CTCF的作用机制，我们确定了能够以完全亲和力结合其靶DNA位点的最小CTCF片段。我们还继续扩展我们早期的研究，表明CTCF分子相互作用。 这为设计能够调节细胞核内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包括CTCF自身相互作用形成二聚体，以及CTCF与粘着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，CTCF与粘着蛋白复合物的相互作用对于在细胞周期的某些阶段将粘着蛋白募集到DNA中至关重要。 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相互作用的粘附素亚基，以及与CTCF相互作用的CTCF结构域。我们还使用质谱分析来鉴定似乎对CTCF绝缘子功能重要的其他辅因子。

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 CTCF self-interaction to form dimers, and 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.