RESUMO
Single nucleotide polymorphisms (SNPs) are the most common type of variation in the human genome. The vast majority of SNPs identified in the human genome do not have any effect on the phenotype; however, some can lead to changes in the function of a gene or the level of its expression. Most SNPs associated with certain traits or pathologies are mapped to regulatory regions of the genome and affect gene expression by changing transcription factor binding sites. In recent decades, substantial effort has been invested in searching for such regulatory SNPs (rSNPs) and understanding the mechanisms by which they lead to phenotypic differences, primarily to individual differences in susceptibility to diseases and in sensitivity to drugs. The development of the NGS (next-generation sequencing) technology has contributed not only to the identification of a huge number of SNPs and to the search for their association (genome-wide association studies, GWASs) with certain diseases or phenotypic manifestations, but also to the development of more productive approaches to their functional annotation. It should be noted that the presence of an association does not allow one to identify a functional, truly disease-associated DNA sequence variant among multiple marker SNPs that are detected due to linkage disequilibrium. Moreover, determination of associations of genetic variants with a disease does not provide information about the functionality of these variants, which is necessary to elucidate the molecular mechanisms of the development of pathology and to design effective methods for its treatment and prevention. In this regard, the functional analysis of SNPs annotated in the GWAS catalog, both at the genome-wide level and at the level of individual SNPs, became especially relevant in recent years. A genome-wide search for potential rSNPs is possible without any prior knowledge of their association with a trait. Thus, mapping expression quantitative trait loci (eQTLs) makes it possible to identify an SNP for which - among transcriptomes of homozygotes and heterozygotes for its various alleles - there are differences in the expression level of certain genes, which can be located at various distances from the SNP. To predict rSNPs, approaches based on searches for allele-specific events in RNA-seq, ChIP-seq, DNase-seq, ATAC-seq, MPRA, and other data are also used. Nonetheless, for a more complete functional annotation of such rSNPs, it is necessary to establish their association with a trait, in particular, with a predisposition to a certain pathology or sensitivity to drugs. Thus, approaches to finding SNPs important for the development of a trait can be categorized into two groups: (1) starting from data on an association of SNPs with a certain trait, (2) starting from the determination of allele-specific changes at the molecular level (in a transcriptome or regulome). Only comprehensive use of strategically different approaches can considerably enrich our knowledge about the role of genetic determinants in the molecular mechanisms of trait formation, including predisposition to multifactorial diseases.
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Cardiovascular diseases (CVDs), the leading cause of death worldwide, generally refer to a range of pathological conditions with the involvement of the heart and the blood vessels. A sizable fraction of the susceptibility loci is known, but the underlying mechanisms have been established only for a small proportion. Therefore, there is an increasing need to explore the functional relevance of trait-associated variants and, moreover, to search for novel risk genetic variation. We have reported the bioinformatic approach allowing effective identif ication of functional non-coding variants by integrated analysis of genome-wide data. Here, the analysis of 1361 previously identif ied regulatory SNPs (rSNPs) was performed to provide new insights into cardiovascular risk. We found 773,471 coding co-segregating markers for input rSNPs using the 1000 Genomes Project. The intersection of GWAS-derived SNPs with a relevance to cardiovascular traits with these markers was analyzed within a window of 10 Kbp. The effects on the transcription factor (TF) binding sites were explored by DeFine models. Functional pathway enrichment and protein-protein interaction (PPI) network analyses were performed on the targets and the extended genes by STRING and DAVID. Eighteen rSNPs were functionally linked to cardiovascular risk. A signif icant impact on binding sites of thirteen TFs including those involved in blood cells formation, hematopoiesis, macrophage function, inf lammation, and vasoconstriction was found in K562 cells. 21 rSNP gene targets and 5 partners predicted by PPI were enriched for spliceosome and endocytosis KEGG pathways, endosome sorting complex and mRNA splicing REACTOME pathways. Related Gene Ontology terms included mRNA splicing and processing, endosome transport and protein catabolic processes. Together, the f indings provide further insight into the biological basis of CVDs and highlight the importance of the precise regulation of splicing and alternative splicing.
