Biomedical subjects

Irina A Udalova

Publications and source records attributed to Irina A Udalova.

9 recordsLinked to original sources

PubMed2006

Localization of a long-range cis-regulatory element of IL13 by allelic transcript ratio mapping.

It appears that, for many genes, the two alleles possessed by an individual may produce different amounts of transcript. When such allelic differences in transcription are observed for some individuals but not others, a plausible explanation is genetic variation in the cis-acting elements that regulate the gene in question. Here we describe a novel analytical approach that uses such observations, combined with genotyping data from the HapMap project, to define the genomic location of cis-acting regulatory elements. When applied to the human 5q31 chromosomal region, where complex regulatory mechanisms are known to exist, we demonstrate the sensitivity of this approach by locating a highly significant cis-regulatory element operating on IL13 at long range from a position 250 kb upstream from the gene (P = 2 x 10(-6)). As this method is unaffected by other sources of variation, such as environmental and trans-acting genetic factors, it provides a tractable approach for dissecting the complexities of genetic variation in gene regulation.

Alleles↗

PubMed2006

Context-specific functional effects of IFNGR1 promoter polymorphism.

We report evidence of a polymorphism in the promoter region of IFNGR1 (encoding interferon-gamma receptor 1) that has opposite functional effects in different cellular contexts. It is a deletion/insertion polymorphism that is found in Africans but not Europeans or Asians, and has been associated with resistance to severe malaria. We find that the IFNGR1-470del allele acts to suppress binding of nuclear proteins to the IFNGR1 promoter region in a manner that is specific for cell type. In B-lymphocytes, the IFNGR1-470del allele suppresses the binding of a approximately 35 kDa nuclear protein and acts to increase reporter gene expression. In epithelial cells, the same allele acts to decrease gene expression and suppresses the binding of approximately 90 kDa STAT-1 and STAT-2 proteins. In T-lymphocytes, this allele causes only subtle differences in nuclear protein binding and has no significant effect on gene expression. These findings suggest a mechanism by which a single genetic variant may cause a broad range of phenotypic consequences.

Cell Line, Tumor↗

PubMed2006

Factor H, a regulator of complement activity, is a major determinant of meningococcal disease susceptibility in UK Caucasian patients.

Defence against Neisseria meningitidis involves complement-mediated bactericidal activity. Factor H (fH) down-regulates complement activation. A putatively functional single-nucleotide-polymorphism (SNP) exists within a presumed nuclear-factor-kappa-B responsive element (NF-kB) in the fH gene (C-496T). Genetic and functional investigations were carried out to determine whether C-496T has a role in meningococcal disease (MD) susceptibility. Genetic susceptibility was investigated in 2 independent studies, a case-control and family-based transmission-disequilibrium-test (TDT), using 2 separate cohorts of UK Caucasian patients. MD susceptibility was both genetically associated with the C/C homozygous genotype (OR = 2.0, 95% CI 1.3 - 3.2, p = 0.001) and linked to the C allele (p = 0.04), the association being most significant in serogroup C infected patients (OR = 2.9, 95% CI 1.6 - 5.5, p = 0.0002). FH serum concentrations were also associated with C-496T genotype, with highest fH concentrations in C/C homozygous individuals (p = 0.01). Functional studies showed NF-kappa-B binding to the C-496T-containing region and that pre-incubation of fH with meningococci reduced bactericidal activity and increased meningococci B and C survival in blood. This study shows that C-496T is both associated and linked with MD and that individuals possessing the fH C-496T C/C genotype are more likely to have increased serum fH protein levels, have reduced bactericidal activity against meningococci and be at an increased risk of contracting MD.

Adolescent↗

PubMed2006

Quantitative profiling of protein-DNA binding on microarrays.

Recent studies on genome-wide localization of transcription factor (TF) binding to DNA have shown that a large proportion of identified sequences do not contain consensus motifs predicted by databases of transcription factor binding sites, such as TRANSFAC. The main limitation of these databases is that they are based on a literature search of published examples of binding; consequently the data are not from a systematic survey and may be subject to sampling biases if investigators focused on particular motifs. Thus, there is an urgent need for systematic profiling of vertebrate transcription factor binding to DNA. We have developed a high-throughput platform for the quantitative analysis of protein-DNA interactions based on microarray technology.

Base Sequence↗

PubMed2004

Quantitative high-throughput analysis of transcription factor binding specificities.

We present a general high-throughput approach to accurately quantify DNA-protein interactions, which can facilitate the identification of functional genetic polymorphisms. The method tested here on two structurally distinct transcription factors (TFs), NF-kappaB and OCT-1, comprises three steps: (i) optimized selection of DNA variants to be tested experimentally, which we show is superior to selecting variants at random; (ii) a quantitative protein-DNA binding assay using microarray and surface plasmon resonance technologies; (iii) prediction of binding affinity for all DNA variants in the consensus space using a statistical model based on principal coordinates analysis. For the protein-DNA binding assay, we identified a polyacrylamide/ester glass activation chemistry which formed exclusive covalent bonds with 5'-amino-modified DNA duplexes and hindered non-specific electrostatic attachment of DNA. Full accessibility of the DNA duplexes attached to polyacrylamide-modified slides was confirmed by the high degree of data correlation with the electromobility shift assay (correlation coefficient 93%). This approach offers the potential for high-throughput determination of TF binding profiles and predicting the effects of single nucleotide polymorphisms on TF binding affinity. New DNA binding data for OCT-1 are presented.

