|ognizant Communication Corporation|
VOLUME 10, NUMBER 5/6
Gene Expression, Vol. 10, pp. 201-211
1052-2166/02 $20.00 + .00
Copyright © 2002 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
R. A. Fillmore,1 D. A. Dean,2 and W. E. Zimmer1
Departments of 1Cell Biology and Neuroscience and 2Microbiology and Immunology, University of South Alabama, Mobile, AL 36688
Nkx 3.1 is an evolutionarily conserved vertebrate homolog of the Drosophila Nk-3 homeodomain gene bagpipe that is expressed by a variety of cells during early mammalian development and has been shown to be a critical factor for prostate development and function. Previous studies utilizing a heterologous cell transfection strategy from our laboratory identified the smooth muscle g-actin (SMGA) gene as a novel molecular target of Nkx 3.1 regulatory activity. In the studies presented here, SMGA gene activity and regulation were evaluated in normal and cancerous prostate epithelial cells. SMGA transcripts were demonstrated in prostate epithelia and SMGA mRNA levels were increased in androgen-responsive LNCaP cancer and normal prostate epithelial cells. SMGA gene transcriptional activity was androgen responsive in these cells and required a segment of the human SMGA promoter containing NKE and SRF (serum response factor) binding elements. This region of the human SMGA proximal promoter is well conserved across species and is synergistically activated by coexpression of Nkx 3.1 and SRF in heterologous CV-1 cells. SMGA transcription was not responsive to steroid in PC-3 prostate epithelial cancer cells, which do not express Nkx 3.1. However, SMGA transcription was influenced by expression of androgen receptor in these cells, a situation that allows the androgen-dependent expression of Nkx 3.1. Furthermore, SMGA gene activity was influenced by direct Nkx 3.1 expression in the PC-3 cells. Thus, SMGA gene activity in prostate epithelia is due, in part, to the androgen-dependent expression of Nkx 3.1. As such, our studies provide the initial description of Nkx 3.1 target gene regulatory activity in the prostate.
Key words: Developmental gene regulation; Androgen receptor; Prostate cancer; Tissue-specific expression; Transcriptional regulation; Nk-homeodomain; Serum response factor
Address correspondence to W. E. Zimmer, Department of Cell Biology and Neuroscience, University of South Alabama College of Medicine, MSB 1201, Mobile, AL 36688. Tel: (251) 460-7982; Fax: (251) 414-8241; E-mail: firstname.lastname@example.org
Current address: Division of Pulmonary and Critical Care Medicine, Northwestern University, Tarry 14-707, 300 E. Superior Avenue, Chicago, IL 60611.
A DNA Microarray-Based Approach to Elucidate the Effects of the Immunosuppressant SR31747A on Gene Expression in Saccharomyces cerevisiae
Elisa Cinato,1 Annick Péléraux,1 Sandra Silve,2 Sylvaine Galiègue,1 Christiane Dhers,3 Claudine Picard,3 Omar Jbilo,1 Gérard Loison,2 and Pierre Casellas1
1Immunology-Oncology Department, Sanofi-Synthelabo, F-34184
Montpellier cedex 04, France
2Molecular and Functional Genomics Department, Sanofi-Synthelabo, F-31676 Labège Innopole cedex, France
3Analytical Research Department, Sanofi-Synthelabo, F-31036 Toulouse cedex, France
SR31747A is an immunosuppressive agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae. In this microorganism, SR31747A was shown to inhibit the ERG2 gene product, namely the D8-D7 sterol isomerase, involved in the ergosterol biosynthesis pathway. Although previous genetic experiments pointed to this enzyme as the target for SR31747A in yeast, the existence of other potential targets could not be ruled out. To enlighten this issue, we undertook a DNA microarray-based approach in which the expression profile of SR31747A-treated wild-type cells defining the "drug signature" was compared with the "mutant signature," the expression profile of the corresponding ERG2-deleted strain. We observed that treatment of ERG2-positive cells with SR31747A resulted in the modulation of mRNA levels of numerous genes. Among them, 121 were also affected in untreated ERG2-disrupted cells compared with wild-type cells. By contrast, drug exposure did not induce any significant transcriptional change in the ERG2 null mutant. These results were consistent with SR31747A being an inhibitor of the sterol isomerase and demonstrated the absence of any additional SR31747A target. The detailed analysis of the observed 121 modulated genes provides new insights into the cellular response to ergosterol deprivation induced by SR31747A through inhibition of the ERG2 gene product.
