Search

Earlier, it was reported that the strong cytomegalovirus enhancer can activate the cytomegalovirus promoter in trans, i.e. as a separate plasmid co-transfected with a promoter-reporter gene construct. Here we demonstrate that the ability of enhancers to activate promoters in trans in transient transfection experiments is a property of not only viral regulatory elements but also of various genomic enhancers and promoters. Enhancer-promoter activation in trans is promoter- and cell type-specific, and accompanied by physical interaction between promoter and enhancer as revealed by chromosome conformation capture assays. Thus, promoter activation in transient co-transfection of promoters and enhancers shares a number of important traits with long-distance promoter activation by enhancers in living cells and may therefore serve as a model of this fundamental cellular process.

The chicken gene for transcription factor CTCF was expressed in COS-1 mammalian cells. The CTCF protein containing polyhistidine tag was partially purified using metallo-affinity and ion-exchange chromatography. The expressed protein localized in the cell nucleus and was shown to be functionally active in the electrophoretic mobility shift assay and specifically interacted with anti-CTCF antibodies.

We used a transient transfection of cultured cells with linearized plasmids to analyze the enhancer-blocking activity of potential insulators including the standard cHS4 chicken beta-globin insulator and several DNA fragments selected from the human genome sequence. About 60-80% of the potential insulators do reveal the enhancer-blocking activity when probed by the transient transfection assay. The activity of different sequences is characterized by certain tissue specificity and by dependence on the orientation of the fragments relative to the promoter. Thus, the transfection model may be used for quantitative analysis of the enhancer-blocking activity of the potential insulators.

Transient expression of a luciferase reporter gene was used to evaluate tissue-specific promoter and enhancer activities of a solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K) in several human and CHO cell lines. The promoter activity of the LTR varied from virtually not detectable (GS and Jurkat cells) to as high as that of the SV40 early promoter (Tera-1 human testicular embryonal carcinoma cells). The negative regulatory element (NRE) of the LTR retained its activity in all cell lines where the LTR could act as a promoter, and was also capable of binding host cell nuclear proteins. The enhancer activity of the LTR towards the SV40 early promoter was detected only in Tera-1 cells and was not observed in a closely related human testicular embryonal carcinoma cell line of different origin, NT2/D1. A comparison of proteins bound to central part of the LTR in nuclear extracts from Tera-1 and NT2/D1 by electrophoretic mobility shift assay revealed striking differences that could be determined by different LTR enhancer activities in these cells. Tissue specificity of the SV40 early promoter activity was also revealed.

We used the 4C-Seq technique to characterize the genome-wide patterns of spatial contacts of several CpG islands located on chromosome 14 in cultured chicken lymphoid and erythroid cells. We observed a clear tendency for the spatial clustering of CpG islands present on the same and different chromosomes, regardless of the presence or absence of promoters within these CpG islands. Accordingly, we observed preferential spatial contacts between Sp1 binding motifs and other GC-rich genomic elements, including the DNA sequence motifs capable of forming G-quadruplexes. However, an anchor placed in a gene/CpG island-poor area formed spatial contacts with other gene/CpG island-poor areas on chromosome 14 and other chromosomes. These results corroborate the two-compartment model of the spatial organization of interphase chromosomes and suggest that the clustering of CpG islands constitutes an important determinant of the 3D organization of the eukaryotic genome in the cell nucleus. Using the ChIP-Seq technique, we mapped the genome-wide CTCF deposition sites in the chicken lymphoid and erythroid cells that were used for the 4C analysis. We observed a good correlation between the density of CTCF deposition sites and the level of 4C signals for the anchors located in CpG islands but not for an anchor located in a gene desert. It is thus possible that CTCF contributes to the clustering of CpG islands observed in our experiments.

Intrinsic lysozyme-like activity was demonstrated for destabilase from the medicinal leech supported by (1) high specific lysozyme activity of the highly purified destabilase, (2) specific inhibition of the lysozyme-like activity by anti-destabilase antibodies, and (3) appreciable lysozyme-like activity in insect cells infected with recombinant baculoviruses carrying cDNAs encoding different isoforms of destabilase. Several isoforms of destabilase constitute a protein family at least two members of which are characterized by lysozyme activity. The corresponding gene family implies an ancient evolutionary history of the genes although the function(s) of various lysozymes in the leech remains unclear. Differences in primary structures of the destabilase family members and members of known lysozyme families allow one to assign the former to a new family of lysozymes. New proteins homologous to destabilase were recently described for Caenorhabditis elegans and bivalve mollusks suggesting that the new lysozyme family can be widely distributed among invertebrates. It remains to be investigated whether the two enzymatic activities (isopeptidase and lysozyme-like) are attributes of one and the same protein.

