Suppression of the Testis-Specific Transcription of the ZBTB32 and ZNF473 Genes in Germ Cell Tumors.

The family of genes containing C2H2 zinc finger domains, which has more than 700 members, is one of the largest in the genome. Of particular interest are C2H2 genes with potential tissue-specific transcription, which determine the functional properties of individual cell types, including those associated with pathological processes. The aim of this work was to identify C2H2 family genes with tissue-specific transcription and analyze changes in their activity during tumor progression. To search for these genes, we used four databases containing data on gene transcription in human tissues obtained by RNA-Seq analysis. The analysis showed that, although the major part of the C2H2 family genes is transcribed in virtually all tissues, a group of genes has tissue-specific transcription, with most of the transcripts being found in the testis. After having compared all four databases, we identified nine such genes. The testis-specific transcription was confirmed for two of them, namely ZBTB32 and ZNF473, using quantitative PCR of cDNA samples from different organs. A decrease in ZBTB32 and ZNF473 transcription levels was demonstrated in germ cell tumors. The studied genes can serve as candidate markers in germ cell tumors.

Correlation between Expression of KLF5 and ZEB1 Transcription Factor Genes in Pancreatic Cancer.

The mRNA content of the transcription factors KLF5 and ZEB1 was studied in pancreatic tumor tissues and in fetal and normal pancreas. Transcription of these factors was not high and similar in normal and fetal pancreatic tissues but greatly increased in the pancreatic ductal adenocarcinoma tissues. A significant positive correlation between the KLF5 and ZEB1 transcription levels in the pancreatic tumor tissues was observed.

Expression of transcription factor genes in cell lines corresponding to different stages of pancreatic cancer progression.

The expression level of six transcription factor genes and the content of their protein products in five pancreatic cancer cell lines with parallel control of expression of three marker genes reflecting epithelial or mesenchymal state of cells was investigated. Cell lines MIA PaCa-2 and Capan-2 represented the best models of quasi-mesenchymal and epithelial, respectively, types of progression of the pancreatic ductal adenocarcinoma, according to the content of E-cadherin and vimentin and the expression of KLF5 and ZEB1 transcription factors.

In trans promoter activation by enhancers in transient transfection.

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.

Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus.

A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently - in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected.

Expression of chicken CTCF gene in COS-1 cells and partial purification of CTCF protein.

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.

Assay of insulator enhancer-blocking activity with the use of transient transfection.

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.

[Affinity capture of specific DNA fragments with the use of short synthetic sequences].

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine and 5-propynyl-2'-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments from mixtures.

Human PSENEN and U2AF1L4 genes are concertedly regulated by a genuine bidirectional promoter.

Head-to-head genes with a short distance between their transcription start sites may constitute up to 10% of all genes in the genomes of various species. It was hypothesized that this intergenic space may represent bidirectional promoters which are able to initiate transcription of both genes, but the true bidirectionality was proved only for a few of them. We present experimental evidence that, according to several criteria, a 269 bp region located between the PSENEN and U2AF1L4 human genes is a genuine bidirectional promoter regulating a concerted divergent transcription of these genes. Concerted transcription of PSENEN and U2AF1L4 can be necessary for regulation of T-cell activity.

Functional dissection of an enhancer-like element located within the second intron of the human U2AF1L4 gene.

A detailed functional and evolutionary analysis of an enhancer element of the human genome (enhancer 12) located in the second intron of the U2AF1L4 gene, which we identified earlier, is presented. Overlapping fragments of the studied genome region were analyzed for enhancer activity, and the site responsible for the activity of this element was identified using transient transfections of HeLa cells. Comparison of the enhancer 12 sequence with orthologous sequences from seven primate species revealed the existence of evolutionarily conserved sequences within this element. One of the identified conservative regions is likely responsible for the enhancer activity and is able to specifically interact in vitro with proteins of HeLa cell nuclear extract. The ability of orthologous primate sequences to compete with enhancer 12 for binding with HeLa cell nuclear extract proteins and to enhance the activity of the reporter gene in transient transfection of HeLa cells is demonstrated.

[Enhancer activity of DNA fragments from FXYD5-COX7A region of human chromosome 19].

