ognizant Communication Corporation



Gene Expression, Vol. 12, pp. 1-11
1052-2166/04 $20.00 + .00
Copyright © 2004 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

A Promoter Polymorphism in the Central MHC Gene, IKBL, Influences the Binding of Transcription Factors USF1 and E47 on Disease-Associated Haplotypes

Alvin Boodhoo,1,3 Agnes M.-L. Wong,2,3 David Williamson,3 Dominic Voon,4 Silvia Lee,2,3 Richard J. N. Allcock,2,3 and Patricia Price2,3

1University of Mauritius, Reduit, Mauritius
2School of Surgery and Pathology, University of Western Australia, Nedlands 6009, Western Australia
3Department of Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, Perth 6001, Western Australia
4GeneStream Pty Ltd, Western Australia

The human major histocompatibility complex (MHC) contains genes that affect susceptibility to numerous immunopathological diseases. We propose that genes in the central MHC between TNFA and HLA-B explain associations between the 8.1 haplotype (HLA-A1, B8, DR3) and disease. IKBL encodes a protein resembling members of the IkB protein family that regulate bioavailability of NFkB. We have identified two polymorphisms in the 500 bp upstream of the transcription start site of IKBL that distinguish the 8.1 haplotype from the resistant 7.1 haplotype (HLA-A3, B7, DR15). A single nucleotide polymorphism at -62 disrupts a putative E-box binding sequence. To investigate binding of transcription factors in vitro, we exposed 32P-labeled DNA fragments carrying both alleles to nuclear extracts, showing allele-specific binding of nuclear proteins from Jurkat cells but not from other lineages. Supershift studies using Jurkat nuclear extract showed that the E-box protein, E47, and ubiquitously expressed transcription factor USF1 bind to the E-box element of the 7.1 haplotype. Transient transfections of luciferase reporter constructs carrying promoter alleles of IKBL into Jurkat cells showed an effect of IKBL-62 alleles. In contrast, alleles at -421 did not affect transcription factor binding or transcription. IKBL was expressed at low levels in Jurkat cells but not in blood mononuclear cells, and expression declined following mitogenic stimulation. The restriction of IKBL expression to Jurkat cells is consistent with evidence that E47 is expressed in thymocytes and suggests IKBL may affect autoimmunity through an effect on T-cell selection.

Keywords: IKBL; MHC; Promoter polymorphism; Transcription factors

Address correspondence to Dr. Patricia Price, Department of Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, Wellington Street, Perth 6001, Western Australia. Tel: +61 8 9224 0378; Fax: +61 8 9224 0204; E-mail: pprice@cyllene.uwa.edu.au

Gene Expression, Vol. 12, pp. 13-27
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Copyright © 2004 Cognizant Comm. Corp.
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Expression of the Peripheral Benzodiazepine Receptor Triggers Thymocyte Differentiation

Pierrick Rochard,* Sylvaine Galiegue,* Norbert Tinel, Annick Peleraux, Annie Bord, Omar Jbilo, and Pierre Casellas

Immunology-Oncology Department, Sanofi-Synthelabo Recherche, 371 rue du Professeur Joseph Blayac, 34184 Montpellier cedex 04, France

In the thymus, during T-cell differentiation, the expression of the peripheral benzodiazepine receptor (PBR) modulates. The protein level decreases between the double negative and double positive stages, and then increases when thymocytes become single positive. We addressed the role played by PBR in T-cell maturation. To this aim, we used Jurkat cells, which are immature T lymphocytes derived from an acute lymphoblastic leukemia. These cells are PBR negative and were stably transfected to achieve PBR levels similar to that in mature T cells. Using the DNA chip technology, we analyzed the PBR expression-dependent gene changes and evidenced that PBR-expressing cells exhibited more mature features than mock-transfected ones. A majority of the modulated genes encode proteins playing direct or indirect roles during the lymphocyte maturation process. In particular, PBR expression induced several differentiation markers (such as CD1, CD6), or key regulating elements (e.g., RAG1, RAG2, CD99, TCR). By contrast, some regulators of TCR signaling were reduced. PBR expression also affected the expression of critical apoptosis regulators: the proapoptotic lipocortin I, galectin-1, and galectin-9 were reduced while the antiapoptotic Bcl-2 was induced. Altogether our results supported the hypothesis that PBR controls T-cell maturation and suggested mechanisms through which PBR may regulate thymocyte-positive selection.

