Study of the in Vivo Functional Role of Mutations in the BTB Domain of the CP190 Protein of Drosophila melanogaster.

The Drosophila transcription factor СР190 is one of the key proteins that determine the activity of housekeeping gene promoters and insulators. CP190 has an N-terminal BTB domain that allows for dimerization. Many of known Drosophila architectural proteins interact with the hydrophobic peptide-binding groove in the BTB domain, which is presumably a mechanisms for recruiting CP190 to regulatory elements. To study the role of the BTB domain in the interaction with architectural proteins, we obtained transgenic flies expressing CP190 variants with mutations in the peptide-binding groove, which disrupts their interaction with architectural proteins. As a result of the studies, it was found that mutations in the BTB domain do not affect binding of the CP190 protein to polytene chromosomes. Thus, our studies confirm the previously obtained data that CP190 is recruited to regulatory elements by several transcription factors interacting, in addition to BTB, with other CP190 domains.

The NCoR Co-repressor Interacts with the Kaiso Transcription Factor through a Mechanism Different from That of BCL6 Interaction.

The vertebrate transcription factor Kaiso binds specifically to methylated DNA sequences using C2H2-type zinc fingers. In addition to C2H2-domains, the BTB/POZ domain, which forms homodimers, is located at the N-terminus of Kaiso. Kaiso, like several other well-studied BTB/POZ proteins, including BCL6, interacts with the NCoR (nuclear co-repressor) protein, which determines the landing of transcriptional repressive complexes on chromatin. Using the yeast two-hybrid system, we have shown that the N-terminal domain of NCoR interacts with the C-terminal zinc fingers of Kaiso, and not with its BTB/POZ domain, as previously assumed. The results obtained demonstrate that NCoR interacts with various transcription factor domains, which can increase the efficiency of attracting NCoR-dependent repressor complexes to regulatory regions of the genome.

Study of the N-Terminal Domain Homodimerization in Human Proteins with Zinc Finger Clusters.

CTCF belongs to a large family of transcription factors with clusters of C2H2-type zinc finger domains (C2H2 proteins) and is a main architectural protein in mammals. Human CTCF has a homodimerizing unstructured domain at the N-terminus which is involved in long-distance interactions. To test the presence of similar N-terminal domains in other human C2H2 proteins, a yeast two-hybrid system was used. In total, the ability of unstructured N-terminal domains to homodimerize was investigated for six human C2H2 proteins with an expression profile similar to CTCF. The data indicate the lack of the homodimerization ability of these domains. On the other hand, three C2H2 proteins containing the structured domain DUF3669 at the N-terminus demonstrated homo- and heterodimerization activity.

Human CTCF Interacts with Drosophila CP190, but not with Kaiso.

Human CTCF (hCTCF) is a major architectural protein in mammals. In Drosophila, the CTCF homologue (dCTCF) interacts with the BTB domain of the CP190 protein, which is involved in the establishment of open chromatin and activity of insulators. Previously, it was shown that the BTB protein Kaiso interacts with hCTCF and regulates its activity. We have carried out a detailed study of the interaction between these proteins in the yeast two-hybrid assay. Surprisingly, Kaiso did not interact with hCTCF and its Drosophila homologue. On the other hand, CP190 interacted with the C-terminus of hCTCF. The results obtained demonstrate that the interaction between CTCF and CP190 proteins is highly conserved. It is likely that humans have other BTB proteins that perform the functions described for the Drosophila CP190.

[Structure of Neuroglobin from Cold-Water Sponge Halisarca dujardinii].

The iron-containing protein neuroglobin (Ngb) involved in the transport of oxygen is generally considered the precursor of all animal globins. In this report, we studied the structure of Ngb of the cold-water sponge Halisarca dujardinii. In sponges, the oldest multicellular organisms, the Ngb gene contains three introns. In contrast to human Ngb, its promoter contains a TATA-box, rather than CG-rich motifs. In sponges, Ngb consists of 169 amino acids showing rather low similarity with its mammalian orthologues. It lacks Glu and Arg residues in positions required for prevention of hypoxia-related apoptosis. Nevertheless, Ngb contains both proximal and distal conserved heme-biding histidines. The primary structure of H. dujardinii neuroglobin predicted by sequencing was confirmed by mass-spectrometry analysis of recombinant Ngb expressed in E. coli. The high level of Ngb expression in sponge tissues suggests its possible involvement in the gas metabolism and presumably in other key metabolic processes in H. dujardinii.

C2H2 Zinc Finger Proteins: The Largest but Poorly Explored Family of Higher Eukaryotic Transcription Factors.

The emergence of whole-genome assays has initiated numerous genome-wide studies of transcription factor localizations at genomic regulatory elements (enhancers, promoters, silencers, and insulators), as well as facilitated the uncovering of some of the key principles of chromosomal organization. However, the proteins involved in the formation and maintenance of the chromosomal architecture and the organization of regulatory domains remain insufficiently studied. This review attempts to collate the available data on the abundant but still poorly understood family of proteins with clusters of the C2H2 zinc finger domains. One of the best known proteins of this family is a well conserved protein known as CTCF, which plays a key role in the establishment of the chromosomal architecture in vertebrates. The distinctive features of C2H2 zinc finger proteins include strong and specific binding to a long and unique DNA recognition target sequence and rapid expansion within various animal taxa during evolution. The reviewed data support a proposed model according to which many of the C2H2 proteins have functions that are similar to those of the CTCF in the organization of the chromatin architecture.

ZAD-Domain Is Essential for Nuclear Localization of Insulator Proteins in Drosophila melanogaster.

Many arthropod zinc-finger transcription factors contain a N-terminal domain called ZAD (Zinc-finger Associated Domain), which consists of four cysteines coordinating a single zinc ion. Dimerization ability has been shown for several ZAD-domains. The functional role of this domain is poorly understood. In this paper, we demonstrate that a point mutation within the ZAD-domain of the Zw5 insulator protein disrupts its nuclear localization without affecting its dimerization ability. The importance of the ZAD-domain for nuclear localization has also been shown for the Pita and Grauzone proteins. Therefore, one of the ZAD-domain functions is control of the nuclear localization of transcription factors.

CTCF and Sgfl1 proteins form alternative complexes with ENY2 proteins.

ENY2 is a multifunctional protein, a component of the SAGA complex (protein complex involved in the regulation of transcription, possessing histone-acetyltransferase and H2B-histone-deubiquitinylating activities [1]) and TREX-2 (complex involved in the mRNA nuclear export [2]). Besides this, the interactions of ENY2 with DNA-binding insulator proteins Su(Hw) and dCTCF have been described as essential for the barrier activity of corresponding insulators [3, 4]. In this study, we described the formation of mutually exclusive complexes of ENY2 with insulator proteins and Sgfl1-a component of the SAGA complex, direct binding partner for ENY2.

[Translation elongation factor EF1α1 interacts with ZAD domains of transcription factors from Drosophila melanogaster].

A large class of arthropod transcription factors is formed by proteins with a characteristic N-terminal Zinc-finger-Associated Domain (ZAD) which contains four cysteine residues that coordinate a zinc ion. The number of putative proteins with ZAD in the Drosophila genome exceeds 90, and the degree of sequence similarity between these domains can be as low as 23%. Efficient binding of ZADs from the proteins Grau, ZIPIC, and Zw5 to the translation elongation factor EF1α1 in nuclear and cytoplasmic extracts has been demonstrated. EF1α1 is probably involved in the regulation of the activity of ZAD-containing transcription factors.