|ognizant Communication Corporation|
VOLUME 9, NUMBER 6
Gene Expression, Vol. 9, pp. 237-248
1052-2166/01 $20.00 + .00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Adenovirus-Mediated Increase of HNF-3 Levels Stimulates Expression of Transthyretin and Sonic Hedgehog, Which Is Associated With F9 Cell Differentiation Toward the Visceral Endoderm Lineage
Yongjun Tan,1 Robert H. Costa,1 Imre Kovesdi,2 and Ronald R. Reichel3
1University of Illinois at Chicago, College of Medicine,
Department of Molecular Genetics, 900 South Ashland Ave, Chicago, IL 60607-7170
2GenVec, Inc., 65 West Watkins Mill Road, Gaithersburg, MD 20878
3Department of Cellular and Molecular Pharmacology, The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064
Retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells toward the visceral endoderm lineage is accompanied by increased expression of the Forkhead Box (Fox) transcription factors hepatocyte nuclear factor 3a (HNF-3a) and HNF-3b, suggesting that they play a crucial role in visceral endoderm development. Retinoic acid stimulation results in a cascade of HNF-3 induction in which HNF-3a is a primary target for retinoic acid action and its increase is required for subsequent induction of HNF-3b expression. Increased expression of HNF-3b precedes activation of its known target genes, including transthyretin (TTR), Sonic hedgehog (Shh), HNF-1a, HNF-1b, and HNF-4a. In order to examine whether increased HNF-3 expression is sufficient to induce expression of its downstream target genes without retinoic acid stimulation, we have used adenovirus-based expression vectors to increase HNF-3 protein levels in F9 cells. We demonstrate that adenovirus-mediated increase of HNF-3a levels in F9 cells is sufficient to induce activation of endogenous HNF-3b levels followed by increased TTR and Shh expression. Furthermore, we show that elevated HNF-3b levels stimulate expression of endogenous TTR and Shh without retinoic acid stimulation. Moreover, ectopic HNF-3 levels in undifferentiated F9 cells are insufficient to induce HNF-3a, HNF-1a, HNF-1b, and HNF-4a expression, suggesting that their transcriptional activation required other regulatory proteins induced by the retinoic acid differentiation program. Finally, our studies demonstrate the utility of cell infections with adenovirus expressing distinct transcription factors to identify endogenous target genes, which are assembled with the appropriate nucleosome structure.
Key words: Hepatocyte nuclear factor 3; Sonic hedgehog; Transthyretin; Retinoic acid stiumlation
Address correspondence to Dr. Yongjun Tan or Dr. Robert H. Costa, Department of Molecular Genetics (M/C 669), University of Illinois at Chicago, College of Medicine, 900 S. Ashland Ave, Rm. 2220 MBRB, Chicago, IL 60607-7170. Office tel: (312) 996-0474; Lab tel: (312) 996-0534; Lab fax: (312) 355-4010; E-mail: firstname.lastname@example.org or RobCosta@uic.edu
Exploring Relationships in Gene Expressions: A Partial Least Squares Approach
Department of Statistics and Mathematics, Georgia State University, Atlanta, GA 30303
Microarray technology has revolutionized the way gene functions are monitored. Analysis of microarray data is a fast growing research area that interfaces various disciplines such as biology, biochemistry, computer science, and statistics. While various clustering and classification techniques have been successfully employed to group genes based on the similarity of their expression patterns, much is yet to be learned about the interrelationship of the expression levels among various genes. We approach this problem with a statistical technique called partial least squares that is capable of modeling a large number of variables each with relatively few observations. This property of the partial least squares methodology appears to be attractive for application to microarray data sets where the simultaneous expression levels of many genes are collected each at a few time points (or individuals). We use it to analyze publicly available microarray data on sporulation of budding yeast (Saccharomyces cerevisiae). We investigate a number of representative genes, one from each temporal group (based on the time of first induction) of positively expressed genes and show that in each case most of the variability was explained by only two partial regression terms based on all remaining genes. Moreover, the predicted expression levels of the representative genes from partial least squares fit very well on the average with the true expression levels over time. Finally, we compare the biological functions of the genes with largest coefficients with those of the predicted genes. In many cases, the genes are involved in similar or related biological functions including negative relationships. We show that this method can identify established gene relationships; we argue that it can be an exploratory tool for identifying potential gene relationships requiring further biological investigation.