Key words: PBR; DNA chips; T lymphocytes; Cellular differentiation; Apoptosis

Address correspondence to Pierre Casellas, Immunology-Oncology Department, Sanofi-Synthelabo Recherche, 371 rue du Professeur Joseph Blayac, 34184 Montpellier cedex 04, France. Tel: 33 4 67 10 62 90; Fax: 33 4 67 10 60 00; E-mail: pierre.casellas@sanofi-synthelabo.com

*These two authors contributed equally to this work.

Gene Expression, Vol. 12, pp. 29-38
1052-2166/04 $20.00 + .00
Copyright © 2004 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Modulation of 3-Hydroxy-3-methylglutaryl-CoA Reductase Gene Expression by CuZn Superoxide Dismutase in Human Fibroblasts and HepG2 Cells

Bruna De Felice,1 Mariarosaria Santillo,2 Rosalba Serù,2 Simona Damiano,2 Gianfranco Matrone,2 Robert Roy Wilson,3 and Paolo Mondola2

1Department of Life Sciences, University of Naples 2, Via Vivaldi, 43, 81100, Caserta, Italy
2Department of Neuroscience, Unit of Physiology, University of Naples "Federico II," Via S. Pansini, 5, 80131, Naples, Italy
3NOAA, 325 Broadway, Boulder, CO

The homeostasis of intracellular cholesterol in animal cells is highly regulated by a complex system in which the microsomal rate-limiting enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase plays a key role in cholesterol synthesis. Substantial evidence has demonstrated that the cytosolic antioxidant enzyme CuZn superoxide dismutase (SOD1) inhibits the HMG-CoA reductase activity in rat hepatocytes and in human fibroblasts by decreasing cholesterol synthesis. Although these data suggest that SOD1 exerts a physiological role in cholesterol metabolism, it is still unclear whether the decrease of HMG-CoA reductase activity is mediated by transcriptional or by posttranscriptional events. The results of the present study, obtained by one-step RT-PCR assay, demonstrated that both SOD1 and the metal-free form of enzyme (Apo SOD1) inhibit HMG-CoA reductase gene expression in hepatocarcinoma HepG2 cells, in normal human fibroblasts, and in fibroblasts of subjects affected by familiar hypercholesterolemia. Accordingly, SOD1 could be used as a potential agent in the treatment of hypercholesterolemia, even in subjects lacking a functional LDL receptor pathway.

Key words: Cholesterol; CuZn superoxide dismutase; Familial hypercholesterolemia; 3-Hydroxy 3-methylglutaryl-CoA reductase; Human fibroblasts; HepG2 cells

Address correspondence to Prof. Paolo Mondola, Department of Neuroscience, Unit of Physiology, University of Naples "Federico II," Via S. Pansini, 5- 80131 Naples, Italy. Tel: +390817463225; Fax: +390817463639; E-mail: mondola@unina.it

Gene Expression, Vol. 12, pp. 39-47
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Copyright © 2004 Cognizant Comm. Corp.
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Gene Expression Profiling of Fibroblasts From a Human Progeroid Disease (Mandibuloacral Dysplasia, MAD #248370) Through cDNA Microarrays

Francesca Amati,1 Michela Biancolella,1 Maria Rosaria D'apice,1 Stefano Gambardella,1 Ruggiero Mango,1 Paolo Sbraccia,2 Monica D'adamo,2 Katia Margiotti,1 Annamaria Nardone,1 Marc Lewis,2 and Giuseppe Novelli14

