ognizant Communication Corporation



Gene Expression, Vol. 12, pp. 137-149
1052-2166/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

An In Vitro Approach to Test the Possible Role of Candidate Factors in the Transcriptional Regulation of the RET Proto-Oncogene

Tiziana Bachetti,1 Silvia Borghini,1 Roberto Ravazzolo,1,2 and Isabella Ceccherini1

1Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy
2Dipartimento di Pediatria e CEBR, Università degli Studi di Genova, Genova, Italy

Neural crest cells arise from the epithelium of the dorsal neural tube and migrate to various districts giving origin, among others, to sympathetic, parasympathetic, and enteric ganglia. It has been shown that the transcription factors HOX11L1, HOX11L2, MASH1, PHOX2A, and PHOX2B are all necessary, to various extents, to the correct development of the autonomic nervous system. To investigate their possible role in the transcriptional regulation of the RET proto-oncogene, a gene playing a crucial role in correct intestinal innervation, we undertook a specific in vitro experimental strategy. Two neuroblastoma cell lines (SK-N-MC and SK-N-BE) were cotransfected with each transcription factor expressing plasmids and sequential deletion constructs of the 5´ c-RET flanking region cloned upstream of the Luciferase reporter gene. Here we show that HOX11L1 enhances the activity of the c-RET promoter in SK-N-MC cell line by stimulating a region between -166 bp and -35 bp. Gel shift assays performed with oligonucleotides spanning this promoter sequence showed a change of the SP1 interaction with its binding sites, consequent to transfection with HOX11L1. While HOX11L2 showed no effect in both the cell lines, we have observed PHOX2A, PHOX2B, and MASH1 triggering a reproducible increase in the Luciferase activity in SK-N-BE cell line. A sequence responsible of the PHOX2A-dependent activation has been identified, while PHOX2B seems to act indirectly, as no physical binding has been demonstrated on c-RET promoter.

Key words: Transcriptional regulation; RET proto-oncogene; Homeobox genes; Reporter gene assay; Autonomic nervous system development

Address correspondence to Isabella Ceccherini, Ph.D., Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, Largo G. Gaslini, 5, 16148 Genova, Italy. Tel: +39 010 5636800; Fax: +39 010 3779797; E-mail: isa.c@unige.it

Gene Expression, Vol. 12, pp. 151-163
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Copyright © 2005 Cognizant Comm. Corp.
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Casein Kinase II Phosphorylation Regulates aNAC Subcellular Localization and Transcriptional Coactivating Activity

Isabelle Quélo,1* Claude Gauthier,1 and René St-Arnaud1,2

1Genetics Unit, Shriners Hospital for Children, Montreal (Quebec) Canada H3G 1A6
2Departments of Medicine, Surgery and Human Genetics, McGill University, Montreal (Quebec), Canada H3A 2T5

The subcellular localization of the aNAC coactivator is regulated, but the signaling pathways controlling its nucleocytoplasmic shuttling and coactivation function are not completely characterized. We report here that casein kinase II (CK2) phosphorylated aNAC on several phosphoacceptor sites, especially in an amino-terminal cluster. Deletion or mutation of the clustered CK2 sites induced nuclear accumulation of aNAC in cells. aNAC also localized to the nucleus when endogenous CK2 activity was inhibited by quercetin or 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB). These observations suggested that phosphorylation by CK2 might play a signaling role in the nuclear export of aNAC. Interestingly, inhibition of the chromosome region maintenance 1 (CRM1) exportin by leptomycin B (LMB) led to accumulation of aNAC in the nucleus. We conclude that CK2 phosphorylation of the N-terminal cluster corresponds to the signal for aNAC's nuclear export via a CRM1-dependent pathway. Finally, the nuclear accumulation of the protein resulting from the lack of CK2 phosphorylation mediated a slight but significant increase of the aNAC coactivating function on AP-1 transcriptional activity. Thus, aNAC's exit from the nucleus and capacity to potentiate transcription appear dependent on its phosphorylation status.

Key words: aNAC; Casein kinase II (CK2); c-Jun; CRM1; Leptomycin B; Nuclear export

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: rst-arnaud@shriners.mcgill.ca

*Current address: Aventis Pharma Deutschland GmbH, Industriepark Hoechst, D-65926 Frankfurt am Main, Germany.