Key words: SR31747; DNA chips; Antiproliferative; Sterol isomerase; Ergosterol pathway
Address correspondence to Pierre Casellas, Sanofi-Synthelabo, 371
rue du Professeur Joseph Blayac, F-34184 Montpellier cedex 04, France.
Tel: (33) 4 67 10 62 90; Fax: (33) 4 67 10 60 00; E-mail: email@example.com
Identification and Characterization of Nine Novel Human Small GTPases Showing Variable Expressions in Liver Cancer Tissues
Hua He, Fangyan Dai, Long Yu, Xingyu She, Yong Zhao, Jianmin Jiang, Xiaosong Chen, and Shouyuan Zhao
State Key Laboratory of Genetic Engineering, Group of Liver Cancer Research, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China
Digestion and detoxification are the two most important functions of the liver, and liver cells always keep a high metabolism level and active vesicular traffic. The malfunction of the vesicular traffic system might be a cause of the abnormal biological behavior of cancerous liver cells. The Ras superfamily is known to regulate various steps of vesicular traffic in eukaryotic cells. It would be significant to determine the change of vesicular transport molecules such as the members of Ras superfamily in carcinogenesis of liver cells. In the present study, we have cloned nine novel genes encoding human small GTPases: RAB1B, RAB4B, RAB10, RAB22A, RAB24, RAB25 ARL5, SARA1, and SARA2, among which the former six belong to the RAB family and the latter three belong to the ARF/SAR1 family. The identification of these new genes has greatly enlarged the pool of the Ras superfamily. It is interesting to find that they are upregulated in most of the 11 hepatocellular carcinoma and 1 cholangiohepatoma cases. Furthermore, the expression in 16 normal human adult tissues, the chromosome loci, and the gene structures of the nine genes are also described. The above findings could be valuable for understanding the vesicular transport system and elucidating the molecular basis of liver cancer carcinogenesis.
Key words: RAB; ARF/SAR1; Cloning; Expression; Liver cancer
Address correspondence to Prof. Long Yu, Institute of Genetics, Fudan University, 220 Handan Road, Shanghai 200433, P.R. China. Tel: 86-21-65642422; Fax: 86-21-65643250; E-mail: firstname.lastname@example.org
Methylphosphate Cap Structure in Small RNAs Reduces the Affinity of RNAs to La Protein
Rajat Bhattacharya, Karthika Perumal,* Krishna Sinha,** Richard Maraia,*** and Ram Reddy
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030
La protein is an abundant 47-kDa phosphoprotein found mostly in the nucleus of eukaryotic cells with a small fraction present in the cytoplasm. Nascent RNA transcripts synthesized by RNA polymerase III are known to be associated with La protein. This binding has been shown to occur to the 3´ end of RNA via RNA recognition motifs and to the 5´ triphosphate via the Walker A motif of the La protein. In this study, we developed an in vitro immunoprecipitation assay to quantitate the 5´ ppp-dependent binding of small RNAs to the human La protein. Using this assay, we found that oligonucleotides five bases or longer bind to the human La protein in a 5´ ppp-dependent manner; pppG did not bind to La protein in this assay. In addition, CH3pppN cap structure present on the 5´ ends of U6 and B2 small RNAs reduced the ability of these RNAs to bind the human La protein. These data show that Walker motif in the human La protein can bind to short RNAs containing 5´ ppp and removal of 5´ ppp from RNAs, or modification of 5´ pppN to CH3pppN or m7GpppN, significantly reduces the ability of small RNAs to bind the human La protein. These data suggest that one of the functions of methylphosphate cap structure in U6 snRNA and B2 RNAs is possibly to reduce the affinity of these RNAs to La protein.
