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Gene Expression, Vol. 12, pp. 223-230
1052-2166/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

An Integrated Strategy for the Optimization of Microarray Data Interpretation

Xinmin Li1 and Richard J. Quigg1,2

1Functional Genomics Facility and 2Section of Nephrology, Division of Biological Sciences, The University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA

The completion of a microarray experiment represents just a starting point toward understanding the biology of interest. A follow-up strategy is needed to fully elucidate the functional significance of microarray-derived measurements of differential expression. Given the fact that no single approach can fully unravel the fundamental biology that is typically quite complex, the follow-up strategy must be integrated at multiple levels encompassing bioinformatics, genomics, and proteomics. In this review, we discuss an integrative approach, which can be used to prioritize microarray-derived candidate genes, define their functions, and place them in the context of the biological system being studied.

Key words: Data analysis; Microarray; siRNA; QTL mapping; Genome database

Address correspondence to Xinmin Li, Ph.D., Functional Genomics Facility, Division of Biological Sciences, The University of Chicago, 5841 S. Maryland Ave., MC5100, Chicago, IL 60637, USA. Tel: (773) 834-8420; Fax: (773) 834-9925; E-mail: xli@medicine.bsd.uchicago.edu

Gene Expression, Vol. 12, pp. 231-243
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Copyright © 2005 Cognizant Comm. Corp.
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Role of Arginine Residues 14 and 15 in Dictating DNA Binding Stability and Transactivation of the Aryl Hydrocarbon Receptor/Aryl Hydrocarbon Receptor Nuclear Translocator Heterodimer

Susanne C. Wache, Erica M. Hoagland, Georgia Zeigler, and Hollie I. Swanson

Department of Molecular and Biomedical Pharmacology, University of Kentucky Medical Center, Lexington, KY 40536, USA

The aryl hydrocarbon receptor (AHR) and its DNA binding partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) are basic helix-loop-helix/PAS proteins. The goal of the current study was to determine the extent to which residues R14 and R15 contained within the basic region of the AHR contribute to the DNA binding affinity and stability of the AHR/ARNT heterodimer. Towards this end, we first performed equilibrium binding and dissociation rate analyses using a single dioxin response element (DRE-1). While the KD and Bmax values obtained from the equilibrium binding analysis were similar for the wild-type AHR (wt AHR) and that containing the substitutions of R14 and R15 with Q residues (Q14Q15 AHR), dissociation rate analyses revealed that the stability of the Q14Q15 AHR DNA binding complex was approximately 10-fold less. Using a two-site DNA binding model, we also found that AHR/ARNT heterodimer does not participate in cooperative binding, as binding of the second dimer appears to be prohibited by occupation of the first. This property was similar regardless of the composition of the amino acids at positions 14 and 15. Finally, reporter assays revealed that the Q14Q15 substitutions severely compromised the ability of the AHR to activate gene expression despite appropriate nuclear localization. The present results revealed that DNA binding stability of the AHR/ARNT heterodimer is an important requirement for its transactivation capabilities and that this stability is governed, in part, by residues R14 and R15 that lie within the basic region of the AHR.

Key words: Aryl hydrocarbon receptor; Basic helix-loop-helix/PAS; DNA binding

Address correspondence to Hollie I. Swanson, University of Kentucky Medical Center, MS-305, 800 Rose St., Lexington, KY 40536, USA. Tel: (859) 323-1463; Fax: (859) 323-1981; E-mail: hswan@uky.edu

Gene Expression, Vol. 12, pp. 245-257
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Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Distinct Gene Expression Profile of Human Mesenchymal Stem Cells in Comparison to Skin Fibroblasts Employing cDNA Microarray Analysis of 9600 Genes

Cornelia Brendel,1* Larissa Kuklick,1* Oliver Hartmann,2 Theo Daniel Kim,1 Ulrich Boudriot,3 Dagmar Schwell,1 and Andreas Neubauer1

1Department of Hematology, Oncology and Immunology, Philipps-University Marburg, Baldingerstrasse, Marburg, Germany
2Institute of Medical Biometry and Epidemiology, Faculty of Medicine and Hospital, Philipps-University Marburg, Bunsenstrasse 3, Marburg, Germany
3Department of Orthopaedics, Philipps-University Marburg, Baldingerstrasse, Marburg, Germany