We previously detected in salivary gland secretions of the medicinal leech (Hirudo medicinalis) a novel enzymatic activity, endo-epsilon(gamma-Glu)-Lys isopeptidase, which cleaves isopeptide bonds formed by transglutaminase (Factor XIIIa) between glutamine gamma-carboxamide and the epsilon-amino group of lysine. Such isopeptide bonds, either within or between protein polypeptide chains are formed in many biological processes. However, before we started our work no enzymes were known to be capable of specifically splitting isopeptide bonds in proteins. The isopeptidase activity we detected was specific for isopeptide bonds. The enzyme was termed destabilase. Here we report the first purification of destabilase, part of its amino acid sequence isolation and sequencing of two related cDNAs derived from the gene family that encodes destabilase proteins, and the detection of isopeptidase activity encoded by one of these cDNAs cloned in a baculovirus expression vector. The deduced mature protein products of these cDNAs contain 115 and 116 amino acid residues, including 14 highly conserved Cys residues, and are formed from precursors containing specific leader peptides. No homologous sequences were found in public databases.

For a 140-kb human genome locus, an analysis of the distribution of Dam methylase accessible sites, DNase I sensitive and resistant regions, unmethylated CpG sites and acetylated histone H3 molecules was performed and compared with transcriptional activity of the genes within the locus. A direct correlation was found between the extent of Dam methylation and C5 cytosine (CpG) methylation. It was also demonstrated that promoter regions of all highly and moderately transcribed genes are highly accessible to methylation by Dam methylase. In contrast, promoters of non-transcribed genes showed a very low extent of Dam methylation. Promoter regions of non-transcribed genes were also highly CpG methylated, and the promoter and more distant 5'-regions of the housekeeping gene COX6B1 were substantially CpG-demethylated. Some highly Dam methylase accessible regions are present in the intergenic regions of the locus suggesting that the latter contain either unidentified non-coding transcripts or extended regulatory elements like locus control regions.

The publication of the human and other metazoan genome sequences opened up the possibility for mapping and analysis of genomic regulatory elements. Unfortunately, experimental data on genomic positions of such sequences as enhancers, silencers, insulators, transcription terminators, and replication origins are very limited, especially at the whole genome level. As most genomic regulatory elements (e.g., enhancers) are generally gene-, tissue-, or cell-specific, the prediction of these elements in silico is often ambiguous. Therefore, the development of high-throughput experimental approaches for identification and mapping of genomic functional elements is highly desirable. In this review we discuss novel approaches to high-throughput experimental identification of mammalian genomes cis-regulatory elements which is a necessary step toward the complete genome annotation.

Epigenetic elements of the genome, i.e. elements that determine stably inherited changes in gene expression without changes in the genomic DNA sequence, are essential tools of genetic regulation in higher eukaryotes. The complete sequencing of the human and other genomes allowed studies to be started on positioning of these elements within long multigenic regions of the genome, which is a prerequisite for a comprehensive functional annotation of genomes. This mini-review considers some recent experimental approaches to the high-throughput identification and mapping of epigenetic elements of mammalian genomes, including the mapping of methylated CpG sites, open and closed chromatin regions, and DNase I hypersensitivity sites.

Identification of insulators is one of the most difficult problems in functional mapping of genomes. For this reason, up to now only a few insulators have been described. In this article we suggest an approach that allows direct isolation of insulators by a simple positive-negative selection based on blocking enhancer effects by insulators. The approach allows selection of fragments capable of blocking enhancers from mixtures of genomic fragments prepared from up to 1-Mb genomic regions. Using this approach, a 1-Mb human genome locus was analyzed and eight potential insulators were selected. Five of the eight sequences were positioned in intergenic regions and two were within introns. The genes of the alpha-polypeptide H+/K+ exchanging ATPase (ATP4A) and amyloid beta (A4) precursor-like protein 1 (APLP1) within the locus studied were found to be flanked by insulators on both sides. Both genes are characterized by distinct tissue-specific expression that differs from the tissue specificity of the surrounding genes. The data obtained are consistent with the conception that insulators subdivide genomic DNA into loop domains that comprise genes characterized by similar expression profiles. Using chromatin immunoprecipitation assay, we demonstrated also that at least six of the putative insulators revealed in this work could bind the CTCF transcription factor in vivo. We believe that the proposed approach could be a useful instrument for functional analysis of genomes.