Key words: La protein; Methylphosphate cap structure; Walker motif
Address correspondence to Ram Reddy, Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel: (713) 798-7906; Fax: (713) 798-3145; E-mail: email@example.com
*Present address: UTMB, Galveston, TX.
**Present address: Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX.
***Present address: Laboratory of Molecular Growth Regulation, NICHHD, National Institutes of Health, Bethesda, MD 20892.
Isabelle Quélo,1 Mélanie Hurtubise,1,2 and René St-Arnaud1,2,3
1Genetics Unit, Shriners Hospital for Children, and Departments of 2Human Genetics and 3Surgery, McGill University, Montreal, Quebec, Canada H3G 1A6
aNAC is a transcriptional coactivator known to interact with the N-terminal activation domain of the c-Jun transcription factor. In this article, we describe the identification of the c-Jun interaction domain within the aNAC protein. Deletion analysis of aNAC indicated that the c-Jun binding site was located in the middle part of the protein, between residues 89 and 129. The deletion of the C-terminal end of aNAC, including the c-Jun interacting domain, induced a nuclear translocation of the mutated coactivator. Despite its presence in the nucleus, this deletion mutant did not retain the capacity to coactivate an AP-1 response. These results demonstrate that the interaction between aNAC and c-Jun was necessary for the potentiation of the AP-1 transcriptional activity. These data are consistent with a mechanism by which aNAC acts as a coactivator for c-Jun-dependent transcription by interacting with the c-Jun N-terminal activation domain.
Key words: aNAC; c-Jun; Coactivation; Transcriptional activation
Address correspondence to René St-Arnaud, Genetics Unit, Shriners
Hospital for Children, 1529 Cedar Avenue, Montreal, Quebec, Canada H3G
1A6. Tel: 514-282-7155; Fax: 514-842-5581; E-mail: firstname.lastname@example.org
Zhongmin Xiang,1 Lap Ho,1 Shrishailam Yemul,1 Zhong Zhao,1 Patrick Pompl,1 Kevin Kelley,2 Anju Dang,3 Weiping Qing,1 David Teplow,4 and Giulio Maria Pasinetti1
1Neuroinflammation Research Laboratories, Department of Psychiatry,
and 2Department of Molecular, Cellular and Developmental Biology,
Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY
3Ciphergen Biosystems, Inc., 6611 Dumbarton Circle, Fremont, CA 94555
4Center for Neurologic Diseases, Brigham and Woman's Hospital, 77 Avenue Louis Pasteur, Boston, MA 02115
Several epidemiologic studies have reported that cyclooxygenase (COX) inhibitors prevent/delay the onset of Alzheimer's disease (AD). Recent experimental studies suggest that these compounds can also diminish amyloid-b (Ab) neuropathology in rodent models of AD. To explore the relationship of COX expression to Ab neuropathology, we crossed mice expressing both mutant amyloid precursor protein [K670N/M671L (APPswe)] and mutant PS1 (A246E) with mice expressing human COX-2 selectively in neurons. We show here that human COX-2 expression in APPswe/PS1/COX-2 mice induces potentiation of brain parenchymal amyloid plaque formation and a greater than twofold increase in prostaglandin E2 production, at 24 months of age. This increased amyloid plaque formation coincided with a preferential elevation of Ab1-40 and Ab1-42 with no change in total amyloid precursor protein (APP) expression/content in the brain. Collectively these data suggest that COX-2 influences APP processing and promotes amyloidosis in the brain.