Key words: Microarray; Gene expression data; Partial least squares; Yeast; Modeling
Address correspondence to Susmita Datta, Department of Mathematics and Statistics, Georgia State University, Atlanta, GA 30303. Tel: (404) 651-0643; Fax: (404) 651-2246; E-mail: email@example.com
Reversal of Inhibition by the T7 Concatemer Junction Sequence on Expression From a Downstream T7 Promoter
Li Cheng and Emanuel Goldman
Department of Microbiology & Molecular Genetics, University of Medicine & Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103
We have previously reported that placement of the phage T7 concatemer junction (CJ) just upstream of another gene on a plasmid in a T7 system proved to be inhibitory to expression of the downstream gene. We had hypothesized that the inhibition was a result of a readthrough transcript of the CJ element interacting with the translation start region of the downstream gene; also that in the absence of a T7 termination signal, transcription continued around the plasmid multiple times ("rolling circle" transcription), always juxtaposing the inhibitory CJ sequence proximal to the downstream gene mRNA. Two strong predictions were made from this model: 1) that introduction of a spacer sequence between the CJ element and the downstream gene should alleviate the inhibition, and 2) that reintroduction of a T7 transcription terminator should prevent rolling circle transcription, thereby reversing the inhibition by allowing some transcripts to be generated originating from the downstream promoter that did not contain the inhibitory CJ element upstream. We report here that both of these predictions have been fulfilled. However, the reversal of inhibition was only partial in the construct where the T7 terminator was reintroduced, indicating that there remains a residual inhibitory effect of the CJ element on expression of the downstream gene. A possible explanation is that the CJ element, acting as a pause site for transcription, blocks access to the downstream T7 promoter, thereby reducing transcription from that promoter. If this explanation is correct, steric hindrance of transcription starts resulting from an upstream RNA polymerase pause site may represent a previously unrecognized mechanism of transcriptional control.
Key words: T7 expression systems; T7 transcription terminator; RNA polymerase pause sites; Steric hindrance of transcription starts
Address correspondence to Emanuel Goldman, Department of Microbiology & Molecular Genetics, University of Medicine & Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103. Tel: (973) 972-4367; Fax: (973) 972-3644; E-mail: firstname.lastname@example.org
Antagonistic Regulation of Dlx2 Expression by PITX2 and Msx2: Implications for Tooth Development
Patrick D. Green,1 Tord A. Hjalt,2 Dianne E. Kirk,1 Lillian B. Sutherland,3 Bethan L. Thomas,4 Paul T. Sharpe,4 Malcolm L. Snead,5 Jeffrey C. Murray,2 Andrew F. Russo,3 and Brad A. Amendt1
1Department of Biological Science, The University of Tulsa,
Tulsa, OK 74104-3189
Departments of 2Pediatrics, and 3Physiology and Biophysics, The University of Iowa
4Department of Craniofacial Development, GKT Dental Institute, King's College, University of London
5The Center for Craniofacial Molecular Biology, The University of Southern California, School of Dentistry
The transcriptional mechanisms underlying tooth development are only beginning to be understood. Pitx2, a bicoid-like homeodomain transcription factor, is the first transcriptional marker observed during tooth development. Because Pitx2, Msx2, and Dlx2 are expressed in the dental epithelium, we examined the transcriptional activity of PITX2 in concert with Msx2 and the Dlx2 promoter. PITX2 activated while Msx2 unexpectedly repressed transcription of a TK-Bicoid luciferase reporter in a tooth epithelial cell line (LS-8) and CHO cell line. Surprisingly, Msx2 binds to the bicoid element (5´-TAATCC-3´) with a high specificity and competes with PITX2 for binding to this element. PITX2 binds to bicoid and bicoid-like elements in the Dlx2 promoter and activates this promoter 45-fold in CHO cells. However, it is only modestly activated in the LS-8 tooth epithelial cell line that endogenously expresses Msx2 and Pitx2. RT-PCR and Western blot assays reveal that two Pitx2 isoforms are expressed in the LS-8 cells. We further demonstrate that PITX2 dimerization can occur through the C-terminus of PITX2. Msx2 represses the Dlx2 promoter in CHO cells and coexpression of both PITX2 and Msx2 resulted in transcriptional antagonism of the Dlx2 promoter. Electrophoretic mobility shift assays demonstrate that factors in the LS-8 cell line specifically interact with PITX2. Thus, Dlx2 gene transcription is regulated by antagonistic effects between PITX2, Msx2, and factors expressed in the tooth epithelia.
