Laboratory of human genes structure and functions

Department of Genetics and Postgenomic Technologies

Head: Eugeny Sverdlov, member of the academy of sciences

Human and bacterial functional genomics, molecular mechanisms of cancer, host-pathogen interactions, diagnostics and gene therapy of tumors

Tatyana L. Azhikina Group

The members of Dr. Azhikina's group actively conduct research in two main areas:

1.  Functional genomics and RNomics of mycobacteria, including Mycobacterium tuberculosis, the causative agent of human tuberculosis.

A new method for transcriptome analysis of intracellular pathogens has been developed in our group. It allows to study pathogens in infected tissues and can be used for research projects like search for virulence factors and drug therapy targets, or design of disease monitoring strategies (Azhikina et al., 2010; Skvortsov et al., 2013; Ignatov et al., 2010). By far, this method is the only approach using deep sequencing for analysis of pathogen’s transcriptome directly in the infected tissue. We applied this unique approach to investigate transcriptome changes occurring in pathogenic (M.tuberculosis) and opportunistic (M.avium) mycobacterial strains during their adaptation to infection in mice with genetically different susceptibility to tuberculosis. A comparative study of the M.tuberculosis transcriptome in vivo was performed to determine particular features of mycobacterial gene expression leading to the progressive disease (Skvortsov et al., 2013).

Our group is also focused on qualitative and quantitative description of the mycobacterial transcriptomes. For instance, we performed comprehensive analysis of M.avium transcriptome in culture using RNA-seq and determined transcription start sites and relative transcription levels of all genes, and identified 86 novel small RNAs. Some of M.avium small non-coding RNAs were also shown to be present in M.tuberculosis genome. Furthermore, several small RNAs are differently regulated in the two mycobacterial strains, which may contribute to the differences in metabolic features of these bacteria. Several small RNAs specific only for M.avium were also identified (Ignatov et al., 2013).

Finally, a study of M.tuberculosis transcriptome in the non-culturable state model (dormancy model) has also been conducted. We found that a 50-100 fold decrease of the mRNA quantity takes place in the dormant cells compared with the cells in the logarithmic phase. The genes whose level of expression changes during transition to dormancy state have also been identified. Moreover, we found that several small RNAs are transcribed at a level higher than that of the protein-coding genes (Ignatov et al., 2015).

At present, our work is focused on elucidating mechanisms of small RNAs function and their role in the establishment and maintenance of dormancy.


2. Investigation of the role that highly conservative Piwi proteins play in the development and maintenance of germ cell tumours.

Our group is focused on studying testicular germ cell tumors (TGCTs), which are heterogeneous cancers classified into less invasive seminomas and more aggressive nonseminomas. TGCTs are believed to be caused by both genetic and environmental factors and are deemed to arise because of failure to undergo normal spermatogenesis. One of the key players in spermatogenesis is piRNA/PIWI pathway responsible for epigenetic silencing of retrotransposons . Importantly, PIWI proteins have been also implicated in development of other types of cancers. Recently, we have demonstrated that PIWIL2 is expressed in TGCTs as its short 60 kDa isoform (PL2L60A) (Gainetdinov et al, Plos One, 2014). We have also found that spermatogenesis is retained in premalignant testis tissues adjacent to nonseminoma, but not those adjacent to seminomas. Moreover, this pattern is followed by expression of PIWI pathway genes and DNA methylation level over LINE-1 promoters. This finding could be used as a novel diagnostic marker, possibly, noninvasively (Gainetdinov et al, Oncotarget, 2016). Current work is aimed at elucidating detailed mechanisms of PIWI/piRNA pathway deregulation in the course of TGCT development and its clinical implications.

Genomics of cis-regulatory elements (L.G. Nikolaev, S.B. Akopov, I.P. Chernov, T.L. Azhikina)

Publication of nucleotide sequences of several eukaryotic genomes opened up a possibility to study the regulatory mechanisms determining the phenotypic variability of living organisms. Mammalian genome contains up to 30,000 protein-coding genes, and much larger portion of the genome is transcribed. Both genes and transcribed genome regions of a multicellular organism are participate in complex regulated network that determines the existence of many specialized cell types. Transcription of genes and non-coding sequences is regulated at several levels: linear genome architecture represented by DNA cis-regulatory elements, modifications of DNA, chromatin structure, compartmental organization of the nucleus etc.

Despite the achievements in a study of individual regulatory systems, we are still far away from complete understanding of the mechanisms that regulate the genome as a whole. Even in the relatively simple cases of cis-regulators identification we encounter serious problems which demand great efforts from many researchers to be solved. For example, it is predicted that human genome may contain up to 100,000 enhancers and silencers, but only small part of them is characterized to date.