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The most popular model for the search of ChIP-seq data for transcription factor binding sites (TFBS) is the positional weight matrix (PWM). However, this model does not take into account dependencies between nucleotide occurrences in different site positions. Currently, two recently proposed models, BaMM and InMoDe, can do as much. However, application of these models was usually limited only to comparing their recognition accuracies with that of PWMs, while none of the analyses of the co-prediction and relative positioning of hits of different models in peaks has yet been performed. To close this gap, we propose the pipeline called MultiDeNA. This pipeline includes stages of model training, assessing their recognition accuracy, scanning ChIP-seq peaks and their classification based on scan results. We applied our pipeline to 22 ChIP-seq datasets of TF FOXA2 and considered PWM, dinucleotide PWM (diPWM), BaMM and InMoDe models. The combination of these four models allowed a significant increase in the fraction of recognized peaks compared to that for the sole PWM model: the increase was 26.3 %. The BaMM model provided the main contribution to the recognition of sites. Although the major fraction of predicted peaks contained TFBS of different models with coincided positions, the medians of the fraction of peaks containing the predictions of sole models were 1.08, 0.49, 4.15 and 1.73 % for PWM, diPWM, BaMM and InMoDe, respectively. Thus, FOXA2 BSs were not fully described by only a sole model, which indicates theirs heterogeneity. We assume that the BaMM model is the most successful in describing the structure of the FOXA2 BS in ChIP-seq datasets under study.
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Chronic stress is the leading risk factor of a broad range of severe psychopathologies. Nonetheless, the molecular mechanisms triggering these pathological processes are not well understood. In our study, we investigated the effects of 15-day social defeat stress (SDS) on the genome-wide landscape of trimethylation at the 4th lysine residue of histone H3 (H3K4me3) and on the transcriptome in the prefrontal cortex of mice that were reared normally (group SDS) or subjected to maternal separation early in life (group MS+SDS). The mice with the history of stress early in life showed increased susceptibility to SDS in adulthood and demonstrated long-lasting genome-wide alterations in gene expression and splicing as well as in the H3K4me3 epigenetic landscape in the prefrontal cortex. Thus, the high-throughput techniques applied here allowed us to simultaneously detect, for the first time, genome-wide epigenetic and transcriptional changes in the murine prefrontal cortex that are associated with both chronic SDS and increased susceptibility to this stressor.
Assuntos
Epigênese Genética/fisiologia , Histonas/metabolismo , Privação Materna , Córtex Pré-Frontal/metabolismo , Derrota Social , Estresse Psicológico/metabolismo , Fatores Etários , Processamento Alternativo/fisiologia , Animais , Corticosterona/genética , Corticosterona/metabolismo , Feminino , Expressão Gênica , Histonas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Psicológico/genética , Estresse Psicológico/psicologiaRESUMO
H3K4me3 is typically found in the promoter region of genes and is a mark associated with an open chromatin state and active gene transcription. Nonetheless, the role of H3K4me3 in the regulation of transcription is still debated. To improve the understanding of the connection between H3K4me3 density in promoters and gene expression, we assessed the correlation between these two parameters. We utilized genome-wide high-throughput RNA sequencing (RNA-seq) data and H3K4me3-based chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq), carried out on the same samples of the prefrontal cortex from 10 male C57Bl6 mice with different stress experience [Social defeat stress in adult mice causes alterations in gene expression, alternative splicing, and the epigenetic landscape of H3K4me3 in the prefrontal cortex: an impact of early-life stress, 1]. In addition, we assessed the correlation between H3K4me3 density and gene expression in datasets of cell-specific genes. Altogether, the results are useful for the elucidation of H3K4me3 involvement in the regulation of transcription in the murine prefrontal cortex.
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We analyzed association of potentially regulatory polymorphisms (rs590352, rs11542583, rs3829202, rs207258, and rs4796672) with breast cancer. A significant association was found between this disease and rs2072580T>A (p=0.001) located in the overlapping promoter regions of the SART3 and ISCU genes. In women with AA and AT genotypes, the risk of breast cancer is higher by 6.7 times (p=0.001) and 12 times (p=0.001), respectively, in comparison with TT genotype. Under a codominant model of inheritance (AT vs AA+TT), the risk of breast cancer was increased by 4.2 times (Ñ=0.001) for the AT genotype. Under a recessive model of inheritance (TT vs AA+TT), the risk of disease was 10-fold higher (Ñ=0.001) for the TT genotype. It has been demonstrated that the T>A substitution affects the binding properties of transcription factors CREB1 and REST.
