N-terminal domain of the architectural protein CTCF has similar structural organization and ability to self-association in bilaterian organisms

Abstract

ctcf is the main architectural protein found in most of the examined bilaterian organisms. the cluster of the C2H2 zinc-finger domains involved in recognition of long DNA-binding motif is only part of the protein that is evolutionarily conserved, while the N-terminal domain (NTD) has different sequences. Here, we performed biophysical characterization of CTCF NTDs from various species representing all major phylogenetic clades of higher metazoans. With the exception of Drosophilides, the N-terminal domains of CTCFs show an unstructured organization and absence of folded regions in vitro. In contrast, NTDs of Drosophila melanogaster and virilis ctcfs contain unstructured folded regions that form tetramers and dimers correspondingly in vitro. Unexpectedly, most NTDs are able to self-associate in the yeast two-hybrid and co-immunoprecipitation assays. These results suggest that NTDs of CTCFs might contribute to the organization of ctcf-mediated long-distance interactions and chromosomal architecture. Chromosomes in the genomes of all higher eukaryotes have a highly organized architecture and consist of discrete topologically associated domains (TADs) 1-5. TADs often also include smaller domains (sub-TADs) that are flanked by short boundary elements or longer regions (inter-TADs) that contain active chromatin and housekeeping genes. In addition, promoters, enhancers, silencers and insulators form a network of specific distance interactions that properly regulate gene transcription 6-9. Until now, the unresolved question remains how specific distance interactions between remote regulatory elements are established and maintained through the cell cycle 10. Currently, the best-characterized protein involved in the organization of chromosome architecture is CTCF, which was initially found as a transcriptional repressor 11. It is believed that CTCF is the main architectural protein in mammals, which is responsible for the organization of TAD boundaries and distance interactions between enhancers and promoters 12-16. CTCF was found in most of the higher eukaryotes including all studied bilateral organisms but is absent in yeast and plants 17,18. Usually CTCFs from different organisms contain the cluster of eleven C2H2 zinc-finger domains (ZF) localized in the central part of the protein. In human CTCF, ZFs from 3 to 7 recognize specific 15 bp consensus 19. The DNA-binding ZFs are the most evolutionary conserved among CTCFs that bind to similar sites in most higher eukaryotic genomes 20. Moreover, it was found that even several chromatin domains controlled by CTCF are conserved in distant species 21. Other ZF domains are usually less conserved

Mechanisms of Enhancer-Promoter Interactions in Higher Eukaryotes

“…Five C2H2 domains in CTCF specifically bind to an extended DNA motif that is conserved in most animals [ 66 ]. The N-termini of CTCF homologs from representative bilaterian species feature unstructured domains that are capable of homodimerization [ 67 ]. A motif that interacts with the cohesin SA2-SCC1 sub-complex was identified between the N-terminal homodimerization domain and the C2H2 cluster ( Figure 3 A) [ 68 ].…”

CTCF as a boundary factor for cohesin-mediated loop extrusion: evidence for a multi-step mechanism

“…CTCF is an 11-Zinc Finger (ZF) DNA-Binding protein that is conserved across most animals, but absent from plants, C. elegans and yeast [ 66 , 67 ]. Mammalian CTCF has unstructured N- and C-terminal domains flanking the 11-ZF DNA-binding domain [ 68 , 69 ] ( Figure 2b ). Depending on the antibody used and the bioinformatic threshold, CTCF binds ~40,000–90,000 sites in mammalian genomes, of which ~30-60% are cell-type specific and with around half in intergenic regions and the other half at promoters, in introns or exons [ 39 , 66 , 70 – 72 ].…”

Mechanisms of Enhancer-Promoter Interactions in Higher Eukaryotes

“…Five C2H2 domains in CTCF specifically bind to an extended DNA motif that is conserved in most animals [64]. The N-termini of CTCF homologs from representative bilaterian species feature unstructured domains that are capable of homodimerization [65]. A motif that interacts with the cohesin SA2-SCC1 sub-complex was identified between the N-terminal homodimerization domain and the C2H2 cluster [66].…”

“…CTCF can block the progression of the cohesin complex when the interaction between its N-terminal domain and the SA2-SCC1 sub-complex [66] is oriented correctly relative to the moving cohesin complex. The dimerization between the N-terminal domains of the CTCF protein is thought to stabilize the formation of chromatin loops [65].…”

“…In transgenic model systems, the pairing between two copies of repetitive binding sites for dCTCF, Su(Hw), or Pita was able to bring the yeast GAL4-dependent activator region and the reporter gene promoter in close proximity, resulting in the transcription of the reporter gene [145][146][147]. The dCTCF N-terminus contains an unstructured domain that can form tetrameric complexes [65,148], which can contribute to distance interactions. Similarly, Pita contains an Nterminal zinc-finger-associated domain (ZAD) capable of forming homodimers [147].…”