A very important part of genome regulatory machinery are epigenetic elements, i.e. elements determining stably inherited changes in gene expression level without change of DNA sequence. Among them are sites of DNA methylation, regions containing modified histones etc. In mammals, methylation of CpG sites plays a key role in gene regulation by means of either modification of cis-regulatory elements or modification of long genome region which is associated with chromatin conformation change. To elucidate the mechanisms of methyl-dependent regulation of expression the detailed maps of methylation of long genomic regions are necessary. A comparison of these maps for different types of cells may reveal how DNA methylation affects gene expression.

In our lab several methods are developed allowing a high-throughput identification and genome mapping of non-methylated CpG dinucleotides, transcription start sites, and several genomic regulatory elements — nuclear matrix attachment sites (S/MARs), insulators, enhancers, open and closed chromatin regions, and binding sites of DNA-recognizing proteins including transcription factors, within long (1–2 megabases) DNA sequences. The data obtained are integrated within relatively small (1 megabase) region of human chromosome 19. Also, we construct maps of polygenic genome regions for normal and tumor tissues which are necessary for study of genome regulation and for practical use in medical genetics.

Integrated map of regulatory elements positions within 500 kb human chromosome 19 region. Vertical color lines indicate positions of corresponding regulatory elements.

Structure and functional study of Mycobacterium tuberculosis, Burkholderia pseudomallei and Burkholderia mallei pathogenesis (T.L. Azhikina, G.S. Monastyrskaya)

Tuberculosis — a chronic infection caused by Gram-positive bacteria Mycobacterium tuberculosis — is one of ten world’s main causes of premature death and second among infections. Mycobacterial genome is extremely variable that complicates tuberculosis diagnostics and therapy. We developed the experimental method of comparative analysis of the genomes — the RFLP subtractive hybridization — which allows comparing genomes of closely related bacterial strains. With the use of this system a comparison of the genomes of different M. tuberculosis strains circulating in Russia and causing different clinical manifestations of the disease was performed. Basing on the differential sequences obtained the experimental approach was developed allowing to study an insertion-deletion polymorphism within Russian population of M. tuberculosis (ID-typing).

M. tuberculosis caused human infection with a wide spectrum of clinical manifestations that depends on complex and not completely studied interactions between host and bacterium. We are studying a full spectrum of transcripts of the M. tuberculosis genes during primary infection and after the transition into chronic and latent forms. This will help to reveal the hoe the pathogen escapes from host response. In collaboration with the immunogenetics lab of Central Institute for Tuberculosis (Head Prof. A.S. Apt) we developed a method allowing for analysis of the intracellular pathogen’s transcriptome directly in infected tissues. The method may be useful for search of virulence factors, targets for drug therapy and for development of the epidemiological monitoring strategy for any pathogen.

In collaboration with State Scientific Center of Applied Microbiology (I. Abaev) the whole-genome comparison of pathogenic Burkholderia pseudomallei C—141 and Burkholderia mallei C5 strains causing melioidosis and glanders was performed. The library was constructed that contains DNA fragment that differs between B. pseudomallei C141 and B. mallei C5 and maybe used for diagnostics of melioidosis and glanders.

Position of differential fragments found (circles) on the M. tuberculosis deletion genome map. The map reflects non-random distribution of the deleted regions. The numbers indicate genomic distance (in megabases), 0 — replication start site.

Approaches to diagnostics and gene therapy of cancer (T.V. Vinogradova, E.P. Kopantzev, I.P. Chernov)

Currently much attention is paid to development of new generation therapy of cancer. Within the framework of the State program «Priority fields in science and technology in Russia during 2007–2012» our lab participated in the integrated project «Development and production of the prototype of novel and effective therapies and diagnostics of the post-genome generation for use in tumor therapy». The aim of this project was the development and clinical tests of first gene therapy based preparations for treatment of lung and esophagus cancers. The approaches used in this project are based on the fundamental and methodical research performed in the laboratory. The project integrates efforts of the major scientific centers of Russia: Institute of Molecular genetics RAS, Blokhin Cancer Research Center RAMS, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Novosibirsk State University, Institute of gene biology RAS, Gamaleya Research Institute of Epidemiology and Microbiology RAMS, and biotechnology companies Evrogen and Binnofarm. Within the framework of this project the constructions allowing for specific expression of therapeutic genes in tumor cells and systems for delivery of the therapeutic genes into tumors were developed.