Assuntos
Antígenos de Neoplasias/genética , Neoplasias da Mama/genética , Proteínas Ferro-Enxofre/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Proteínas de Ligação a RNA/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/epidemiologia , Estudos de Casos e Controles , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Pessoa de Meia-Idade , Fatores de Risco , Federação Russa/epidemiologia , Adulto JovemRESUMO
Exposure to stress activates the hypothalamic-pituitary-adrenal axis and leads to increased levels of glucocorticoid (GC) hormones. Prolonged elevation of GC levels causes neuronal dysfunction, decreases the density of synapses, and impairs neuronal plasticity. Decreased sensitivity to glucocorticoids (glucocorticoid resistance) that develops as a result of chronic stress is one of the characteristic features of stress-induced psychopathologies. In this article, we reviewed the published data on proposed molecular mechanisms that contribute to the development of glucocorticoid resistance in brain, including changes in the expression of the glucocorticoid receptor (GR) gene, biosynthesis of GR isoforms, and GR posttranslational modifications. We also present data on alterations in the expression of the FKBP5 gene encoding the main component of cell ultra-short negative feedback loop of GC signaling regulation. Recent discoveries on stress- and GR-induced changes in epigenetic modification patterns as well as normalizing action of antidepressants are discussed. GR and FKBP5 gene polymorphisms associated with stress-induced psychopathologies are described, and their role in glucocorticoid resistance is discussed.
Assuntos
Glucocorticoides/metabolismo , Transtornos Mentais/metabolismo , Transdução de Sinais , Estresse Psicológico/metabolismo , Animais , Humanos , Transtornos Mentais/genética , Transtornos Mentais/fisiopatologia , Polimorfismo Genético , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Estresse Psicológico/genética , Estresse Psicológico/fisiopatologia , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Steroidogenic factor 1 (SF-1) belongs to a small group of the transcription factors that bind DNA only as a monomer. Three different approaches-Sitecon, SiteGA, and oPWM-constructed using the same training sample of experimentally confirmed SF-1 binding sites have been used to recognize these sites. The appropriate prediction thresholds for recognition models have been selected. Namely, the thresholds concordant by false positive or negative rates for various methods were used to optimize the discrimination of steroidogenic gene promoters from the datasets of non-specific promoters. After experimental verification, the models were used to analyze the ChIP-seq data for SF-1. It has been shown that the sets of sites recognized by different models overlap only partially and that an integration of these models allows for identification of SF-1 sites in up to 80% of the ChIP-seq loci. The structures of the sites detected using the three recognition models in the ChIP-seq peaks falling within the [-5000, +5000] region relative to the transcription start sites (TSS) extracted from the FANTOM5 project have been analyzed. The MATLIGN classified the frequency matrices for the sites predicted by oPWM, Sitecon, and SiteGA into two groups. The first group is described by oPWM/Sitecon and the second, by SiteGA. Gene ontology (GO) analysis has been used to clarify the differences between the sets of genes carrying different variants of SF-1 binding sites. Although this analysis in general revealed a considerable overlap in GO terms for the genes carrying the binding sites predicted by oPWM, Sitecon, or SiteGA, only the last method elicited notable trend to terms related to negative regulation and apoptosis. The results suggest that the SF-1 binding sites are different in both their structure and the functional annotation of the set of target genes correspond to the predictions by oPWM+Sitecon and SiteGA. Further application of Homer software for de novo identification of enriched motifs in ChIP-Seq data for SF-1ChIP-seq dataset gave the data similar to oPWM+Sitecon.