Broad differentiation capacity has been described for mesenchymal stem cells (MSC) from human bone marrow. We sought to identify genes associated with the immature state and pluripotency of this cell type. To prove the pluripotent state of the MSC, differentiation into osteocytes, adipocytes, and chondrocytes was performed in vitro. In contrast, normal skin cells did not harbor these differentiation abilities. We compared the expression profile of human bone marrow MSC with cDNA from one primary human skin cell line as control, using a cDNA chip providing 9600 genes. The identity of all relevant genes was confirmed by direct sequencing. Data of gene array expression were corroborated employing quantitative PCR analysis. About 80 genes were differently expressed more than threefold in MSC compared to mature skin fibroblasts. Interestingly, primary human MSC were found to upregulate a number of genes important for embryogenesis such as distal-less homeo box 5, Eyes absent homolog 2, inhibitor of DNA binding 3, and LIM protein. In contrast, mesenchymal lineage genes were downregulated in MSC in comparison to skin cells. We also detected expression of some genes involved in neural development, indicating the broad differentiation capabilities of MSC. We conclude that human mesenchymal stem cells harbor an expression profile distinct from mature skin fibroblast, and genes associated with developmental processes and stem cell function are highly expressed in adult mesenchymal stem cells.

Key words: Mesenchymal stem cells; Microarray; Differentiation; Gene expression profile

Address correspondence to Cornelia Brendel, M.D., Department of Hematology, Oncology and Immunology, Philipps-University Marburg, Baldingerstrasse, 35033 Marburg, Germany. Tel: ++49-6421-2865061; Fax: ++49-6421-2866358; E-mail: BrendelC@mailer.uni-marburg.de

*Both authors contributed equally to this work.

Gene Expression, Vol. 12, pp. 259-271
1052-2166/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Association of TIF-IA and Polymerase I Mediates Promoter Recruitment and Regulation of Ribosomal RNA Transcription in Acanthamoeba castellanii

Joseph C. Gogain1 and Marvin R. Paule2

1Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262, USA
2Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA

Large amounts of energy are expended for the construction of the ribosome during both transcription and processing, so it is of utmost importance for the cell to efficiently regulate ribosome production. Understanding how this regulation occurs will provide important insights into cellular growth control and into the coordination of gene expression mediated by all three transcription systems. Ribosomal RNA (rRNA) transcription rates closely parallel the need for protein synthesis; as a cell approaches stationary phase or encounters conditions that negatively affect either growth rate or protein synthesis, rRNA transcription is decreased. In eukaryotes, the interaction of RNA polymerase I (pol I) with the essential transcription initiation factor IA (TIF-IA) has been implicated in this downregulation of transcription. In agreement with the first observation that rRNA transcription is regulated by altering recruitment of pol I to the promoter in Acanthamoeba castellanii, we show here that pol I and an 80-kDa homologue of TIF-IA are found tightly associated in pol I fractions competent for specific transcription. Disruption of the pol I-TIF-IA complex is mediated by a specific dephosphorylation of either pol I or TIF-IA. Phosphatase treatment of TIF-IA-containing A. castellanii pol I fractions results in a downregulation of both transcriptional activity and promoter binding, reminiscent of the inactive pol I fractions purified from encysted cells. The fraction of pol I competent for promoter recruitment is enriched in TIF-IA relative to that not bound by immobilized promoter DNA. This downregulation coincides with an altered electrophoretic mobility of TIF-IA, suggesting at least it is phosphorylated.

Key words: Growth control; Polymerase I; Rrn3; rRNA; Transcription; Regulation

Address correspondence to Marvin R. Paule, Department of Biochemistry and Molecular Biology, Colorado State University, 1870 Campus Delivery, Fort Collins, CO 80523-1870, USA. Tel: (970) 491-6748; Fax: (907) 491-0494; E-mail: mpaule@lamar.colostate.edu

Gene Expression, Vol. 12, pp. 273-287
1052-2166/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Wound Site Neutrophil Transcriptome in Response to Psychological Stress in Young Men

Sashwati Roy,1 Savita Khanna,1 Pier-En Yeh,2 Cameron Rink,1 William B. Malarkey,2,3,4,5,6 Janice Kiecolt-Glaser,4 Bryon Laskowski,2 Ronald Glaser,2,5,6 and Chandan K. Sen1

