Insulator function and CTCF

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

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) separating active and inactive genomic regions. 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. . 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 in nucleoli, with the goal of identifying the properties that determine that occupancy. Results show that there is a wide divergence of gene identity among cell types, with a subset of cell lines sharing significant properties.. 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.