Assuntos
Fator Esteroidogênico 1/metabolismo , Animais , Sítios de Ligação , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Masculino , Ratos , Ratos Wistar , Fator Esteroidogênico 1/químicaRESUMO
The regulatory region of the Trl gene was analyzed using the mutation Trl 3609 , resulting from the insertion of the P-element into the promoter region of the gene as well as mutations obtained on its basis. It is shown that two last transcription start sites, which are most often used in vitro in S2 cells, are almost not used in vivo. Experimental data indicate that transcription terminators in transposons play an important role in the decrease in the transcription level of the recipient gene.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tecido Adiposo/metabolismo , Alelos , Animais , Northern Blotting , Linhagem Celular , Elementos de DNA Transponíveis , Drosophila melanogaster , Gânglios dos Invertebrados/metabolismo , Regulação da Expressão Gênica , Técnicas Genéticas , Discos Imaginais/metabolismo , Larva , Mutagênese Insercional , Regiões Promotoras Genéticas , Glândulas Salivares/metabolismoRESUMO
Key aspects of gene transcription regulation in multicellular organisms, including the characteristics of their promoters, transcription-factor binding sites, and composition elements are reviewed. The functional role of transcription regulatory proteins (basal factors and regulatory transcription factors), and the mechanisms responsible for regulation of their activity are also discussed. Furthermore, we describe the importance of DNA-encoded nucleosome organization and chromatin modifications in the course of transcription regulation, as well as some mechanisms that regulate the activity of transcription factors associated with genetic networks. The current outlook on regulatory gene expression codes in eukaryotes is presented.
Assuntos
Regulação da Expressão Gênica , Genoma , Transcrição Gênica , Animais , Cromatina/metabolismo , Eucariotos/genética , Humanos , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The transcription factor GAGA, encoded by the gene Trl, controls expression of many Drosophila melanogaster genes. We have compiled the presently largest sample (120 sites) of published nucleotide sequences with experimentally confirmed binding to GAGA protein. Analysis of the sample has demonstrated that despite an apparent structural diversity of the GAGA sites, they fall into four distinct groups, namely, (1) the sites containing two GAG trinucleotides with no more than one nucleotide substitution in each and separated by spacers with a length of 1 or 3 nucleotides (GAGnGAG and GAGnnnGAG); (2) the sites containing a single GAGAG motif; (3) (GA)(3-9) microsatellite repeats; and (4) the sites corresponding to three and more direct repeats of GAG trinucleotide homolog and its inverted repeats separated by spacers of various lengths. Using the software package SITECON, the methods were elaborated for recognizing the sites of GAGnGAG (method 1) and GAGnnnGAG (method 2) types in DNA sequences. Experimental verification confirmed the ability to interact with the GAGA factor for 72% of the sites predicted using method 1 and 94.5% of the sites predicted by method 2. Application of the experimentally verified methods to analyzing the localization of potential GAGA binding sites in the target genes of this transcription factor has demonstrated that the 5'-untranslated regions (5'UTRs) and first introns are enriched for these sites (two-threefold relative to the average occurrence frequency in the D. melanogaster genome) as compared with a moderate enrichment (not exceeding 1.5-fold) of promoter regions (-4000/+200 bp or -1000/+100 bp).
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes de Insetos/genética , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Regiões 5' não Traduzidas/genética , Animais , Sequência de Bases , Sítios de Ligação , DNA/genética , Proteínas de Drosophila/metabolismo , Íntrons/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/genéticaAssuntos
Proteínas da Membrana Bacteriana Externa/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Receptores de Superfície Celular/genética , Proteínas da Membrana Bacteriana Externa/química , Sequência de Bases/genética , Sítios de Ligação/genética , Ligação Proteica/genética , Receptores de Superfície Celular/química , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Transcription factors of the FoxA family (forkhead box A) regulate cell metabolism and differentiation and maintain specificity of liver cell proteome and phenotype of mature hepatocytes. The relationship between hepatocarcinogenicity of azo compounds o-aminoazotoluene (OAT) and 3'-methyl-4-dimethylaminobenzene (3'MeDAB) for GR mice and one of the early events, modulation of the DNA-binding activity of FoxA transcription factor, was studied. Single injection of 3'MeDAB to 12-day-old mice caused liver tumors in 100% males and females similarly as OAT, a well-known mouse hepatocarcinogene. The DNA-binding activity of FoxA in the liver decreased 2.5-3 times by OAT, this resulting in a 40% reduction of glucocorticoid induction of tyrosine aminotransferase (liver-specific gene). In contrast to these, 3'MeDAB did not modify FoxA protein activities or the degree of glucocorticoid induction of tyrosine aminotransferase.
