Insulator function and CTCF

我们对 CTCF 蛋白很感兴趣，几年前我们首次发现它具有绝缘体的特性，当增强子和启动子放置在增强子和启动子之间时，可以阻止它们之间的相互作用。 我们证明了这种活性在调节 Igf2/H19 印迹位点的亲本等位基因特异性基因表达中发挥着重要作用。 近年来的工作表明，CTCF 的主要作用模式是稳定 DNA 上 CTCF 结合位点之间的相互作用，从而导致环结构域的形成。 根据相互作用的几何形状，此类环可以排除增强子，导致绝缘，或者使增强子和启动子更靠近，从而导致激活。 在寻找 CTCF 相互作用蛋白的过程中，我们在体外和体内发现了 CTCF 和着丝粒蛋白 CENP-E 之间的强烈相互作用。 我们能够在着丝粒周围区域的重复 DNA 序列中识别出招募 CENP-E 的不寻常 CTCF 位点。 CENP-E 的结合依赖于 CTCF 的结合。 这些结果表明 CTCF 在将 CENP-E 招募到着丝粒方面具有​​新功能，这些不寻常的 CTCF 结合位点具有新作用，并且可能是一种以 CTCF 为中心的新颖且功能重要的组织结构。 我们最近报道了 CTCF 和 DEAD 盒解旋酶 p68 之间的相互作用。 我们发现，与 p68 形成复合物的长非编码 RNA SRA 对于这种相互作用至关重要。我们研究了 p68-SRA 相互作用的性质，以及 RNA 在 CTCF 绝缘体活性中的作用。 我们现在已将这些研究扩展到对 SRA 功能的详细检查。 许多长非编码RNA募集Polycomb复合物PRC2（它传递与基因表达沉默相关的组蛋白修饰）或Trithorax组相关复合物之一（它传递激活修饰）。 我们发现单个 SRA 分子可以同时结合两个复合物，这是长非编码 RNA 的一个新功能。 这一结果表明 DNA 上 SRA 结合位点可能同时显示沉默和激活标记。已知此类二价位点存在于多能干细胞中，我们正在探索 SRA 参与此类位点创建的可能性。 所有这些结果都与染色质结构、组蛋白修饰和基因组的长程组织在细胞功能中的作用有关，进而与正常和异常细胞代谢和细胞分裂的问题有关。

We have been interested in the protein CTCF, which we first identified some years ago as having properties of an insulator, blocking interaction between enhancers and promoters when placed between them. We demonstrated that this activity plays an important role in regulating parent of origin allele-specific gene expression at the Igf2/H19 imprinted locus. Work in recent years has shown that a principal mode of action of CTCF is to stabilize interactions between CTCF binding sites on DNA, leading to formation of loop domains. Depending on the geometry of the interactions such loops can either exclude an enhancer leading to insulation, or bring enhancer and promoter closer together, leading to activation. In the course of searching for CTCF interacting proteins we identified a strong interaction, both in vitro and in vivo, between CTCF and the centromeric protein CENP-E. We were able to identify, within the repeat DNA sequences of the pericentromeric region, unusual CTCF sites that recruited CENP-E. Binding of CENP-E is dependent on binding of CTCF. These results suggest a novel function for CTCF in recruiting CENP-E to the centromere, a novel role for these unusual CTCF binding sites, and perhaps a novel and functionally important organized structure centered on CTCF. We have recently reported an interaction between CTCF and the DEAD box helicase, p68. We found that a long non-coding RNA, SRA, which forms a complex with p68, is essential for this interaction. We have investigated the nature of the p68-SRA interaction, and the role of the RNA in insulator activity of CTCF. We have now extended these studies to a detailed examination of SRA function. Many long non-coding RNAs recruit either the Polycomb complex, PRC2, which delivers histone modifications that are associated with silencing of gene expression, or one of the Trithorax group related complexes, which delivers activating modifications. We find that a single SRA molecule can bind both complexes simultaneously, a novel function for long non-coding RNAs. This result suggests that sites on DNA at which SRA is bound could display both silencing and activating marks. Such bivalent sites are known to occur in pluripotent stem cells and we are exploring the possibility that SRA is involved in creation of such sites. All of these results relate to the role of chromatin structure, histone modifications, and long range organization of the genome in cell function, and are in turn related to questions of normal and abnormal cell metabolism and cell division.