The enhancer activity of four previously identified within the one megabase region of human chromosome 19 DNA fragments was investigated. All four fragments had similar tissue-specific profile--maximum of enhancer activity was observed in HEK293 and minimum in HeLa cells. Enhancers obtained had pronounced specificity toward cytomegalovirus promoter compared with SV40 promoter. Functional dissection of one of the fragments (enhancer 14) demonstrated that only its inner 127 b.p. part possessed enhancer activity. The negative regulators, i.e. silencers or insulators are probably located in flanking regions of enhancer 14 and limit its effect on promoter. At the same time, enhancer activity of enhancer 14 depends on its orientation relative to promoter that isn't typical to majority of enhancer elements. Inner 127 b.p. fragment contains 11 transcription factor binding sites; 8 of them are factors that take part in immune system regulation. Enhancer 14 is located 500 b.p. upstream of transcription start site of TYROBP (DAP12) gene that codes for of T-killer cells activator protein and possibly functions as tissue-specific enhancer for this gene.

Identification and mapping of ten new potential insulators in the FXYD5-COX7A1 region of human chromosome 19q13.12.

A positive-negative selection system revealed 10 potential insulators able to block enhancer interaction with promoter in the 10(6) bp human chromosome 19 region between genes FXYD5 and COX7A1. Relative positions of insulators and genes are in accord with the hypothesis that insulators subdivide genomic DNA into independently regulated loop domains.

Vertebrate Protein CTCF and its Multiple Roles in a Large-Scale Regulation of Genome Activity.

The CTCF transcription factor is an 11 zinc fingers multifunctional protein that uses different zinc finger combinations to recognize and bind different sites within DNA. CTCF is thought to participate in various gene regulatory networks including transcription activation and repression, formation of independently functioning chromatin domains and regulation of imprinting. Sequencing of human and other genomes opened up a possibility to ascertain the genomic distribution of CTCF binding sites and to identify CTCF-dependent cis-regulatory elements, including insulators. In the review, we summarized recent data on genomic distribution of CTCF binding sites in the human and other genomes within a framework of the loop domain hypothesis of large-scale regulation of the genome activity. We also tried to formulate possible lines of studies on a variety of CTCF functions which probably depend on its ability to specifically bind DNA, interact with other proteins and form di- and multimers. These three fundamental properties allow CTCF to serve as a transcription factor, an insulator and a constitutive dispersed genome-wide demarcation tool able to recruit various factors that emerge in response to diverse external and internal signals, and thus to exert its signal-specific function(s).

Identification of recognition sites for myc/max/mxd network proteins by a whole human chromosome 19 selection strategy.

In this study, we have identified 20 human sequences containing Myc network binding sites in a library from the whole human chromosome 19. We demonstrated binding of the Max protein to these sequences both in vitro and in vivo. The majority of the identified sequences contained one or several CACGTG or CATGTG E-boxes. Several of these sites were located within introns or in their vicinity and the corresponding genes were found to be up- or down-regulated in differentiating HL-60 cells. Our data show the proof of principle for using this strategy in identification of Max target genes, and this method can also be applied for other transcription factors.

Methods for identification of epigenetic elements in mammalian long multigenic genome sequences.

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 and mapping of cis-regulatory elements within long genomic sequences].

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.

Tissue specificity of methylation of cytosines in regulatory regions of four genes located in the locus FXYD5-COX7A1 of human chromosome 19: correlation with their expression level.

In this study, we compared degree of methylation of selected CpG sites in CCGG sequences located in promoter regions of four human genes with expression level of these genes in several human cell lines and tissues. These genes were subdivided into two groups according to the dependence of their expression on CpG methylation in the 5 -regions. The first group, characterized by clear correlation of methylation with the transcription level, includes housekeeping gene COX6B (the absence of methylation unambiguously correlates with expression) and urothelium-specific uroplakin gene (the methylation coincides with absence of expression). The second group includes genes that are expressed in many, but not all tissues and cells. For these genes (LEAP-1 and ATP4A), there was no correlation between methylation and expression. It is possible that methylation provides some basal level of gene repression, which is overcome by binding of tissue-specific transcription factors, whereas lack of methylation gives the opportunity for gene expression in various cells and tissues.

[Regulatory potential of S/MAR elements in transient expression].

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.

Tissue specificity of enhancer and promoter activities of a HERV-K(HML-2) LTR.

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.

[Activation of the RIG-I gene, coding for DEXH/D-protein in infection of RH cells by tick-borne encephalitis virus]. / Aktivatsiia gena RIG-I, kodiruiushchego DEXH/D-belok pri infektsii kletok RH virusom kleshchevogo éntsefalita.

It was demonstrated by subtractive hybridization that the infection of a human embryonic kidney cell line with tick-borne encephalitis virus causes an approximately tenfold transcription activation of the RIG-1 gene, which encodes a protein of the DExH/D-box-containing RNA helicase family. A possible involvement of the protein in antiviral cell systems is discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.