Assuntos
Compostos Azo/toxicidade , Derivados de Benzeno/toxicidade , Fatores Nucleares de Hepatócito/metabolismo , Neoplasias Hepáticas/induzido quimicamente , o-Aminoazotolueno/toxicidade , Animais , Compostos Azo/farmacologia , Derivados de Benzeno/farmacologia , Feminino , Glucocorticoides , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas c-ets/metabolismo , Ativação Transcricional/efeitos dos fármacos , o-Aminoazotolueno/farmacologiaRESUMO
The carcinogenic effects of estragole in mice of the earlier unexplored strain ICR has been studied. It has been shown that there is a distinct correlation between the extent of inhibition of glucocorticoid-mediated induction of tyrosine aminotransferase and trypthophan oxygenase after acute administration of estragole and the frequency of liver tumors after estragole exposure. Estragole inhibits the induction of these enzymes only in female mice, but not in male mice and rats. DNA-binding activities of liver-enriched transcription factors were investigated on carcinogen-susceptible and -resistant animals. Estragole decreases the HNF4 (hepatic nuclear factor 4) and FOXA DNA-binding activities only in susceptible female mice, but not in nonsusceptible male mice and rats and does not influence the C/EBP and HNF1 activities. Pentachlorophenol, which prevents the hepatocarcinogenic effect of estragole, abolishes its inhibitory effect on tyrosine aminotransferase and trypthophan oxygenase glucocorticoid induction and restores the FOXA and HNF4 DNA-binding activities. The parallelism between the hepatocarcinogenic effects of estragole and the inhibition of FOXA and HNF4 DNA-binding activities serves as an additional argument for the involvement of these factors in the mechanisms of tumor suppression in the liver.
Assuntos
Anisóis/toxicidade , Carcinógenos/toxicidade , Glucocorticoides/farmacologia , Fator 4 Nuclear de Hepatócito/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/enzimologia , Proteínas de Neoplasias/metabolismo , Derivados de Alilbenzenos , Animais , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Neoplasias Hepáticas/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos ICR , Especificidade de Órgãos/efeitos dos fármacos , Pentaclorofenol/farmacologia , Ratos , Ratos Wistar , Caracteres SexuaisAssuntos
Predisposição Genética para Doença , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , o-Aminoazotolueno/farmacologia , Alanina Transaminase/sangue , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Hepatectomia , Hepatócitos/metabolismo , Hepatócitos/patologia , Fígado/anatomia & histologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Regeneração Hepática/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Tamanho do Órgão/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
It is known that the carcinogenic effect of estragole, a component of essential oils of many spicy plants, is characterized by species, tissue, and sex specificity. It causes mainly liver tumors in female mice but is not carcinogenic for male mice and for rats. In this work, the estragole hepatocarcinogenicity was shown for female mice of previously not studied ICR line. The strict correlation between estragole hepatocarcinogenicity and its ability to decrease the level of glucocorticoid induction of liver-specific enzymes tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) was found. Inhibition of TAT and TO inducibility by estragole takes place only in female mice but not in male mice and in rats. Studying the estragole effect on DNA-binding activity of transcription factors, present mainly in liver and regulating expression of genes encoding liver-specific proteins, has shown that estragole decreases FOXA and HNF4 activities but not activities of C/EBP and HNF1, and this happens only in female mice, for which this substance is hepatocarcinogen, but not in male mice and in rats. Pentachlorophenol, preventing hepatocarcinogenic effect of estragole, abolishes inhibitory influence of the latter on the TAT and TO glucocorticoid induction and restores DNA-binding activity of FOXA and HNF4. Thus, a correlation was revealed between the estragole hepatocarcinogenic effect and decrease in DNA-binding activity of transcription factors FOXA and HNF4, which might be indicative of the role of these factors in tumor suppression mechanisms in liver.