Hence, the bases were established for the development and production of novel effective medical preparations and diagnostics based on gene therapy approach and biologically active proteins.

Cancer and development (M.V. Zinovyeva, T.V. Vinogradova, G.S. Monastyrskaya)

Demonstration of the fact that genes that are activated during development of lung and esophagus in embryogenesis are switched off in normal cells when they are being transformed into cancer cells and conversely is one the major achievements of the project. This confirms the hypothesis that process of cancer transformation and progression of normal cells may be compared with embryonal development or self-reproduction and differentiation of stem cells of certain tissues. The genes that express differentially in cancer and development probably play key role in tumorigenesis. Study of such genes allows carrying out a rational choice of therapeutic targets because systems regulating embryonal development and damaged in cancer are most perspective targets.

Modified oligonucleotides (V.K. Potapov)

Development of new methods of production and synthesis of modified oligonucleotides with changed structure of the base or of sugar-phosphate skeleton was the mainstream of the group through the last three years. Working out of approaches to solid phase synthesis of morpholino-analogues of oligonucleotides, which have morpholine ring in their structure instead of ribose, is the most perspective direction of the research. Such analogues are uncharged molecules and resistant to nucleases. They preserve DNA structure and form stable complementary bonds with native oligonucleotides.

Synthesis of oligonucleotides bearing spacers of specified length with thiol group in certain sites is the second direction of the work. These oligonucleotides are used in study of their complexes with integrases of HIV and ASV by X-ray diffraction analysis by prof. Vlodover group (NCI, Frederic).

Eugeny Sverdlov, member of the academy of sciencesdepart.
Galina Monastyrskaya, Ph.D., senior research associatel. r.
Viktor Potapov, D.Scl. r.
Sergej Akopov, Ph.D.s. r.
Irina Boni, Ph.D.s. r.
Tat'jana Vinogradova, Ph.D.s. r.
Ljudmila Zavalova, Ph.D.s. r.
Evgenij Kopancev, Ph.D.s. r.
Lev Nikolaev, D.Scs. r.
Evgeny Snezhkov, Ph.D.s. r.
Nataliya Uspenskaya, Ph.D.s. r.
Timofey Skvortsov, Ph.D.r.
Dmitry Didych, Ph.D.r.
Svetlana Bulanenkova, Ph.D.r. f.
Nina Beljaeva, Ph.D.r. f.
Marina Zinov'eva, Ph.D.r.
Kirill Kashkin, Ph.D.r.
Nadezhda Skapcovar. f.+7(495)330-65-47
Ol'ga Filjukova, Ph.D.r. f.+7(495)330-69-92
Igor' Chernov, Ph.D.r.
Irina Alekseenko, Ph.D.r.
Leonid Aseev, Ph.D.r.
Ildar Gainetdinov, Ph.D.r.
Ljudmila Koledinskajaj. r. f.+7(495)3306329
Rudolf Pogosyanj. r.
Aleksandr Sassj. r. f.+7(495)330-70-29
Sofia KondratievaPhD
Liya KondratyevaPhD
Dina AntonovaPhD
Arina MazurovaPhD
Dmitrij GnatenkoPhD
Ljudmila Averinaeng.+7(495)330-63-29
Zoja Kravchenkores. eng.+7(495)3306329
Tat'jana Levitanres.
Natalija Medvedevares. eng.+7(495)3306329

Former members:

Tatyana Azhikina, D.Scl. r.

Selected publications

  1. Gainetdinov I.V., Kondratieva S.A., Skvortsova Y.V., Zinovyeva M.V., Stukacheva E.A., Klimov A., Tryakin A.A., Azhikina T.L. (2016). Distinguishing epigenetic features of preneoplastic testis tissues adjacent to seminomas and nonseminomas. Oncotarget , [+]

    PIWI pathway proteins are expressed during spermatogenesis where they play a key role in germ cell development. Epigenetic loss of PIWI proteins expression was previously demonstrated in testicular germ cell tumors (TGCTs), implying their involvement in TGCT development. In this work, apart from studying only normal testis and TGCT samples, we also analyzed an intermediate stage, i.e. preneoplastic testis tissues adjacent to TGCTs. Importantly, in this study, we minimized the contribution of patient-to-patient heterogeneity by using matched preneoplastic/TGCT samples. Surprisingly, expression of germ cell marker DDX4 suggests that spermatogenesis is retained in premalignant testis tissues adjacent to nonseminoma, but not those adjacent to seminoma. Moreover, this pattern is followed by expression of PIWI pathway genes, which impacts one of their functions: DNA methylation level over LINE-1 promoters is higher in preneoplastic testis tissues adjacent to nonseminomas than those adjacent to seminomas. This finding might imply distinct routes for development of the two types of TGCTs and could be used as a novel diagnostic marker, possibly, noninvasively. Finally, we studied the role of CpG island methylation in expression of PIWI genes in patient samples and using in vitro experiments in cell line models: a more complex interrelation between DNA methylation and expression of the corresponding genes was revealed.

