Insulator function and CTCF

我们一直对蛋白质CTCF感兴趣，几年前我们首次确定它具有绝缘体的特性，当放在增强剂和启动子之间时，它可以阻止增强剂和启动子之间的作用。我们证明了这种活性在调节原发源亲本的等位基因特异性基因在Igf2/H19印迹基因座上的表达中起着重要作用。近年来的工作表明，CTCF的一个主要作用模式是稳定DNA上CTCF结合位点之间的相互作用，导致环状结构域的形成。根据相互作用的几何形状，这种环可以排除导致绝缘的增强子，或者使增强子和启动子更紧密地结合在一起，导致激活。 在寻找CTCF相互作用蛋白的过程中，我们发现CTCF与着丝粒蛋白CENP-E在体外和体内都有很强的相互作用。我们能够在着丝粒周围区域的重复DNA序列中鉴定出招募CENP-E的不寻常的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募集到着丝粒上具有新的功能，这些不寻常的CTCF结合位点可能具有新的作用，并且可能是以CTCF为中心的新的、具有重要功能的组织结构。 我们最近报道了CTCF与死盒解旋酶p68之间的相互作用。我们发现，一个长的非编码RNA SRA，它与p68形成一个复合体，对这种相互作用是必不可少的。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。我们现在已经将这些研究扩展到对SRA功能的详细检查。许多长的非编码RNA招募的要么是多梳复合体PRC2，它提供与基因表达沉默相关的组蛋白修饰，要么是三胸基团相关复合体之一，它提供激活修饰。我们发现，一个单一的SRA分子可以同时结合这两个复合物，这是一个新的功能，对于长的非编码RNA。这一结果表明，DNA上SRA结合的位置可能同时显示沉默和激活标记。已知在多能干细胞中存在这样的二价位点，我们正在探索SRA参与创建这样的位点的可能性。 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，并反过来与正常和异常的细胞代谢和细胞分裂问题有关。

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.