Assuntos
Anisóis/farmacologia , Carcinógenos/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Glucocorticoides/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Derivados de Alilbenzenos , Animais , Feminino , Fatores de Transcrição Forkhead/genética , Fator 4 Nuclear de Hepatócito/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Ratos , Ratos Wistar , Caracteres Sexuais , Especificidade da Espécie , Triptofano Oxigenase/metabolismo , Tirosina Transaminase/metabolismoRESUMO
To reveal the mechanism of tumor-suppressing activity of FOXA proteins in liver, a search for potential target genes of these transcription factors involved in proliferation control was carried out. In the first step, we have used data from the literature concerning gene expression in mouse liver (high content of FOXA proteins) and kidney (FOXA expression is absent) obtained by hybridization on microchips. A search for FOXA binding sites in regulatory regions of forty differentially expressing genes involved in proliferation control was carried out using the computer method SITECON. Eleven genes containing clusters of potential FOXA sites incorporating 3-6-fold repeats of TTTG were revealed. The FOXA-specific interaction with such microsatellite sites was confirmed by gel-retardation technique using the GST-fused protein containing the DNA-binding domain of FOXA2. Six genes containing clusters of confirmed binding sites--Cul2, Cdc73, Ptk, Pdcd, Creb, and Ppp2r5d--were selected. The effect of hepatocarcinogen orthoaminoazotoluene (OAT), which lowers the FOXA activity, on expression of these genes was studied by the real-time PCR. OAT was shown to increase sharply the level of mRNA of the Cul2 and Cdc73 genes.
Assuntos
Expressão Gênica , Fatores Nucleares de Hepatócito/metabolismo , Fígado/metabolismo , Animais , Sítios de Ligação , Carcinógenos/farmacologia , Proliferação de Células , Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Camundongos , RNA Mensageiro/biossíntese , Elementos Reguladores de Transcrição , o-Aminoazotolueno/farmacologiaRESUMO
Using electrophoretic mobility shift assay we first revealed in the nuclear extracts of the rodent malaria parasite Plasmodium berghei (P. berghei) proteins, which bind specifically to the double-stranded oligonucleotides reproducing the binding sites of the transcription factors of AP1 family, NF-IL6 and SP1 involved in the up-regulation of human multidrug resistance (mdr1) gene and to the oligonucleotide corresponding to the element responsive for the stimulation by serum (SRE). The nuclear proteins isolated from the P. berghei strains with various chloroquine sensitivity bound differently to the most of the oligonucleotide probes used. Mutations in the consensus sequences of AP1, NF-IL6 and SRE led to the loss of some DNA-protein complexes, suggesting the existence of malaria parasite nuclear proteins, whose DNA-binding domains are similar to DNA-binding domains of NF-IL6, SRF1, and AP1 family members. These proteins exhibit greater activities in chloroquine resistant strains. The results obtained denote profound alterations in the plasmodium regulatory apparatus occurred as the result of selection on chloroquine resistance.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , DNA/química , Resistência a Medicamentos/genética , Proteínas Nucleares/química , Plasmodium berghei/metabolismo , Proteínas de Protozoários/química , Animais , Antimaláricos/farmacologia , Cloroquina/farmacologia , Sequência Consenso , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Mutação , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Oligonucleotídeos/química , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Elementos Reguladores de Transcrição/genética , Fatores de Transcrição/metabolismoAssuntos
Algoritmos , Sequências Reguladoras de Ácido Nucleico , Fator Esteroidogênico 1/metabolismo , Fatores de Transcrição/metabolismo , Interface Usuário-Computador , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Bases de Dados Factuais , Internet , Conformação de Ácido Nucleico , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ratos , Alinhamento de Sequência , Análise de Sequência de DNA , Fator Esteroidogênico 1/genética , Suínos , Fatores de Transcrição/genéticaRESUMO
Selective increase of DNA-binding activity of constitutive androstane receptor was detected in rat and mouse liver in response to aminoazo dyes exhibiting hepatocarcinogenic activity for these species (ortho-aminoazotoluene for mice and 3'-methyl-4-dimethylaminobenzene for rats). Competition of azo dyes with 3H-5alpha-androst-16-ene-3alpha-ol (a well-known ligand of constitutive androstane receptor) for binding to liver cell cytosol proteins was studied. Ortho-aminoazotoluene and 3'-methyl-4-dimethylaminobenzene were better competitors for cytosol proteins from mouse and rat liver, respectively.