Gene Expression, Vol. 12, pp. 165-176
1052-2166/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Tissue, Species, and Environmental Differences in Absolute Quantities of Murine mRNAs Coding for Alpha, Mu, Omega, Pi, and Theta Glutathione S-Transferases

Julia Ruiz-Laguna, Nieves Abril, María-José Prieto-ÁLamo, Juan López-Barea, and Carmen Pueyo

Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, edificio Severo Ochoa, planta 2a, Carretera Madrid-Cádiz Km 396-a, Universidad de Córdoba, 14071-Córdoba, Spain

This article reports the first absolute quantitative analysis of expression patterns of murine transcripts (Gsta1/2, Gsta3, Gsta4, Gstm1, Gstm2, Gstm3, Gsto1, Gstp1/2, Gstt1, Gstt2) coding for most glutathione S-transferases (GSTs) of alpha, mu, omega, pi, and theta classes. We examine how the steady-state numbers of transcripts are modulated in association with: three animal organs (liver, kidney, and lung) where extensive detoxification occurs; two species (Mus musculus and Mus spretus) representing common laboratory and aboriginal mice; and two genetic and animal living conditions (wild-derived inbred animals and free-living mice). Moreover, quantitations performed examine how the pulmonary steady-state Gst mRNA amounts are affected in M. musculus by paraquat (a superoxide generator), and in M. spretus by dwelling at a polluted area. The results point to complex tissue-, species-, and life condition-dependent expression of the investigated transcripts. Among others, they show: i) the ubiquity of most transcripts, except Gstm3 mRNA that was virtually absent or at very low amounts (</=0.001 molecules/pg) in kidney and lung of M. spretus; ii) unique expression profiles for each transcript and mouse organ examined; iii) outstanding species-specific differences in basal amounts of most Gst mRNAs, this effect being most apparent in the case of Gsta1/2, Gsta3, Gstm2, Gsto1, Gstt1, and Gstt2; iv) paraquat-induced upregulation of most Gst mRNAs, with the notable exception of those coding for theta class GSTs; v) a tendency for mice dwelling at a wildlife reserve of having lower and more homogeneous Gsta3 mRNA levels than those collected in an anthropogenic environment.

Key words: Mus spretus; Gst transcript copy numbers; Biomarkers; Oxidative stress; Detoxification; Quantitative RT-PCR

Address correspondence to Carmen Pueyo, Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, edificio Severo Ochoa, planta 2a, Carretera Madrid-Cádiz Km 396-a, Universidad de Córdoba, 14071-Córdoba, Spain. Tel: +34 957 218695; Fax: +34 957 218688; E-mail: bb1pucuc@uco.es

Gene Expression, Vol. 12, pp. 177-192
1052-2166/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Transcriptional Modulations by RXR Agonists Are Only Partially Subordinated to PPARa Signaling and Attest Additional, Organ-Specific, Molecular Cross-Talks

Pascal G. P. Martin,1 Frédéric Lasserre,1 Cécile Calleja,1* Armelle Van Es,1* Alain Roulet,1 Didier Concordet,2 Michela Cantiello,3 Romain Barnouin,1* Béatrice Gauthier,4 and Thierry Pineau1

1Laboratoire de Pharmacologie et Toxicologie, I.N.R.A., BP3, Toulouse, France
2UMR 181, I.N.R.A./E.N.V.T., Toulouse, France
3Dipartimento di Patologia Animale, Universita'degli Studi di Torino, Torino, Italy
4Galderma R&D, Sophia-Antipolis, France

Nuclear hormone receptors (NR) are important transcriptional regulators of numerous genes involved in diverse pathophysiological and therapeutic functions. Following ligand activation, class II NR share the ability to heterodimerize with the retinoid X receptor (RXR). It is established that RXR activators, rexinoids, transactivate several peroxisome proliferator-activated receptor a (PPARa) target genes in a PPARa-dependent manner. We hypothesized that, once activated, RXR might signal through quiescent NR other than PPARa, in an organ-specific manner. To study this putative phenomenon in vivo, we developed an array of 120 genes relevant to the class II NR field. The genes were selected using both published data and high-density screenings performed on RXR or PPARa agonist-treated mice. Wild-type C57BL/6J and PPARa-deficient mice were treated with fenofibrate (PPARa activator) or LGD1069 (RXR activator). Using our customized array, we studied the hepatic, cardiac, and renal expression of this panel of 120 genes and compared them in both murine genotypes. The results obtained from this study confirmed the ability of an RXR agonist to modulate PPARa-restricted target genes in the liver and the kidney. Furthermore, we show that various organ-specific regulations occurring in both genotypes (PPARa +/+ or -/-) are highly indicative of the ability of RXR to recruit other class II NR pathways. Further development of this molecular tool may lead to a better understanding of the permissiveness of class II nuclear receptor dimers in vivo.

