ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Enhancer Elements, Genetic , Plasmids/metabolism , Promoter Regions, Genetic , Trans-Activators/genetics , Transcriptional Activation , Transfection/methods , Animals , Cytomegalovirus/genetics , Cytomegalovirus/metabolism , Fireflies/enzymology , Fireflies/genetics , Genes, Reporter , HeLa Cells , Hep G2 Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Organ Specificity , Plasmids/chemistry , Renilla/enzymology , Renilla/genetics , Trans-Activators/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
Repressor Proteins/isolation & purification , Animals , CCCTC-Binding Factor , COS Cells , Cell Nucleus/metabolism , Chickens , Chlorocebus aethiops , Gene Expression , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
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.
Subject(s)
Enhancer Elements, Genetic , Insulator Elements , Animals , Biological Assay , CHO Cells , Chickens , Cricetulus , DNA/genetics , Genome, Human , HeLa Cells , Hep G2 Cells , Humans , Plasmids , Promoter Regions, Genetic , Transfection , beta-Globins/genetics , beta-Globins/metabolismABSTRACT
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.
Subject(s)
DNA/chemistry , Oligonucleotides/chemical synthesis , Peptide Nucleic Acids/chemical synthesis , Adenosine/analogs & derivatives , Adenosine/chemistry , Base Sequence , DNA/genetics , DNA/isolation & purification , Humans , Oligonucleotides/chemistry , Peptide Nucleic Acids/chemistry , Peptide Nucleic Acids/genetics , Polymerase Chain Reaction , Promoter Regions, Genetic , Repressor Proteins/chemistryABSTRACT
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.
Subject(s)
Amyloid Precursor Protein Secretases/genetics , Gene Expression Regulation , Membrane Proteins/genetics , Nuclear Proteins/genetics , Promoter Regions, Genetic , Ribonucleoproteins/genetics , Amyloid Precursor Protein Secretases/metabolism , Cell Line , Cloning, Molecular , Consensus Sequence , Humans , Membrane Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleotide Motifs , Organ Specificity/genetics , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , Splicing Factor U2AF , Transcription, GeneticABSTRACT
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.
Subject(s)
Enhancer Elements, Genetic , Introns , Nuclear Proteins/genetics , Ribonucleoproteins/genetics , Base Sequence , Conserved Sequence , Evolution, Molecular , Genes, Reporter , HeLa Cells , Humans , Molecular Sequence Data , Nuclear Proteins/metabolism , Protein Binding , Ribonucleoproteins/metabolism , Splicing Factor U2AFABSTRACT
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.
Subject(s)
Chromosome Mapping , Chromosomes, Human, Pair 19/genetics , Electron Transport Complex IV/genetics , Insulator Elements , Membrane Glycoproteins/genetics , Neoplasm Proteins/genetics , Humans , Ion Channels , Microfilament Proteins , Promoter Regions, GeneticABSTRACT
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).
ABSTRACT
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.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Chromosomes, Human, Pair 19/genetics , Genetic Techniques , Proto-Oncogene Proteins c-myc/metabolism , Repressor Proteins/metabolism , Base Sequence , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Binding Sites , Cell Line, Tumor , Chromosomes, Human, Pair 19/chemistry , Chromosomes, Human, Pair 19/metabolism , Humans , Molecular Sequence Data , Protein Binding , Proto-Oncogene Proteins c-myc/genetics , Repressor Proteins/geneticsABSTRACT
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.
Subject(s)
Epigenesis, Genetic , Genomics/methods , Physical Chromosome Mapping/methods , Regulatory Elements, Transcriptional , Animals , Base Sequence , Chromatin/metabolism , CpG Islands/genetics , DNA Methylation , HumansABSTRACT
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.
Subject(s)
Chromosome Mapping , Genome, Human/genetics , Regulatory Elements, Transcriptional/genetics , Sequence Analysis, DNA , Animals , HumansABSTRACT
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.
Subject(s)
Chromosomes, Human, Pair 19 , Cytosine/chemistry , Gene Expression , Promoter Regions, Genetic , Adenosine Triphosphatases/genetics , Antimicrobial Cationic Peptides/genetics , Cell Line , CpG Islands , Electron Transport Complex IV/genetics , Hepcidins , Humans , Ion Channels , Membrane Glycoproteins/genetics , Methylation , Microfilament Proteins , Neoplasm Proteins/genetics , Protein Subunits/genetics , RNA/metabolism , Tissue Distribution , Uroplakin IaABSTRACT
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.
Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation , Matrix Attachment Regions/genetics , Animals , CHO Cells , Chickens , Cricetinae , HumansABSTRACT
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.
Subject(s)
Endopeptidases/metabolism , Escherichia coli Proteins/metabolism , Muramidase/metabolism , Recombinant Proteins/metabolism , Acetylglucosamine/metabolism , Animals , Catalysis , Chromatography, Affinity , Endopeptidases/genetics , Escherichia coli Proteins/genetics , Hirudo medicinalis/enzymology , Muramidase/genetics , Recombinant Proteins/isolation & purification , Spodoptera/genetics , Substrate SpecificityABSTRACT
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.
Subject(s)
Endogenous Retroviruses/genetics , Enhancer Elements, Genetic/physiology , Promoter Regions, Genetic/physiology , Terminal Repeat Sequences/physiology , Animals , CHO Cells , Cricetinae , Genes, Reporter , Humans , Luciferases/genetics , Organ Specificity , Tumor Cells, CulturedABSTRACT
Modern concepts on the chromatin loop-domain organization and the role of the DNA regions specifically binding the nuclear matrix (nuclear scaffold, or S/MARs) in its formation, maintenance, and regulation are discussed. Some S/MAR structural features, properties of binding the nuclear matrix, and probable mechanisms of their involvement in regulation of gene activity are considered. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.
Subject(s)
Nuclear Matrix/chemistry , Nuclear Matrix/physiology , Amino Acid Sequence , Base Sequence , DNA/chemistry , Molecular Sequence Data , Structure-Activity RelationshipABSTRACT
A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.
Subject(s)
Chromatography, Affinity/methods , Endogenous Retroviruses/genetics , Repetitive Sequences, Nucleic Acid , Base Sequence , DNA Primers , Electrophoresis, Polyacrylamide Gel , Humans , Protein Binding , Species SpecificityABSTRACT
The transient expression of the luciferase reporter gene helped us to detect a tissue-specific enhancer activity of the solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K). The LTR was previously mapped to the 19q13.2 locus. It contains a number of potential regulatory elements including TATA box, binding sites for some nuclear factors, and a polyadenylation signal. However, an analysis of the genomic sequences close to the LTR did not reveal any known genes or the expressing marker sequences (EST), whose functioning could be regulated by this LTR. The enhancer activity can be preserved in the solitary LTR due to its involvement in a long-range control of genome functioning or by the absence of functional disruptive mutations within the human-specific LTR, because it is of a relatively young evolutionary age. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.