|ognizant Communication Corporation|
VOLUME 14, NUMBER 1
Gene Expression, Vol. 14, pp. 1-12
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Retinoic Acid Inducibility of the Human PDGF-A Gene Is Mediated by 5´-Distal DNA Motifs That Overlap With Basal Enhancer and Vitamin D Response Elements
Nancy G. Pedigo,* Hongxing Zhang,* Anjali Mishra,* Joseph R. Mccorkle, Angela K. Ormerod, and David M. Kaetzel
Department of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, Lexington, KY, USA
Retinoic acid (RA) upregulates expression of PDGF ligands and receptors in neonatal rat lung fibroblasts, a process likely to promote maturation of the lung alveolus and possibly microstructures of other organs. A mutational analysis of the gene encoding the PDGF-A ligand has identified a complex retinoic acid response element (RARE) located far upstream of the transcription start site, in a 5´-distal enhanceosome region previously shown to harbor basal and vitamin D-inducible enhancer activity. Maximal RA responsiveness (fourfold) was conferred by nucleotide sequence located between -7064 and -6787, with a variety of deletion and point mutations revealing the importance of at least three nuclear receptor half-sites within the enhancer region (-6851 to -6824), as well as nucleotides located further upstream. Recombinant human retinoic acid receptor/retinoid-X receptor heterodimers bound with high affinity and sequence specificity to multiple regions within the RARE, as demonstrated by electrophoretic mobility shift and DNase I footprinting assays. The addition of RARE activity to previously described functions of the 5´-distal enhanceosome underscores the importance of this region as a key integration point for regulatory control of PDGF-A expression.
Key words: Platelet-derived growth factor; Transcription; Enhancer; Retinoic acid response element; Nuclear receptor
Address correspondence to David M. Kaetzel, Department of Molecular and Biomedical Pharmacology, UKMC MS-301, 800 Rose Street, Lexington, KY 40536-0298, USA. Tel: (859) 257-6558; Fax: (859) 323-1981; E-mail: firstname.lastname@example.org
*These authors contributed equally to the work presented in this article.
Cloning, Expression, and Functional Analysis of Rat Liver Cytosolic Inorganic Pyrophosphatase Gene and Characterization of its Functional Promoter
Harekrushna Panda, Ravi S. Pandey, Priya R. Debata, and Prakash C. Supakar
Institute of Life Sciences, Bhubaneswar, India
Inorganic pyrophosphate (PPi) is formed in several metabolic processes and its hydrolysis by the ubiquitously expressed enzyme inorganic pyrophosphatase (iPPase) is essential for the reactions to proceed in the direction of biosynthesis. Recently, we have reported differential expression and activity of cytosolic iPPase in rat liver with aging. In this article we report the cloning of the coding region of rat liver cytosolic iPPase gene in a bacterial expression vector, its expression, purification, and functional analysis by in-gel enzyme assay. SDSPAGE and Western blot analysis of this expressed protein revealed that its molecular weight (MW) is ~33 kDa, while in-gel assay showed that it is functionally active just as the liver cytosolic iPPase. We have determined the genomic organization of this gene by genome blast approach. We have also cloned and characterized its proximal ~1 kb functional promoter (-1009 to +82) by transient transfection and luciferase assay of different 5´-deleted iPPase promoter-luciferase constructs and also established its transcription start site by primer extension analysis, along with protein-DNA interaction studies for a few putative transcription factor binding sites.
Key words: Inorganic pyrophosphatase; Gene expression; In-gel assay; Transcription start site; Protein-DNA interaction
Address correspondence to Prakash C. Supakar, Ph.D., Institute of Life Sciences, Nalco Square, Bhubaneswar 751023, India. Tel: 91-674-230-2783; Fax: 91-674-230-0728; E-mail: email@example.com
Gene Regulation Networks Related to Neural Differentiation of hESC
Jiang F. Zhong,1,2,3 Yahui Song,2 Jing Du,1 Christine Gamache,1 Kathleen A. Burke,2,3 Brett T. Lund,1 and Leslie P. Weiner1
1Department of Neurology, Keck School of Medicine, University
of Southern California, Los Angeles, CA 90033, USA
2Gene Therapy Laboratories, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
3Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
With the unique property of self-renewal and developmental pluripotency, human embryonic stem cells (hESC) provide an opportunity to study molecular aspects of developmental biology. Understanding gene regulation of hESC pluripotency is a critical step toward directing hESC differentiation for regenerative medicine. However, currently little is known about hESC gene regulation of hESC pluripotency. Applying network analysis to microarray gene expression profiling data, we compared gene expression profiles from pluripotent hESC to hESCderived astrocytes and identified potential gene regulation networks. These gene regulation networks suggest that hECS has stringent control of cell cycle and apoptosis. Our data reveal several potential hESC differentiation biomarkers and suggest that IGF2 and A2M could play a role in hESC pluripotency by altering the availability of cytokines at the local environment of hECS. These findings underscore the importance of network analysis among differentially expressed genes, and should facilitate future study for understanding the gene regulation of hESC pluripotency.