  2. Aseev L.V., Chugunov A.O., Efremov R.G., Boni I.V. (2013). A single missense mutation in a coiled-coil domain of Escherichia coli ribosomal protein S2 confers a thermosensitive phenotype that can be suppressed by ribosomal protein S1. J. Bacteriol. 195 (1), 95–104 [+]

    Ribosomal protein S2 is an essential component of translation machinery, and its viable mutated variants conferring distinct phenotypes serve as a valuable tool in studying the role of S2 in translation regulation. One of a few available rpsB mutants, rpsB1, shows thermosensitivity and ensures enhanced expression of leaderless mRNAs. In this study, we identified the nature of the rpsB1 mutation. Sequencing of the rpsB1 allele revealed a G-to-A transition in the part of the rpsB gene which encodes a coiled-coil domain of S2. The resulting E132K substitution resides in a highly conserved site, TKKE, a so-called N-terminal capping box, at the beginning of the second alpha helix. The protruding coiled-coil domain of S2 is known to provide binding with 16S rRNA in the head of the 30S subunit and, in addition, to interact with a key mRNA binding protein, S1. Molecular dynamics simulations revealed a detrimental impact of the E132K mutation on the coiled-coil structure and thereby on the interactions between S2 and 16S rRNA, providing a clue for the thermosensitivity of the rpsB1 mutant. Using a strain producing a leaderless lacZ transcript from the chromosomal lac promoter, we demonstrated that not only the rpsB1 mutation generating S2/S1-deficient ribosomes but also the rpsA::IS10 mutation leading to partial deficiency in S1 alone increased translation efficiency of the leaderless mRNA by about 10-fold. Moderate overexpression of S1 relieved all these effects and, moreover, suppressed the thermosensitive phenotype of rpsB1, indicating the role of S1 as an extragenic suppressor of the E132K mutation.

  3. Ignatov D., Malakho S., Majorov K., Skvortsov T., Apt A., Azhikina T. (2013). RNA-Seq Analysis of Mycobacterium avium Non-Coding Transcriptome. PLoS ONE 8 (9), e74209 [+]

    Deep sequencing was implemented to study the transcriptional landscape of Mycobacterium avium. High-resolution transcriptome analysis identified the transcription start points for 652 genes. One third of these genes represented leaderless transcripts, whereas the rest of the transcripts had 5' UTRs with the mean length of 83 nt. In addition, the 5' UTRs of 6 genes contained SAM-IV and Ykok types of riboswitches. 87 antisense RNAs and 10 intergenic small RNAs were mapped. 6 intergenic small RNAs, including 4.5S RNA and rnpB, were transcribed at extremely high levels. Although several intergenic sRNAs are conserved in M. avium and M. tuberculosis, both of these species have unique intergenic sRNAs. Moreover, we demonstrated that even conserved small RNAs are regulated differently in these species. Different sets of intergenic sRNAs may underlie differences in physiology between conditionally pathogenic M. avium and highly specialized pathogen M. tuberculosis.

  4. Ignatov D., Kondratieva E., Azhikina T., Apt A. (2012). Mycobacterium avium-triggered diseases: pathogenomics. Cell. Microbiol. 14 (6), 808–18 [+]

    The species Mycobacterium avium includes several subspecies representing highly specialized avian and mammalian pathogens, non-obligatory pathogens of immune compromised humans and saprophitic organisms. Recently obtained information concerning the diversity of M. avium genomic structures not only clarified phylogenic relationships within this species, but began to shed light on the question of how such closely related microorganisms adapt to the occupation of distinct ecological niches. In this review we discuss specific features of M. avium genetic composition, as well as genetic and molecular aspects of M. avium hominissuis (MAH)-triggered disease pathogenesis, including virulence, penetration, immune response manipulation and host genetic control.