1Department of Biopathology and Diagnostic Imaging and 2Department of Internal Medicine, Tor Vergata University, Via Montpellier 1, 00133 Roma, Italy
3Department of Psychology, The University of Texas at Austin, Austin, TX, 78703
4Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive disorder caused basically by a missense mutation within the LMNA gene, which encodes for lamin A/C. We have used gene expression profiling to characterize the specificity of molecular changes induced by the prevalent MAD mutation (R527H). A total of 5531 transcripts expressed in human dermis were investigated in two MAD patients, both carrying the R527H mutation, and three control subjects (age and sex matched). Transcription profiles revealed a differential expression in MAD vs. control fibroblasts in at least 1992 genes. Sixty-seven of these genes showed a common altered pattern in both patients with a threshold expression level >±2. Nevertheless, a large number of these genes (43.3%) are ESTs or encode for protein with unknown function; the other genes are involved in biological processes or pathways such as cell adhesion, cell cycle, cellular metabolism, and transcription. Quantitative RT-PCR was applied to validate the microarray results (R2 = 0.76). Analysis of the effect of the prevalent MAD mutation (R527H) over the transcriptional pattern of genes expressed in the human dermis showed that this LMNA gene mutation has pleiotropic effects on a limited number of genes. Further characterization of these effects might contribute to understanding the molecular pathogenesis this disorder.

Key words: Microarray; Lamin A/C; Mandibuloacral dysplasia; QRT-PCR

Address correspondence to Giuseppe Novelli, Department of Biopathology and Diagnostic Imaging, Tor Vergata University, Via Montpellier 1, 00133 Roma, Italy. Tel: +39-06+20900665; Fax: +39-06-20427313; E-mail: novelli@med.uniroma2.it

Gene Expression, Vol. 12, pp. 49-57
1052-2166/04 $20.00 + .00
Copyright © 2004 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Global Analysis of HuR-Regulated Gene Expression in Colon Cancer Systems of Reducing Complexity

Isabel López De Silanes,1 Jinshui Fan,1 Craig J. Galbán,2 Richard G. Spencer,2 Kevin G. Becker,3 and Myriam Gorospe1

1Laboratory of Cellular and Molecular Biology, 2Laboratory of Clinical Investigation, and 3Research Resources Branch, National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, MD 21224

HuR, a protein that binds to target mRNAs and can enhance their stability and translation, is increasingly recognized as a pivotal regulator of gene expression during cell division and tumorigenesis. We sought to identify collections of HuR-regulated mRNAs in colon cancer cells by systematic, cDNA array-based assessment of gene expression in three systems of varying complexity. First, comparison of gene expression profiles among tumors with different HuR abundance revealed highly divergent gene expression patterns, and virtually no changes in previously reported HuR target mRNAs. Assessment of gene expression patterns in a second system of reduced complexity, cultured colon cancer cells expressing different HuR levels, rendered more conserved sets of HuR-regulated mRNAs. However, the definitive identification of direct HuR target mRNAs required a third system of still lower complexity, wherein HuR-RNA complexes immunoprecipitated from colon cancer cells were subject to cDNA array hybridization to elucidate the endogenous HuR-bound mRNAs. Comparison of the transcript sets identified in each system revealed a strikingly limited overlap in HuR-regulated mRNAs. The data derived from this systematic analysis of HuR-regulated genes highlight the value of low-complexity, biochemical characterization of protein-RNA interactions. More importantly, however, the data underscore the broad usefulness of integrated approaches comprising systems of low complexity (protein-nucleic acid) and high complexity (cells, tumors) to comprehensively elucidate the gene regulatory events that underlie biological processes.

Key words: cDNA arrays; RNA-protein complexes; Posttranscriptional regulation; mRNA turnover; Cancer model

Address correspondence to Myriam Gorospe, Box 12, LCMB, NIA-IRP, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224. Tel: (410) 558-8443; Fax: (410) 558-8386; E-mail: myriam-gorospe@nih.gov