S/MARs (scaffold/matrix attachment regions) are the DNA regions that are involved in the interaction with the nuclear matrix and are identified by in vitro methods. According to the available information, S/MARs possess an insulating activity, i.e., the ability to block the interaction between the enhancer and promoter in vivo, and are, probably, intact insulators or their fragments. Nevertheless, there is still no direct proof for this correspondence. To obtain additional information on the insulator activity of S/MARs, we selected five DNA fragments of different lengths and affinities for the nuclear matrix from the previously constructed library of S/MARs and tested their ability to serve as insulators. Two of five elements exhibited an insulator (enhancer-blocking) activity upon the transient transfection of CHO cells. None of the S/MARs displayed either promoter or enhancer/silencer activities in these cells.

Destabilase-lysozyme (DL) from salivary gland secretion of the medicinal leech (Hirudo medicinalis) is as a member of the invertebrate lysozyme family, which sharply differs from other lysozyme families. In this study, DL lysozyme function was confirmed during expression of a gene encoding DL in Escherichia coli. Several constructs of the expression vectors pKK OmpA and pET-3A with or without bacterial, leech, or yeast signal peptides (SP) were used. The use of a construct without signal peptide genes resulted in normal growth of the transformed cells. Transformation of E. coli cells with the constructs containing SP was accompanied by the disruption of the forming cells. The use of the expression vector pET-32 LTC-System for production of DL as a fusion protein with thioredoxin also resulted in normal cell growth. However, specific activity of DL isolated from such cells was significantly lower than that of enzyme purified from extracts of Spodoptera frugiperda cells, which were infected with the baculovirus vector carrying DL cDNA. It is shown that the action mechanism of invertebrate lysozyme does not differ from that of other families: recombinant DL from S. frugiperda extracts catalyzed cleavage of synthetic substrate, hexamer of N-acetylglucosamine, to di- and tetramers, which is typical for enzymatic function of other lysozyme families.

Our previous studies showed a predominance of high molecular weight protein group in tumor nuclear matrices. Contrary to normal cells, proteins of this group are preferentially phosphorylated. Phosphoproteins of hepatoma nuclear matrix are selectively subjected to rapid proteolysis. By alkali treatment and a monoclonal antibody against phosphotyrosyl residue the presence of two high molecular weight bands of phosphotyrosyl-containing proteins was detected in nuclear matrices of tumor but not of normal liver cells. High molecular weight protein group of tumor nuclear matrices revealed also a rapid turnover and preferential incorporation of labeled amino acids selectively inhibited by chloramphenicol.

Solitary long terminal repeats (LTRs) of the human endogenous retroviruses, scattered in several thousand copies throughout the human genome, are potentially capable of affecting the expression of closely located genes. To assess their regulatory potential, the LTR sequences of one of the most abundant HERV families (HERV-K) were screened for the presence of binding sites for the host cell nuclear factors using mobility shift and UV-crosslinking assays. It was shown that the LTR sequences of two subfamilies harbor a specific binding site for a complex consisting of at least three proteins, ERF1, ERF2 and ERF3 of 98, 91 and 88 kDa apparent molecular mass, respectively. This binding site is located in the 5′ region of the LTR U3 element. The preservation of the specific protein binding site in different HERV-K LTR sequences suggests their possible role in regulation of nearby located genes.

