Insulator function and CTCF

我们一直对CTCF蛋白感兴趣，几年前我们首次发现它具有绝缘体的特性，当置于增强子和启动子之间时，它会阻止它们之间的相互作用。我们证明了这种活性在调节Igf 2/H19印迹位点的亲本来源等位基因特异性基因表达中起重要作用。近年来在其他实验室的工作表明，CTCF的主要作用模式是募集粘附素，这反过来又稳定了DNA上CTCF结合位点之间的相互作用，导致形成环结构域。根据相互作用的几何形状，这种环可以排除增强子，导致绝缘，或者使增强子和促进子更接近，导致激活。 细胞核内的DNA被包装成染色质，并进一步组织成拓扑相关结构域（TADs）。TADs的建立和维持需要蛋白质CTCF，我们有兴趣鉴定和研究CTCF与蛋白质和核酸伴侣的相互作用，这些蛋白质和核酸伴侣被招募用于绝缘子功能。我们已经表明，侧面的DNA结合CTCF的11个锌指的N-和C-末端结构域似乎是本质上无序的，部分解释了大量的CTCF结合伙伴在其他研究中确定。目前的工作重点是进一步表征这些结构域的物理性质，确定高亲和力结合的伴侣，并研究形成的复合物。 作为我们对大规模基因组组织的兴趣的一部分，我们已经进行了对转录因子MAZ的研究，该转录因子通常与CTCF位点相邻。 我们已经观察到两种蛋白质之间的相互作用和MAZ对CTCF性质的影响。特别地，我们已经表明，在MAZ与CTCF相邻结合的位点处，CTCF结合亲和力增加，这与两种蛋白质之间有利的相互作用自由能一致。与CTCF一样，MAZ与粘附素亚基物理相互作用，并且可以独立于CTCF阻止粘附素滑动。它还与CTCF共享独立暂停RNA聚合酶II的延伸形式的能力，从而影响RNA选择性剪接。CTCF/MAZ双位点比仅由CTCF占据的位点更有效地螯合粘附素。结合的MAZ也可以导致RNA Pol 2暂停，这意味着它改变了剪接选择。 这项工作现已扩展到其他MAZ结合伙伴的鉴定。许多转录因子几乎总是与被占据的MAZ位点结合，MAZ对它们的结合很重要，似乎MAZ在保护其他转录因子不从其结合位点移位中起着广泛的作用。 在其他研究中，作为我们对基因组组织感兴趣的一部分，我们对多个细胞系中核仁的基因组含量进行了比较研究。 这解决了什么样的非核糖体基因在核仁中存在和表达的问题，目的是确定决定这种占据的特性。 结果表明，有一个广泛的分歧的Pol 2转录和核仁相关基因的细胞类型之间的身份，与一个子集的细胞系共享显着的属性，特别是在RNA聚合酶2转录基因的数量。先前对一小部分细胞系的研究表明，很少有pol 2转录发生在核仁内或核仁表面。我们发现，某些细胞系有相当数量的核仁相关的转录活性基因。 所有这些结果都与染色质结构、组蛋白修饰和基因组在细胞功能中的长距离组织的作用有关，并且反过来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 other laboratories in recent years has shown that a principal mode of action of CTCF is to recruit cohesin which in turn stabilizes 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. DNA within the cell nucleus is packaged into chromatin, and further organized into topologically associated domains (TADs) . The establishment and maintenance of TADs requires the protein CTCF, and we are interested in identifying and studying the interactions of CTCF with the protein and nucleic acid partners recruited for insulator function. We have shown that the N- and C-terminal domains that flank the DNA binding 11 zinc fingers of CTCF appear to be intrinsically disordered explaining, in part, the large number of CTCF binding partners identified in other studies. Current work focuses on further characterizing the physical nature of these domains, identifying partners that bind with high affinity, and studying the complexes formed. As part of our interest in large scale genome organization we have undertaken a study of the transcription factor MAZ, which is often bound next to CTCF sites. We have observed interactions between the two proteins and an effect of MAZ on CTCF properties. In particular, we have shown that at sites where MAZ is bound next to CTCF, CTCF binding affinity is increased, consistent with a favorable free energy of interaction between the two proteins. Like CTCF, MAZ physically interacts with a cohesin subunit and can arrest cohesin sliding independently of CTCF. It also shares with CTCF the ability to independently pause the elongating form of RNA polymerase II, and consequently affects RNA alternative splicing. CTCF/MAZ double sites are more effective at sequestering cohesin than sites occupied only by CTCF. Bound MAZ can also cause RNA Pol2 to pause, implicating it in alteration of splicing choices. This work has now been extended to the identification of other MAZ binding partners. A number of transcription factors bind nearly always next to occupied MAZ sites, and MAZ is important for their binding.It appears that MAZ plays a widespread role in the protection of other transcription factors from displacement from their binding sites. In other studies, as part of our interest in genome organization we have carried out a comparative study of the genomic content of nucleoli in multiple cell lines. This addresses the question of what non-ribosomal genes are present and expressed in nucleoli, with the goal of identifying the properties that determine that occupancy. Results show that there is a wide divergence of identity of Pol2 transcribed and nucleolar associated genes among cell types, with a subset of cell lines sharing significant properties, particularly in the number of RNA polymerase 2 transcribed genes. Previous studies of a small selection of cell lines had indicated that very little pol2 transcription occurred in or at the surface of the nucleolus. We find that certain cell lines have a considerable number of nucleolus associated transcriptionally active genes. 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.