Key words: COX-2; Amyloid; Alzheimer's disease; Inflammation
Address correspondence to Dr. Giulio Maria Pasinetti, Neuroinflammation Research Laboratories, Mount Sinai School of Medicine, Department of Psychiatry, One Gustave L. Levy Place, New York, NY 10029. Tel: (212) 241-5579; Fax: (212) 876-9042; E-mail: email@example.com
Xiaodong Wu and Marthe J. Howard
Department of Anatomy and Neurobiology, Medical College of Ohio, Toledo, OH 43614
Growth and transcription factors provide important developmental cues to neural crest-derived precursors of enteric neurons. The basic helix-loop-helix transcription factors, HAND2 and HAND1, are expressed in the gastrointestinal tract, but neither the growth factors that induce their expression nor the cell types that express them in the gut are known. We show that transcripts encoding HAND2 are expressed in all segments of the developing gut while those encoding HAND1 are confined to the small intestine and colon. Using in situ hybridization combined with immunostaining using cell type-specific antigens, we demonstrate that transcripts encoding HAND2 are expressed in neurons of both the myenteric and submucoasl ganglia. Transcripts encoding HAND1 are expressed by cells in the epithelial lining of the small intestine and colon. The differential localization of HAND2 and HAND1 is reflected in nonoverlapping patterns of regulation by gut-derived factors. The expression of transcripts encoding HAND2 is increased in neural crest-derived cells when cocultured with E4 gut, suggesting a gut-derived factor regulates expression of HAND genes. Exposure of gut-derived neural crest-derived cells to BMP4 significantly increased the expression of HAND2 in all gut segments. In the esophagus and gizzard, where HAND1 is not normally expressed, treatment with BMP4 induced the expression of transcripts encoding HAND1 in nonneural crest-derived cells. GDNF failed to induce consistent expression of transcripts encoding HAND2 in neural crest cells but did support a modest increase in HAND2 expression in gut-derived crest cells obtained from the esophagus and colon. GDNF had no detectable effect on the expression of transcripts encoding HAND1. These results suggest; 1) that HAND2 has a function in the development of enteric neurons, and 2) that BMP and GDNF differentially regulate HAND2 and HAND1 gene expression in the developing gastrointestinal tract.
Key words: HAND2; HAND1; Enteric nervous system; Neural crest; bHLH; Gut development
Address correspondence to Marthe Howard, Department of Anatomy and
Neurobiology, Medical College of Ohio, 3000 Arlington Ave., Toledo, OH
43614. Tel: (419) 383-5439; E-mail: Mhoward@mco.edu
Ephrin-As Cooperate With EphA4 to Promote Trunk Neural Crest Migration
R. McClennan and C. E. Krull
Biological Sciences, University of Missouri-Columbia, Columbia, MO 65211
Trunk neural crest cells delaminate from the dorsal neural tube and migrate on two distinct pathways: a dorsolateral route, between the ectoderm and somites, and a ventromedial route, through the somitic mesoderm. Neural crest cells that migrate ventromedially travel in a segmental manner through rostral half-somites, avoiding caudal halves. Recent studies demonstrate that various molecular cues guide the migration of neural crest cells, primarily by serving as inhibitors to premature pathway entry or by preventing neural crest from entering inappropriate territories. Trajectories of migrating trunk neural crest are well organized and generally linear in nature, suggesting that positive, migration-promoting factors may be responsible for this organized cell behavior. However, the identity of these factors and their function are not well understood. Here we examine the expression of members of the EphA subclass of receptor tyrosine kinases and ephrins using RT-PCR and immunocytochemistry. Neural crest cells express ephrins and EphA4 at distinct stages during their migration. In functional analyses, addition of ephrin-A2-, ephrin-A5-, and EphA4-Fc disrupted the segmental organization of trunk neural crest migration in explants: neural crest cells entered rostral and caudal halves of somites. Finally, to test the specific effects of these factors on cell behavior, neural crest cells were exposed in vitro to substrate-bound EphA and ephrin-As. Surprisingly, neural crest cells avoided ephrin-A2 or ephrin-A5 substrates; this avoidance was abolished by the addition of EphA4. Together, these data suggest that ephrin-As and EphA4 cooperate to positively promote the migration of neural crest cells through rostral half somites in vivo.
Key words: Migration; Guidance; Chicken; Segmentation; Ephrin; EphA4
Address correspondence to Catherine E. Krull, 108 Lefevre, Biological
Sciences, University of Missouri-Columbia, Columbia, MO 65211. Tel: (573)
882-4067; Fax: (573) 884-5020; E-mail: firstname.lastname@example.org