Key words: PITX2; Dlx2; Transcriptional regulation
Address correspondence to Brad A. Amendt, The University of Tulsa, Department of Biological Sciences, 600 S. College Ave., Tulsa, OK 74104-3189. Tel: (918) 631-3328; Fax: (918) 631-2762; E-mail: email@example.com
SpSoxB1 Serves an Essential Architectural Function in the Promoter SpAN, a tolloid/BMP1-Related Gene
Alan P. Kenny,* Lynne M. Angerer, and Robert C. Angerer
Department of Biology, University of Rochester, Rochester, NY 14627
Transcription of SpAN, which encodes a secreted protease related to tolloid and BMP 1, is differentially regulated along the animal-vegetal axis of the sea urchin embryo by a maternally initiated mechanism. Regulatory sites that bind SpSoxB1 and CBF (CCAAT binding factor) are essential for strong transcriptional activity because mutations of these elements reduce promoter activity in vivo 20- and 10-fold, respectively. Here we show that multimerized SpSoxB1 elements cannot activate transcription from the SpAN basal promoter in vivo. However, like other factors containing HMG-class DNA binding domains, SpSoxB1 does induce strong bending of DNA. The CBF binding site lies abnormally far from the transcriptional start site at -200 bp. We show that the SpSoxB1 site is not required if the CCAAT element is moved 100 bp closer to the transcriptional start site, replacing the SpSoxB1 site. This supports a model in which the bending of SpAN promoter DNA by SpSoxB1 facilitates interactions between factors binding to upstream and downstream regulatory elements.
Key words: DNA bending; HMG; CCAAT
Address correspondence to Robert C. Angerer, Department of Biology, University of Rochester, Rochester, NY 14627. Tel: (716) 275-8715; Fax: (716) 275-2070; E-mail: firstname.lastname@example.org
*Current address: School of Medicine and Dentistry, University of Rochester, Rochester, NY 14634.
Identification of Novel Peroxisome Proliferator-Activated Receptor a (PPARa) Target Genes in Mouse Liver Using cDNA Microarray Analysis
Mustapha Cherkaoui-Malki,1* Kirstin Meyer,1 Wen-Qing Cao,1 Norbert Latruffe,2 Anjana V. Yeldandi,1 M. Sambasiva Rao,1 Christopher A. Bradfield,3 and Janardan K. Reddy1
1Department of Pathology, Northwestern University Medical
School, Chicago, IL 60611-3008
2Laboratoíre de Biologie Moléculaire et Cellulaire, Universite de Bourgogne, BP138, 21004 Dijon, France
3McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, WI 53706
Peroxisome proliferators, which function as peroxisome proliferator-activated receptor-a (PPARa) agonists, are a group of structurally diverse nongenotoxic hepatocarcinogens including the fibrate class of hypolipidemic drugs that induce peroxisome proliferation in liver parenchymal cells. Sustained activation of PPARa by these agents leads to the development of liver tumors in rats and mice. To understand the molecular mechanisms responsible for the pleiotropic effects of these agents, we have utilized the cDNA microarray to generate a molecular portrait of gene expression in the liver of mice treated for 2 weeks with Wy-14,643, a potent peroxisome proliferator. PPARa activation resulted in the stimulation of expression (fourfold or greater) of 36 genes and decreased the expression (fourfold or more decrease) of 671 genes. Enhanced expression of several genes involved in lipid and glucose metabolism and many other genes associated with peroxisome biogenesis, cell surface function, transcription, cell cycle, and apoptosis has been observed. These include: CYP2B9, CYP2B10, monoglyceride lipase, pyruvate dehydrogenase-kinase-4, cell death-inducing DNA-fragmentation factor-a, peroxisomal biogenesis factor 11b, as well as several cell recognition surface proteins including annexin A2, CD24, CD39, lymphocyte antigen 6, and retinoic acid early transcript-g, among others. Northern blotting of total RNA extracted from the livers of PPARa-/- mice and from mice lacking both PPARa and peroxisomal fatty acyl-CoA oxidase (AOX), that were fed control and Wy-14,643-containing diets for 2 weeks, as well as time course of induction following a single dose of Wy-14,643, revealed that upregulation of genes identified by microarray procedure is dependent upon peroxisome proliferation vis-à-vis PPARa. However, cell death-inducing DNA-fragmentation factor-a mRNA, which is increased in the livers of wild-type mice treated with peroxisome proliferators, was not enhanced in AOX-/- mice with spontaneous peroxisome proliferation. These observations indicate that the activation of PPARa leads to increased and decreased expression of many genes not associated with peroxisomes, and that delayed onset of enhanced expression of some genes may be the result of metabolic events occurring secondary to PPARa activation and alterations in lipid metabolism.
Key words: Peroxisome proliferator-activated receptor-a (PPARa); Peroxisome proliferator; Liver DNA microarray analysis
Address correspondence to Janardan K. Reddy, Department of Pathology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611-3008. Tel: (312) 503 8144; Fax: (312) 503 8249; E-mail: email@example.com
*Present address: Laboratoíre de Biologie Moléculaire et Cellulaire, Universite de Bourgogne, BP138, 21004 Dijon, France.