Depending on the ionic environment the replicative complex of silkworm Bombyx mori, containing DNA polymerase alpha and primase, catalyzes on single-stranded DNA of phage M13 a NTP-dependent synthesis or elongation of preformed primers. In the presence of NTPs and dNTPs at conditions optimal for the NTP-dependent synthesis the replicative complex synthesizes on M13 DNA oligoribonucleotides of 9-11 residues, which serve as primers for polymerization of DNA. The length of RNA-primers synthesized by primase of the complex depends on concentration of dNTP but does not depend on activity of DNA polymerase alpha. During elongation of exogenic primers annealed to M13 DNA the complex is processive synthesizing DNA fragments of dozens residues without dissociation from the template. Double-stranded structures in DNA such as "hairpins" appear to be barriers for driving of the complex along the template and cause pauses in elongation. DNA-binding proteins the SSB of Escherichia coli or the p32 of phage T4 destabilize double-stranded regions in DNA and eliminate elongation pauses corresponding to these regions. The replicative complex is able to fill in single-stranded gaps in DNA completely and to perform slowly the synthesis with displacement of one of parent strands in duplexes via repeated cycles of binding to the primer-template, limited elongation and dissociation.

Induction of heat shock proteins (HSP's) synthesis in human lymphocytes in vitro was studied using 1D- and 2D-polyacrylamide gel electrophoresis. High resolution of the O'Farrell's procedure revealed the existence of several isoforms of HSP70. The same major HSP's (70 kDa and 88 kDa) were found in rabbit lymphocytes in vivo under whole body hyperthermia. Freshly isolated lymphocytes seem to offer a new approach to the investigation of HSP synthesis induction in mammals under whole body hyperthermia.

SDS-polyacrylamide gel electrophoresis of the nuclear matrix proteins showed a predominance of high molecular weight and low molecular weight polypeptides in the nuclear matrices of hepatomas and cultured Chinese hamster fibroblasts as compared to quiescent and regenerating rat liver. These features were more prominent in solid hepatoma 27 than in Zajdela ascites hepatoma or in cultured cells. In proliferating cells (tumors, regenerating liver, log phase cell culture) a polypeptide band of 150 kD and lamin B were conspicuous at the expence of lamins A and C.

Heating of Chinese hamster fibroblasts (46 degrees C, 10 min) results in sharp inhibition of protein biosynthesis in the homogenate, nuclei and, in a lesser degree, in the nuclear matrix. The ratio of specific radioactivity of nuclear matrix 35S-proteins to the homogenate radioactivity taken for 100% increases after the heat shock 2,5-fold. Thus, protein biosynthesis in the nuclear matrix is more stable to the damaging action of heat shock than that in the homogenate and nuclei. The nuclear matrix was shown to contain heat shock proteins with Mr of 82-84, 70 and 26 kD, the 70 kD polypeptide being predominant. This polypeptide revealed in 4 hours is intensively accumulated at the 6th hour and remains unchanged by the 17-24th hours after cell heating. The 70 kD heat shock polypeptide can be separated by two-dimensional electrophoresis into two subfractions differing in terms of pI from other proteins revealed within this time interval in the unheated cells.

The increase in rat body temperature by 2-3 degrees as a result of overheating (45 degrees C, 22% humidity) over 90 and 120 min is accompanied by changes in the rate of labeled precursors incorporation into rat liver protein fractions. The incorporation of labeled amino acids into liver nuclear matrix proteins within the first 90 min of overheating is somewhat decreased, whereas 120 min thereafter it exceeds by 30% the corresponding values in control animals kept at room temperature. The polypeptide pattern of the nuclear matrix in hyperthermia is characterized by an increased relative content of polypeptide components around Mr 100, 55, 40 and 30 kDa against a decreased level of several polypeptides as compared to the control.

The thermal shock proteins with molecular mass of 86 kD and pI 5.5 as well as of 70 kD and pI 5.2-5.4 were isolated by means of two-dimensional electrophoresis in polyacrylamide gel from nuclear matrix of chinese hamster fibroblasts after heating of the animals. Incorporation of 35S-methionine into the thermal shock proteins was completely inhibited by actinomycin D (2 micrograms/ml), if the antibiotic was added into the cell incubation medium before heating, while the incorporation of methionine was decreased only slightly when the antibiotic was added after heating of the cells. Thus, biosynthesis of mRNA of the thermal shock proteins of 86 and 70 kD in nuclear matrix was induced by means of an increase in temperature during the incubation of the cells. Biosynthesis of the thermal shock proteins in nuclear matrix was distinctly inhibited by cycloheximide (1 microgram/ml) and was not practically inhibited by puromycin (60 micrograms/ml).