  5. Azhikina T., Kozlova A., Skvortsov T., Sverdlov E. (2011). Heterogeneity and degree of TIMP4, GATA4, SOX18, and EGFL7 gene promoter methylation in non-small cell lung cancer and surrounding tissues. Cancer Genet 204 (9), 492–500 [+]

    We used methylation-sensitive high resolution melting analysis to assess methylation of CpG islands within the promoters of the TIMP4, GATA4, SOX18, and EGFL7 genes in samples of non-small cell lung cancer and surrounding apparently normal tissue and noncancerous lung tissues. We found that the promoter methylation was heterogeneous in both tumor and surrounding normal tissue. This is in contrast to healthy lung tissue, where the promoters were normally either non- or hypomethylated, and the heterogeneity of methylation was low. An increased heterogeneity of methylation in the normal tissues surrounding the tumor may suggest an early start of epigenetic processes preceding genetic and morphologic changes and can be used as a biomarker of early cancerization events. This analysis is an easy and sensitive tool for studying epigenetic heterogeneity and could be used in clinical practice.

  6. Azhikina T., Skvortsov T., Radaeva T., Mardanov A., Ravin N., Apt A., Sverdlov E. (2010). A new technique for obtaining whole pathogen transcriptomes from infected host tissues. BioTechniques 48 (2), 139–44 [+]

    We propose a novel experimental approach based on coincidence cloning for analyzing sequences of bacterial intracellular pathogens specifically transcribed in affected tissues. Co-denaturation and co-renaturation of excess bacterial genomic DNA with the cDNA prepared on total RNA of the infected tissue allows one to select the bacterial fraction of the cDNA sample. We used this technique for preparing and characterizing the Mycobacterium tuberculosis cDNA pool, representing the transcriptome of infected mouse lungs in the chronic phase of infection. A cDNA pool enriched in fragments of mycobacterial cDNA was analyzed by the high-throughput 454 sequencing procedure. We demonstrated that its composition corresponded to what can be expected in the chronic phase of infection and, after the adaptation of M. tuberculosis to the host immune system, was characterized by an active lipid metabolism and switched from aerobic to anaerobic respiration. The technique is universal and requires no prior knowledge of the pathogen genome sequence. Pools of transcribed sequences obtained by this technique retain the main characteristics of the genome-wide gene transcription pattern within infected tissue, and can be used for in vivo analysis of gene expression of a wide spectrum of infection agents, such as viruses, bacteria, and protista.

  7. Kopantzev E.P., Monastyrskaya G.S., Vinogradova T.V., Zinovyeva M.V., Kostina M.B., Filyukova O.B., Tonevitsky A.G., Sukhikh G.T., Sverdlov E.D. (2008). Differences in gene expression levels between early and later stages of human lung development are opposite to those between normal lung tissue and non-small lung cell carcinoma. Lung Cancer 62 (1), 23–34 [+]

    We, for the first time, directly compared gene expression profiles in human non-small cell lung carcinomas (NSCLCs) and in human fetal lung development. Previously reported correlations of gene expression profiles between lung cancer and lung development, deduced from matching data on mouse development and human cancer, have brought important information, but suffered from different timing of mouse and human gene expression during fetal development and fundamental differences in tumorigenesis in mice and humans. We used the suppression subtractive hybridization technique to subtract cDNAs prepared from human fetal lung samples at weeks 10-12 and 22-24 and obtained a cDNA library enriched in the transcripts more abundant at the later stage. cDNAs sequencing and RT-PCR analysis of RNAs from human fetal and adult lungs revealed 12 differentially transcribed genes: ADH1B, AQP1, FOLR1, SLC34A2, CAV1, INMT, TXNIP, TPM4, ICAM-1, HLA-DRA, EFNA1 and HLA-E. Most of these genes were found up-regulated in mice and rats at later stages than in human lung development. In surgical samples of NSCLC, these genes were down-regulated as compared to surrounding normal tissues and normal lungs, thus demonstrating opposite expression profiles for the genes up-regulated during fetal lung development.

  8. Chernov I.P., Timchenko K.A., Akopov S.B., Nikolaev L.G., Sverdlov E.D. (2007). Identification of tissue-specific DNA-protein binding sites by means of two-dimensional electrophoretic mobility shift assay display. Anal. Biochem. 364 (1), 60–6 [+]

    We developed a technique of differential electrophoretic mobility shift assay (EMSA) display allowing identification of tissue-specific protein-binding sites within long genomic sequences. Using this approach, we identified 10 cell type-specific protein-binding sites (protein target sites [PTSs]) within a 137-kb human chromosome 19 region. In general, tissue-specific binding of proteins from different nuclear extracts by individual PTSs did not follow the all-or-nothing principle. Most often, PTS-protein complexes were formed in all cases, but they were different for different nuclear extracts used.