1Laboratory of Molecular Medicine, Department of Surgery, Davis Heart & Lung Research Institute and Ohio State Comprehensive Wound Center, 2Department of Molecular Virology, Immunology and Medical Genetics, 3Department of Internal Medicine, 4Department of Psychiatry, 5Ohio State Institute for Behavioral Medicine Research, and 6Comprehensive Cancer Center, The Ohio State University Medical Center, Columbus, OH 43210, USA

Communication between the central nervous and the immune system occurs through chemical messengers secreted by nerve cells, endocrine organs, or immune cells. Psychological stressors can disrupt these networks. We have previously observed that disruption of the neuroendocrine immune system adversely influences a broad range of physiological processes including wound healing. Migration of neutrophils to the wound site is an early event that induces a transcriptional activation program, which regulates cellular fate and function, and promotes wound healing. In this study, we have sought to identify stress-sensitive transcripts in wound site neutrophils. A skin blister model was used to collect wound fluid and wound site neutrophils from four young men, experiencing or not examination stress. Self-reported stress was recorded using the Beck Depression Inventory. Stress decreased growth hormone levels at the wound site and was related to impaired wound healing in all subjects. High density microarray analyses were performed using RNA from wound site neutrophils. Results show that psychological stress had an overall suppressive effect on the neutrophil transcriptome. Of the 22,283 transcripts screened, 0.5% were downregulated whereas only under 0.3% were induced by stress in all four out of four subjects. Functionally, stress tilted the genomic balance towards genes encoding proteins responsible for cell cycle arrest, death, and inflammation. Further effort to gain a more comprehensive understanding of the functional significance of such behavior-genome interaction is warranted.

Key words: Wound healing; Skin; Microarray; Clinical; Gene

Address correspondence to Dr. Chandan K. Sen, 512 Davis Heart & Lung Research Institute, 473 West 12th Avenue, The Ohio State University Medical Center, Columbus, OH 43210, USA. Tel: 614-247-7658; Fax: 614-247-7818; E-mail: sen-1@medctr.osu.edu

Gene Expression, Vol. 12, pp. 289-303
1052-2166/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

A Selective Role for MRF4 in Innervated Adult Skeletal Muscle: NaV 1.4 Na+ Channel Expression Is Reduced in MRF4-Null Mice

Amy L. Thompson,1 Gregory Filatov,2 Connie Chen,3 Isaac Porter,1 Yingjie Li,2 Mark M. Rich,2 and Susan D. Kraner1

1Department of Molecular and Biomedical Pharmacology, University of Kentucky Medical Center, Lexington, KY 40536, USA
2Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322, USA
3Biological Basis of Behavior, University of Pennsylvania, Philadelphia, PA, 19104, USA

The factors that regulate transcription and spatial expression of the adult skeletal muscle Na+ channel, NaV 1.4, are poorly understood. Here we tested the role of the transcription factor MRF4, one of four basic helix-loop-helix (bHLH) factors expressed in skeletal muscle, in regulation of the NaV 1.4 Na+ channel. Overexpression of MRF4 in C2C12 muscle cells dramatically elevated NaV 1.4 reporter gene expression, indicating that MRF4 is more efficacious than the other bHLH factors expressed at high levels endogenously in these cells. In vivo, MRF4 protein was found both in extrajunctional and subsynaptic muscle nuclei. To test the importance of MRF4 in NaV 1.4 gene regulation in vivo, we examined Na+ channel expression in MRF4-null mice using several techniques, including Western blotting, immunocytochemistry, and electrophysiological recording. By all methods, we found that expression of the NaV 1.4 Na+ channel was substantially reduced in MRF4-null mice, both in the surface membrane and at neuromuscular junctions. In contrast, expression of the acetylcholine receptor, and in particular its a subunit, was unchanged, indicating that MRF4 regulation of Na+ channel expression was selective. Expression of the bHLH factors myf-5, MyoD, and myogenin was increased in MRF4-null mice, but these factors were not able to fully maintain NaV 1.4 Na+ channel expression either in the extrajunctional membrane or at the synapse. Thus, MRF4 appears to play a novel and selective role in adult muscle.