Key words: Nuclear receptor; RXR; PPAR; Retinoid; Rexinoid; Fibrate; Microarray; PPARa -/-; Cutaneous T-cell lymphoma; Vitamin D

Address correspondence to Thierry Pineau, Laboratoire de Pharmacologie et Toxicologie, Institut National de la Recherche Agronomique, 180 Chemin de Tournefeuille, BP3, F 31931 Toulouse, Cedex 9, France. Tel: (33) 561 28 53 95; Fax: (33) 561 28 53 10; E-mail: tpineau@toulouse.inra.fr

*Present address: IGBMC, Illkirch, France.

Gene Expression, Vol. 12, pp. 193-212
1052-2166/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

NF-Y Behaves as a Bifunctional Transcription Factor That Can Stimulate or Repress the FGF-4 Promoter in an Enhancer-Dependent Manner

Cory T. Bernadt,1,.2 Tamara Nowling,1* Matthew S. Wiebe,1,2** and Angie Rizzino1,2

1Eppley Institute for Research in Cancer and Allied Diseases, and the 2Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198-6805

NF-Y is a bifunctional transcription factor capable of activating or repressing transcription. NF-Y specifically recognizes CCAAT box motifs present in many eukaryotic promoters. The mechanisms involved in regulating its activity are poorly understood. Previous studies have shown that the FGF-4 promoter is regulated positively by its CCAAT box and NF-Y in embryonal carcinoma (EC) cells where the distal enhancer of the FGF-4 gene is active. Here, we demonstrate that the CCAAT box functions as a negative cis-regulatory element when cis-regulatory elements of the FGF-4 enhancer are disrupted, or after EC cells differentiate and the FGF-4 enhancer is inactivated. We also demonstrate that NF-Y mediates the repression of the CCAAT box and that NF-Y associates with the endogenous FGF-4 gene in both EC cells and EC-differentiated cells. Importantly, we also determined that the orientation and the position of the CCAAT box are critical for its role in regulating the FGF-4 promoter. Together, these studies demonstrate that the distal enhancer of the FGF-4 gene determines whether the CCAAT box of the FGF-4 promoter functions as a positive or a negative cis-regulatory element. In addition, these studies are consistent with NF-Y playing an architectural role in its regulation of the FGF-4 promoter.

Key words: NF-Y; F9-differentiated cells; EC cells; Transcription; Differentiation; Enhancer; Immunoprecipitation

Address correspondence to Angie Rizzino, Eppley Institute for Research in Cancer and Allied Diseases, 986805 Nebraska Medical Center, Omaha, NE 68198-6508, USA. Tel: (402) 559-6338; Fax: (402) 559-3339; E-mail: arizzino@unmc.edu

*Current address: Department of Medicine, Medical University of South Carolina, Charleston, SC.
**Current address: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI.

Gene Expression, Vol. 12, pp. 213-
1052-2166/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Allele-Specific Conditional Destabilization of Glutamine Repeat mRNAs

Andrew B. Crouse1 and Peter J. Detloff1,2

1Department of Biochemistry and Molecular Genetics and 2Department of Neurobiology, University of Alabama, Birmingham, AL, USA

Several late-onset neurological diseases are caused by the inheritance of an expanded CAG repeat coding for polyglutamine. To date there is no effective means of halting the progression of these diseases, and their underlying molecular mechanisms remain a mystery. Strategies designed to specifically reduce the levels of long repeat mRNA might provide an effective therapy for these diseases. An emphasis on allele specificity is necessary to avoid the potential toxicities associated with reduction of expression. The experiments described here are based on the relationship between translation and mRNA stability and the idea that translation of a repeated codon might be extremely sensitive to reductions in levels of cognate aminoacylated tRNA. Consistent with this hypothesis, we have discovered that reduced glutamine concentration destabilizes mRNAs coding for long glutamine repeats while sparing short repeat versions of the same mRNAs. These results suggest therapy might be attained with existing compounds or environmental conditions known to decrease free glutamine levels.

Key words: Polyglutamine; Huntington's disease; Glutamine; tRNA; Translation; mRNA stability; Rational therapeutic

Address correspondence to Dr. Peter J. Detloff, University of Alabama at Birmingham, 540 KAUL, 720 20th Street South, Birmingham, AL 35242, USA. Tel: (205)-975-8157; Fax: (205)-975-2188; E-mail: pdetloff@bmg.bhs.uab.edu