  9. Bulanenkova S., Snezhkov E., Nikolaev L., Sverdlov E. (2007). Identification and mapping of open chromatin regions within a 140 kb polygenic locus of human chromosome 19 using E. coli Dam methylase. Genetica 130 (1), 83–92 [+]

    Using transient expression of the E. coli Dam methylase gene and analysis of the distribution of methylated GATC sites, we studied the distribution of open chromatin regions within a 140 kb long human genome segment in HEK-293 cells. Dam methylated sites were found in gene introns, exons, and intergenic regions, and their distribution along DNA was uneven. There were regions of high and low density of Dam methylated GATC sites, presumably corresponding to "open" and "closed" chromatin regions, respectively, and to the functional profile of the genomic locus under study. The Dam methylation profile was also generally in agreement with transcriptional activity of genes in the locus. Moreover, DNA regions accessible to Dam methylase apparently coincided with those hypersensitive to DNase I.

  10. Illarionova A.E., Vinogradova T.V., Sverdlov E.D. (2007). Only those genes of the KIAA1245 gene subfamily that contain HERV(K) LTRs in their introns are transcriptionally active. Virology 358 (1), 39–47 [+]

    Insertion of LTRs into some genome locations might seriously affect regulation of the neighboring genes expression. This hypothesis is widely accepted but, however, not confirmed directly. Earlier, we have identified a family of closely related genes highly similar to the KIAA1245 mRNA counterpart. This family included a subfamily of genes some of which contained and the others lacked an LTR in their structure. We compared transcription of several closely related genes of the subfamily differing in the presence or absence of LTRs. Only LTR-containing genes were transcribed in transformed cell lines, tumorous and embryonic human tissues, whereas LTR-lacking genes remained silent. Since the genes were in the same intracellular microenvironment, we suggested that this effect was most probably due to intrinsic cis-characteristics of integrated LTRs and confirmed this by demonstrating high enhancer activity of KIAA1245 LTRs. The expression of the LTR-containing genes in embryonic tissues might suggest their involvement in evolutionary events during primate speciation.

  11. Buzdin A., Kovalskaya-Alexandrova E., Gogvadze E., Sverdlov E. (2006). At least 50% of human-specific HERV-K (HML-2) long terminal repeats serve in vivo as active promoters for host nonrepetitive DNA transcription. J. Virol. 80 (21), 10752–62 [+]

    We report the first genome-wide comparison of in vivo promoter activities of a group of human-specific endogenous retroviruses in healthy and cancerous germ line tissues. To this end, we employed a recently developed technique termed genomic repeat expression monitoring. We found that at least 50% of human-specific long terminal repeats (LTRs) possessed promoter activity, and many of them were up- or downregulated in a seminoma. Individual LTRs were expressed at markedly different levels, ranging from approximately 0.001 to approximately 3% of the housekeeping beta-actin gene transcript level. We demonstrated that the main factors affecting the LTR promoter activity were the LTR type (5'-proviral, 3' proviral, or solitary) and position with regard to genes. The averaged promoter strengths of solitary and 3'-proviral LTRs were almost identical in both tissues, whereas 5'-proviral LTRs displayed two- to fivefold higher promoter activities. The relative content of promoter-active LTRs in gene-rich regions was significantly higher than that in gene-poor loci. This content was maximal in those regions where LTRs "overlapped" readthrough transcripts. Although many promoter-active LTRs were mapped near known genes, no clear-cut correlation was observed between transcriptional activities of genes and neighboring LTRs. Our data also suggest a selective suppression of transcription for LTRs located in gene introns.

  12. Akopov S.B., Ruda V.M., Batrak V.V., Vetchinova A.S., Chernov I.P., Nikolaev L.G., Bode J., Sverdlov E.D. (2006). Identification, genome mapping, and CTCF binding of potential insulators within the FXYD5-COX7A1 locus of human chromosome 19q13.12. Mamm. Genome 17 (10), 1042–9 [+]

    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.

  13. Fushan A., Monastyrskaya G., Abaev I., Sverdlov E. (2006). Genomic fingerprinting of Burkholderia pseudomallei and B. mallei pathogens with DNA array based on interspecies sequence differences obtained by subtractive hybridization. Res. Microbiol. 157 (7), 684–92 [+]