Key words: Synaptogenesis; Skeletal muscle; Sodium channel; Basic helix-loop-helix; Transcription; MRF4; Neuromuscular junction

Address correspondence to Amy L. Thompson, Department of Molecular and Biomedical Pharmacology, UKMC MS-306, 800 Rose Street, Lexington, KY 40536-0298, USA. Tel: (859) 323-4875; Fax: (859) 323-1981; E-mail: althom7@uky.edu

Gene Expression, Vol. 12, pp. 305-313
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Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Testosterone-Dependent Transgene Expression in the Liver of the CAG-lacZ Transgenic Rat

Amornpun Sereemaspun,1,4* Koichi Takeuchi,1* Yuki Sato,2 Sadahiko Iwamoto,3 Takeshi Inakagi,1 Shigeo Ookawara,1 Yoji Hakamata,2 Takashi Murakami,2 and Eiji Kobayashi2

1Department of Anatomy, Jichi Medical School, 2Division of Organ Replacement Research, Center of Molecular Medicine, and 3Department of Legal Medicine and Human Genetics. Jichi Medical School, Tochigi, Japan
4Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

Many endogenous gene expressions in the liver are well known to be predominant in males, compared with those of females. In contrast, the fate of hepatic transgene expression between sexes is not fully understood. Here we studied whether sex hormones changed hepatic transgene expression in the ubiquitous CAG promoter-driven lacZ transgenic (Tg) rat. Both sexes of CAG-lacZ Tg rats received gonadectomy. Liver biopsy was taken weekly to determine the change of transgene expression. Histological result of adult males showed mosaic lacZ expression but it was negative in adult females, while livers in neonatal stage showed comparable expression of lacZ. Other organs exhibited equal expression in both sexes. At 2 weeks after castration, lacZ expression in male liver was significantly decreased and became negative after 4 weeks while no significant difference was observed in the lacZ expression pattern in other organs. After ovariectomy, lacZ expression in female liver remained undetectable. Moreover, testosterone treatment to gonadectomized rats of both sexes could enhance lacZ expression in the liver. In summary, we report that CAG-lacZ Tg rats demonstrate sexual dimorphism of transgene expression specifically only in the liver. Testosterone administration mediated upregulation of liver lacZ expression. Our findings suggested that androgen, especially testosterone, plays an important role in the hepatic transgene expression.

Key words: Transgene expression; lacZ; CAG promoter; Testosterone; Gonadectomy

Address correspondence to Koichi Takeuchi, M.D., Ph.D., Department of Anatomy, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Tochigi 329-0498, Japan. Tel: +81-285-58-7316; Fax: +81-285-40-6587; E-mail: koichi-t@jichi.ac.jp

*These authors contributed equally to this work.

Gene Expression, Vol. 12, pp. 315-323
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Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Lack of the DNA Repair Enzyme OGG1 Sensitizes Dopamine Neurons to Manganese Toxicity During Development

Fernando Cardozo-Pelaez, David P. Cox, and Celeste Bolin

Center for Environmental Health Sciences, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT 59812, USA

Onset of Parkinson's disease (PD) and Parkinson-like syndromes has been associated with exposure to diverse environmental stimuli. Epidemiological studies have demonstrated that exposure to elevated levels of manganese produces neuropathological changes localized to the basal ganglia, including neuronal loss and depletions in striatal dopamine content. However, understanding the mechanisms associated with manganese neurotoxicity has been hampered by the lack of a good rodent model. Elevated levels of 8-hydroxy-2´-deoxyguanosine (oxo8dG) have been found in brain areas affected in PD. Whether increased DNA damage is responsible for neuronal degeneration or is a mere epiphenomena of neuronal loss remains to be elucidated. Thus, by using mice deficient in the ability to remove oxo8dG we aimed to determine if dysregulation of DNA repair coupled to manganese exposure would be detrimental to dopaminergic neurons. Wild-type and OGG1 knockout mice were exposed to manganese from conception to postnatal day 30; in both groups, exposure to manganese led to alterations in the neurochemistry of the nigrostriatal system. After exposure, dopamine levels were elevated in the caudate of wild-type mice. Dopamine was reduced in the caudate of OGG1 knockout mice, a loss that was paralleled by an increase in the dopamine index of turnover. In addition, the reduction of dopamine in caudate putamen correlated with the accumulation of oxo8dG in midbrain. We conclude that OGG1 function is essential in maintaining neuronal stability during development and identify DNA damage as a common pathway in neuronal loss after a toxicological challenge.

Key words: DNA damage; DNA repair; Manganese; Development; Parkinson's disease

Address correspondence to Fernando Cardozo-Pelaez, Ph.D., Center for Environmental Health Sciences, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT 59812, USA. Tel: (406) 243-4025; Fax: (406) 243-2807; E-mail: fernando.cardozo@umontana.edu