Key words: Pluripotent stem cells; Regulation of gene expression; Neuroglia; Cell differentiation; Microarray analysis
Address correspondence to Jiang F. Zhong, Department of Neurology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA. Tel: (323) 442-2371; Fax: (323) 442-2311; E-mail: firstname.lastname@example.org
Gene Expression Variability in Subcutaneous and Omental Adipose Tissue of Obese Men
Yonghua Zhang,1 Yohan Bossé ,2 Picard Marceau,4 Simon Biron,4 Stephan Lebel,4 Denis Richard,5 Marie-Claude Vohl,1,3 and André Tchernof1,3
1Molecular Endocrinology and Oncology Research Center, Laval
University Medical Research Center, Québec, Canada
2McGill University and Genome Quebec Innovation Center, Montreal, Canada
3Department of Food Science and Nutrition, Laval University, Québec, Canada
4Department of Surgery, Laval University, Québec, Canada
5Cardiology Institute, Lava Hospital, Québec, Canada
We investigated interindividual variability in gene expression in abdominal subcutaneous (SC) and omental (OM) adipose tissue of 10 massively obese men. Affymetrix human U133A microarrays were used to measure gene expression levels. A total of 6811 probesets generated significant signal in both depots in all samples. Interindividual variability in gene expression was rather low, with more than 90% of transcripts showing a coefficient of variation (CV) lower than 23.6% and 21.7% in OM and SC adipose tissues, respectively. The distributions of CV were similar between the two fat depots. A set of highly variable genes was identified for both tissues on the basis of a high CV and elevated gene expression level. Among the set of highly regulated genes, 18 transcripts were involved in lipid metabolism and 28 transcripts were involved in cell death for SC and OM samples, respectively. In conclusion, gene expression interindividual variability was rather low and globally similar between fat compartments, and the adipose tissue transcriptome appeared as relatively stable, although specific pathways were found to be highly variable in SC and OM depots.
Key words: Adipose tissue; Omental; Subcutaneous; Microarrays; Obese men
Address correspondence to Andre´ Tchernof, Ph.D., Laval University Medical Research Center, 2705 Laurier Blvd. (T3-67), Québec, (Québec), Canada G1V 4G2. Tel: (418) 654-2296; Fax: (418) 654-2761; E-mail: email@example.com
Complete Deletion of All a-Dystrobrevin Isoforms Does Not Reveal New Neuromuscular Junction Phenotype
Dongqing Wang,1* Bridget B. Kelly,1* Douglas E. Albrecht,2 Marvin E. Adams,2 Stanley C. Froehner,2 and Guoping Feng1.3
1Department of Neurobiology, Duke University Medical Center,
Durham, NC 27710, USA
2Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA
3Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA
The dystrophin glycoprotein complex (DGC) is critical for muscle stability, and mutations in DGC proteins lead to muscular dystrophy. The DGC also contributes to the maturation and maintenance of the neuromuscular junction (NMJ). The gene encoding the DGC protein a-dystrobrevin undergoes alternative splicing to produce at least five known isoforms. Isoform-specific antibody staining and reverse transcription PCR in mutant mice with a deletion of exon 3 of the a-dystrobrevin gene suggested the existence of a remaining synaptic isoform, which might be compensating for a-dystrobrevin function. To test this possibility and to more completely understand the synaptic function of a-dystrobrevin, we used a two-step homologous recombination strategy combined with in vivo Cre-mediated excision to generate mice with a large deletion of the ?-dystrobrevin gene to disrupt all isoforms. However, these mice did not exhibit a more severe NMJ phenotype than that observed in the exon 3-deleted mice. Nonetheless, these mice not only eliminate possible compensation by remaining isoforms of a-dystrobrevin, but also offer a conditional allele that could be used to identify tissue-specific and developmental functions of a-dystrobrevin. This work also demonstrates a successful strategy to achieve deletion of a large genomic sequence, which can be a valuable tool for functional studies of genes encoding multiple isoforms that span a large genomic region.
Key words: Dystrophin glycoprotein complex; Neuromuscular junction; Isoform diversity; Alternative splicing; Homologous recombination
Address correspondence to Guoping Feng, Department of Neurobiology, Box 3209, Duke University Medical Center, Durham, NC 27710, USA. Tel: +1-919-668-1657; Fax: +1-919-668-1891; E-mail: firstname.lastname@example.org
*These authors contributed equally to this work.