    The ability to rapidly and efficiently identify causative agents of dangerous human and animal diseases is a prerequisite to diagnosis, prophylaxis and therapy. Such identification systems can be developed based on DNA markers enabling differentiation between various bacterial strains. One source of these markers is genetic polymorphism. An efficient method for detecting the most stable polymorphisms without knowledge of genomic sequences is subtractive hybridization. In this work we report an approach to typing of Burkholderia pseudomallei and B. mallei that cause melioidosis and glanders, respectively. Typing is based on hybridization of bacterial genomes with a DNA array of genomic markers obtained using subtractive hybridization. The array comprised 55 DNA fragments which distinguished the genomes of B. pseudomallei C-141 and B. mallei C-5 strains, and it was used to test 28 radioactively labeled B. pseudomallei strains and 8 B. mallei strains. Each strain was characterized by a specific hybridization pattern, and the results were analyzed using cluster analysis. 18 patterns specific to B. pseudomallei and 6 patterns specific to B. mallei were found to be unique. The data allowed us to differentiate most studied B. pseudomallei variants from one another and from B. mallei strains. It was concluded that DNA markers obtained by subtractive hybridization can be potentially useful for molecular typing of B. pseudomallei and B. mallei strains, as well as for their molecular diagnosis. The method reported can be easily adapted for use both with DNA arrays and DNA microarrays with fluorescent probes.

  14. Vetchinova A.S., Akopov S.B., Chernov I.P., Nikolaev L.G., Sverdlov E.D. (2006). Two-dimensional electrophoretic mobility shift assay: identification and mapping of transcription factor CTCF target sequences within an FXYD5-COX7A1 region of human chromosome 19. Anal. Biochem. 354 (1), 85–93 [+]

    An approach for fast identification and mapping of transcription factor binding sites within long genomic sequences is proposed. Using this approach, 10 CCCTC-binding factor (CTCF) binding sites were identified within a 1-Mb FXYD5-COX7A1 human chromosome 19 region. In vivo binding of CTCF to these sites was verified by chromatin immunoprecipitation assay. CTCF binding sites were mapped within gene introns and intergenic regions, and some of them contained Alu-like repeated elements.

  15. Azhikina T., Gainetdinov I., Skvortsova Y., Sverdlov E. (2006). Methylation-free site patterns along a 1-Mb locus on Chr19 in cancerous and normal cells are similar. A new fast approach for analyzing unmethylated CCGG sites distribution. Mol. Genet. Genomics 275 (6), 615–22 [+]

    We describe a newly developed technique for rapid identification of positions of genomic DNA breaks, preexisting or introduced by specific digestion, in particular, by restriction endonucleases (RIDGES). We applied RIDGES in analyzing unmethylated CCGG sites distribution along a 1-Mb long genome region (D19S208-COX7A1 on chromosome 19) in cancerous and normal lung tissues. Both tissues were characterized by a profoundly uneven density of unmethylated sites along the fragment. Interestingly, the distribution of hypomethylated regions did not correlate with gene locations within the fragment, and one of the most hypomethylated areas contained practically no genes. We also demonstrated that the methylation pattern of a long genome DNA fragment was rather stable and practically unchanged in human lung cancer tissue as compared with its normal counterpart, in accordance with the suggestion (Ross et al. in Nat Genet 24:227-235, 2000) that cell lines of common origin have typically similar transcription profiles. An analogous suggestion might probably be made for global methylation patterns of genomic DNA.

  16. Azhikina T., Gvozdevsky N., Botvinnik A., Fushan A., Shemyakin I., Stepanshina V., Lipin M., Barry C. 3rd, Sverdlov E. (2006). A genome-wide sequence-independent comparative analysis of insertion-deletion polymorphisms in multiple Mycobacterium tuberculosis strains. Res. Microbiol. 157 (3), 282–90 [+]

    We applied an enhanced version of subtractive hybridization for comparative analyses of indel differences between genomes of several Mycobacterium tuberculosis strains widespread in Russian regions, and the H37Rv reference strain. A number of differences were detected and partially analyzed, thus demonstrating the practicality of the approach. A majority of the insertions found were shared by all Russian strains, except for strain 1540 that revealed the highest virulence in animal tests. This strain possesses a number of genes absent from other clinical strains. Two of the differential genes were found to encode putative membrane proteins and are presumed to affect mycobacterial interaction with the host cell, thus enhancing virulent properties of the isolate. The method used is of general application, and enables the elaboration of a catalogue of indel polymorphic genomic differences between closely related strains.

