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。近年来其他实验室的工作表明，CTCF 的主要作用模式是招募粘连蛋白，粘连蛋白反过来又稳定 DNA 上 CTCF 结合位点之间的相互作用，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。 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 (TAD)。 TAD 的建立和维持需要蛋白质 CTCF，我们有兴趣识别和研究 CTCF 与为绝缘子功能而招募的蛋白质和核酸伙伴的相互作用。我们已经证明，CTCF 11 个锌指 DNA 结合侧翼的 N 端和 C 端结构域似乎本质上是无序的，这在一定程度上解释了其他研究中发现的大量 CTCF 结合伴侣。目前的工作重点是进一步表征这些结构域的物理性质，识别以高亲和力结合的伙伴，并研究形成的复合物。 作为我们对大规模基因组组织感兴趣的一部分，我们对转录因子 MAZ 进行了研究，该因子通常与 CTCF 位点结合。 我们观察到两种蛋白质之间的相互作用以及 MAZ 对 CTCF 特性的影响。特别是，我们已经证明，在 MAZ 与 CTCF 相邻的结合位点，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 与粘连蛋白亚基发生物理相互作用，并且可以独立于 CTCF 阻止粘连蛋白滑动。它还与 CTCF 一样具有独立暂停 RNA 聚合酶 II 延伸形式的能力，从而影响 RNA 选择性剪接。 CTCF/MAZ 双位点在隔离粘连蛋白方面比仅由 CTCF 占据的位点更有效。结合的 MAZ 还会导致 RNA Pol2 暂停，这意味着它会改变剪接选择。 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在占据的 MAZ 位点附近结合，而 MAZ 对于它们的结合很重要。看来 MAZ 在保护其他转录因子免于从其结合位点移位方面发挥着广泛的作用。 在其他研究中，作为我们对基因组组织兴趣的一部分，我们对多个细胞系中核仁的基因组含量进行了比较研究。 这解决了核仁中存在和表达哪些非核糖体基因的问题，目的是确定决定该占据的特性。 结果表明，不同细胞类型的 Pol2 转录基因和核仁相关基因的身份存在很大差异，其中一部分细胞系具有相同的显着特性，特别是在 RNA 聚合酶 2 转录基因的数量方面。先前对一小部分细胞系的研究表明，核仁内部或表面很少发生 pol2 转录。我们发现某些细胞系具有相当数量的核仁相关转录活性基因。 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 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.