Acrylic Resins↗

PubMed2003

Comparing the fine specificity of DNA binding by NF-kappaB p50 and p52 using principal coordinates analysis.

Principal coordinates analysis has been proposed as an efficient way of predicting the binding affinity of a transcription factor to different DNA motifs, as it can model complex interactions that are difficult to represent with standard position-weight matrices. Here we evaluate its ability to distinguish the DNA binding properties of two closely related proteins, the homodimeric forms of NF-kappaB p50 and p52. When tested experimentally against 50 different variants of the generalised NF-kappaB motif GGRRNNYYCC, the binding specificities of p50 and p52 were similar but not identical (correlation rho = 0.86). These experimental data can be modelled accurately with six principal coordinates that are similar for p50 and p52, plus one principal coordinate that is significantly stronger for p52 than for p50, relating to the inner positions of the binding site. These findings are compatible with crystallographic data showing that p52 has greater ability than p50 to form water molecule-mediated hydrogen bonds with inner nucleotide positions of the binding site.

Base Sequence↗

PubMed2002

Quantitative prediction of NF-kappa B DNA-protein interactions.

We describe a general method based on principal coordinates analysis to predict the effects of single-nucleotide polymorphisms within regulatory sequences on DNA-protein interactions. We use binding data for the transcription factor NF-kappaB as a test system. The method incorporates the effects of interactions between base pair positions in the binding site, and we demonstrate that such interactions are present for NF-kappaB. Prediction accuracy is higher than with profile models, confirmed by crossvalidation and by the experimental verification of our predictions for additional sequences. The binding affinities of all potential NF-kappaB sites on human chromosome 22, together with the effects of known single-nucleotide polymorphisms, are calculated to determine likely functional variants. We propose that this approach may be valuable, either on its own or in combination with other methods, when standard profile models are disadvantaged by complex internucleotide interactions.

Base Sequence↗

PubMed2002

Inflammatory bowel disease is associated with a TNF polymorphism that affects an interaction between the OCT1 and NF(-kappa)B transcription factors.

Tumour necrosis factor-alpha (TNF) expression is increased in inflammatory bowel disease (IBD), and TNF maps to the IBD3 susceptibility locus. Transmission disequilibrium and case-control analyses, in two independent Caucasian cohorts, showed a novel association of the TNF(-857C) promoter polymorphism with IBD (overall P=0.001 in 587 IBD families). Further genetic associations of TNF(-857C) with IBD sub-phenotypes were seen for ulcerative colitis and for Crohn's disease, but only in patients not carrying common NOD2 mutations. The genetic data suggest a recessive model of inheritance, and we observed ex vivo lipopolysaccharide-stimulated whole-blood TNF production to be higher in healthy TNF(-857C) homozygotes. We show the transcription factor OCT1 binds TNF(-857T) but not TNF(-857C), and interacts in vitro and in vivo with the pro-inflammatory NF(-kappa)B transcription factor p65 subunit at an adjacent binding site. Detailed functional analyses of these interactions in gut macrophages, in addition to further genetic mapping of this gene-dense region, will be critical to understand the significance of the observed association of TNF(-857C) with IBD.

Animals↗

PubMed2002

Evidence for a dual mechanism for IL-10 suppression of TNF-alpha production that does not involve inhibition of p38 mitogen-activated protein kinase or NF-kappa B in primary human macrophages.

IL-10 is a potent anti-inflammatory cytokine and inhibitor of TNF-alpha production. The molecular pathways by which IL-10 inhibits TNF-alpha production are obscure, with diverse mechanisms having been published. In this study, a new approach has been taken for the study of human cells. Adenovirus was used to deliver TNF-alpha promoter-based luciferase reporter genes to primary human monocytic cells. The reporter genes were highly responsive to macrophage activation and appeared to mirror the behavior of the endogenous TNF-alpha gene. When added, either with or after the stimulus, IL-10 required the 3' untranslated region of the TNF-alpha gene to inhibit luciferase mRNA and protein expression, indicating a posttranscriptional mechanism. However, if macrophages were incubated with IL-10 before activation, inhibition of gene expression was also mediated by the 5' promoter, suggesting a transcriptional mechanism. To our knowledge, this is the first time that a dual mechanism for IL-10 function has been demonstrated. Studies to elucidate the mechanisms underlying the inhibition of TNF-alpha production addressed the effect of IL-10 on the activation of p38 mitogen-activated protein kinase and NF-kappaB. However, these studies could demonstrate no requirement for the inhibition of p38 mitogen-activated protein kinase or NF-kappaB activation as potential mechanisms. Overall, these results may explain the diversity previously ascribed to the complex mechanisms of IL-10 anti-inflammatory activity.

3' Untranslated Regions↗