  17. Kovalskaya E., Buzdin A., Gogvadze E., Vinogradova T., Sverdlov E. (2006). Functional human endogenous retroviral LTR transcription start sites are located between the R and U5 regions. Virology 346 (2), 373–8 [+]

    Human endogenous retroviruses (HERVs) occupy about 5% of human DNA and are thought to be remnants of ancient retroviral infections of human ancestors' germ cells. HERVs can modify expression of host cell genes through their cis-regulatory elements concentrated in their long terminal repeats (LTRs). Although numerous HERV-related RNAs were identified in the human transcriptome, for most of them, it remains unclear whether they are LTR-promoted or read-through products initiated from neighboring genomic promoters. Here, we describe mapping of transcriptional start sites within solitary and proviral LTRs of the HERV-K (HML-2) human-specific subfamily of endogenous retroviruses. Surprisingly, the transcription was initiated predominantly from the very 3' termini of the LTR R regions. The data presented here may shed light on adaptive coevolution of human endogenous retroviruses with their host cells.

  18. Buzdin A., Kovalskaya-Alexandrova E., Gogvadze E., Sverdlov E. (2006). GREM, a technique for genome-wide isolation and quantitative analysis of promoter active repeats. Nucleic Acids Res. 34 (9), e67 [+]

    We developed a technique called GREM (Genomic Repeat Expression Monitor) that can be applied to genome-wide isolation and quantitative analysis of any kind of transcriptionally active repetitive elements. Briefly, the technique includes three major stages: (i) generation of a transcriptome wide library of cDNA 5' terminal fragments, (ii) selective amplification of repeat-flanking genomic loci and (iii) hybridization of the cDNA library (i) to the amplicon (ii) with subsequent selective amplification and cloning of the cDNA-genome hybrids. The sequences obtained serve as 'tags' for promoter active repetitive elements. The advantage of GREM is an unambiguous mapping of individual promoter active repeats at a genome-wide level. We applied GREM for genome-wide experimental identification of human-specific endogenous retroviruses and their solitary long terminal repeats (LTRs) acting in vivo as promoters. Importantly, GREM tag frequencies linearly correlated with the corresponding LTR-driven transcript levels found using RT-PCR. The GREM technique enabled us to identify 54 new functional human promoters created by retroviral LTRs.

  19. Mamedov I., Batrak A., Buzdin A., Arzumanyan E., Lebedev Y., Sverdlov E.D. (2002). Genome-wide comparison of differences in the integration sites of interspersed repeats between closely related genomes. Nucleic Acids Res. 30 (14), e71 [+]

    New whole-genome approach to comparison of LTR distribution in genomes of related species was presented. The eleven LTR insertions distinguishing human genome from chimpanzee ones were described at the first time.

  20. Buzdin A., Khodosevich K., Mamedov I., Vinogradova T., Lebedev Y., Hunsmann G., Sverdlov E. (2002). A technique for genome-wide identification of differences in the interspersed repeats integrations between closely related genomes and its application to detection of human-specific integrations of HERV-K LTRs. Genomics 79 (3), 413–22 [+]

    We have developed a method of targeted genomic difference analysis (TGDA) for genomewide detection of interspersed repeat integration site differences between closely related genomes. The method includes a whole-genome amplification of the flanks adjacent to target interspersed repetitive elements in both genomic DNAs under comparison, and subtractive hybridization (SH) of the selected amplicons. The potential of TGDA was demonstrated by the detection of differences in the integration sites of human endogenous retroviruses K (HERV-K) and related solitary long terminal repeats (LTRs) between the human and chimpanzee genomes. Of 55 randomly sequenced clones from a library enriched with human-specific integration (HSI) sites, 33 (60%) represented HSIs. All the human-specific (Hs) LTRs belong to two related evolutionarily young groups, suggesting simultaneous activity of two master genes in the hominid lineage. No deletion/insertion polymorphism was detected for the LTR HSIs for 25 unrelated caucasoid individuals. We also discuss the possible research applications for TGDA research.

  21. Sverdlov E.D. (2000). Retroviruses and primate evolution. BioEssays 22 (2), 161–171 [+]

    Human endogenous retroviruses (HERVs), probably representing footprints of ancient germ-cell retroviral infections, occupy about 1% of the human genome. HERVs can influence genome regulation through expression of retroviral genes, either via genomic rearrangements following HERV integrations or through the involvement of HERV LTRs in the regulation of gene expression. Some HERVs emerged in the genome over 30 MYr ago, while others have appeared rather recently, at about the time of hominid and ape lineages divergence. HERVs might have conferred antiviral resistance on early human ancestors, thus helping them to survive. Furthermore, newly integrated HERVs could have changed the pattern of gene expression and therefore played a significant role in the evolution and divergence of Hominoidea superfamily. Comparative analysis of HERVs, HERV LTRs, neighboring genes, and their regulatory interplay in the human and ape genomes will help us to understand the possible impact of HERVs on evolution and genome regulation in the primates.


Eugeny Sverdlov

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