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Gene Expression, Vol. 8, pp. 197-206, 1999
1052-2166/99 $20.00 + .00
Copyright © 1999 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Differential Upregulation of p53-Responsive Genes by Genotoxic Stress in Hematopoietic Cells Containing Wild-Type and Mutant p53

Bendi Gong and Alex Almasan

Department of Cancer Biology, Lerner Research Institute, and Department of Radiation Oncology, The Cleveland Clinic Foundation, Cleveland, OH 44195

Cells respond to genotoxic stress by activation of many genes, including the tumor suppressor p53. p53 activates transcriptionally target genes, such as p21waf1 and gadd45, which can lead to cell cycle arrest, or bax, which can lead to cell death. We examined the response to genotoxic stress in two hematopoietic cell lines that harbor either wild-type (MOLT-4) or a mutant p53 with a codon 161 mutation (U266). We adapted a multiprobe RNase protection assay (RPA) to determine the steady-state RNA levels, and in combination with nuclear runoff assays, transcriptional rates of multiple stress-induced genes. We found a differential activation of growth arrest and cell death-specific p53 target genes in cells with wild-type or mutant p53. Our results show that genotoxic stress can activate the p21waf1 and gadd45 genes in both cell lines. However, the bax gene was not induced in U266 cells. Bax and gadd45 gene induction could be efficiently blocked by pretreating the cells with the antioxidant compound pyrrolidine dithiocarbamate, suggesting that oxidative stress was involved in these responses. Induction of all three genes in MOLT-4 cells was clearly at the transcriptional level, because we detected transcriptional activity by nuclear runoff RPA assays, and transfection with a consensus p53 binding sequence. U266 cells did not activate the same reporter, in spite of the upregulation of p21waf1 and gadd45 RNA levels. However, the p21waf1-reporter constructs containing 0.9 to 2.4 kb of the native p21 promoter were potently activated in U266 cells. These results indicate a differential regulation of p53 target genes in cells containing wild-type or codon 161 mutant p53.

Key Words: RNase protection; Ionizing radiation; p21/waf1; gadd45; bax; Cell cycle

Address correspondence to Alex Almasan, Department of Cancer Biology, The Cleveland Clinic Foundation NB40, Cleveland, OH 44195. Tel: (216) 444-9970; Fax: (216) 445-6269; E-mail: almasaa@ccf.org

Gene Expression, Vol. 8, pp. 207-217, 1999
1052-2166/99 $20.00 + .00
Copyright © 1999 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

LIM Domain-Containing Protein Trip6 Can Act as a Coactivator for the v-Rel Transcription Factor

Marieke Koedood Zhao,1* Yuan Wang,1 Kerry Murphy,1** Jinseong Yi,2 Mary C. Beckerle,2 and Thomas D. Gilmore1

1Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215
2Department of Biology and Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112

The retroviral oncoprotein v-Rel is a transcriptional activator in the Rel/NF-kB family of eukaryotic transcription factors. v-Rel malignantly transforms a variety of cell types in vitro and in vivo, and its transforming activity is dependent on the ability of v-Rel to bind to DNA and activate transcription. In this report, we used the yeast two-hybrid assay to identify proteins that interact with C-terminal sequences of v-Rel that are needed for transcriptional activation and transformation. One protein, Trip6, that we identified in this screen was previously identified as a thyroid hormone receptor-interacting protein. Trip6 is a member of a subfamily of LIM domain-containing proteins that are thought to transport intracellular signals from the cell surface to the nucleus. By several criteria, we show that sequences from Trip6, which include the LIM domains, behave as a coactivator for transcriptional activation by v-Rel. That is, a GAL4-Trip6 fusion protein can activate transcription in yeast and chicken cells, Trip6 can enable C-terminal sequences of v-Rel to activate transcription in yeast, and Trip6 can enhance activation by v-Rel from a kB site reporter plasmid in yeast. Although full-length Trip6 localizes to adhesion plaques, deletion of N-terminal sequences allows human Trip6 to enter the nucleus of chicken cells. Lastly, Northern blotting shows that Trip6 mRNA is expressed in many human tissues. Coexpression of Trip6 does not affect the transforming activity of v-Rel. Taken together, our results indicate that Trip6 may be a protein that is important for the ability of v-Rel to activate transcription and transform cells, and may represent a potential target for blocking Rel-mediated oncogenesis and transcriptional activation.

Key Words: v-Rel; Retroviral oncogene; Trip6; Coactivator; LIM domain; Transcription factor; Malignant transformation; NF-kB; Two-hybrid assay

*Present address: Genetics Institute, Andover, MA 01810.
**Present address: Channing Labs, Brigham and Women's Hospital, Boston, MA 02115.
Address correspondence to Thomas D. Gilmore, Boston University, Biology Department, 5 Cummington Street, Boston, MA 02215. Tel: (617) 353-5444; Fax: (617) 353-6340; E-mail: gilmore@bio.bu.edu

Gene Expression, Vol. 8, pp. 219-229, 1999
1052-2166/99 $20.00 + .00
Copyright © 1999 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Rel/NF-kB Represses bcl-2 Transcription in pro-B Lymphocytes

U. Shivraj Sohur,1 Mrinalini N. Dixit,2 Chih-Li Chen,1 Mike W. Byrom,1 and Lawrence D. Kerr1,2

Departments of 1Microbiology & Immunology and 2Cell Biology, Vanderbilt University School of Medicine, 1161 21st Ave. South, Nashville, TN 37232-2363

The mechanisms controlling programmed cell death (PCD) during early B cell development are not well understood. Members of both the Bcl-2 family of apoptosis-related proteins and the nuclear factor-kappa B/Rel (NF-kB/Rel) family of transcription factors are expressed differentially during B cell development. To date, however, no direct interactions between these two families have been demonstrated. The FL5.12 cell line represents a model for progenitor B cell development. Such cells reproducibly undergo PCD upon IL-3 withdrawal. The signal to enter the apoptotic pathway is mediated by a shift in the ratio of Bcl-2:Bax. While bax levels remain constant, bcl-2 transcription rate, steady-state mRNA, and protein levels decrease. Analysis of the bcl-2 promoter reveals 3 kB sites functionally able to bind kB factors from FL5.12 nuclear extracts. Cotransfection studies demonstrate that NF-kB factors can repress bcl-2 transcription and that site-directed mutagenesis of the kB motifs abolishes this repression. These studies suggest that NF-kB mediates PCD in pro-B cells through transcriptional repression of the survival gene bcl-2, thus shifting the bcl-2:bax ratio in favor of death-promoting complexes.

Key Words: B cell development; Progenitor B cells; Programmed cell death or apoptosis; Transcription; Nuclear factor kappa B; BCL-2

Address correspondence to Lawrence D. Kerr, Vanderbilt University, School of Medicine, MCN A-4314, 1161 21st Ave. South, Nashville, TN 37232-2363. Tel: (615) 343-2568; Fax: (615) 343-2569; E-mail: lawrence.kerr@mcmail.vanderbilt.edu

Gene Expression, Vol. 8, pp. 231-246, 1999
1052-2166/99 $20.00 + .00
Copyright © 1999 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Cloning and Expression of the Mouse Deoxyuridine Triphosphate Nucleotidohydrolase Gene: Differs From the Rat Enzyme in That it Lacks Nuclear Receptor Interacting LXXLL Motif

Lixin Kan,* Sanjay Jain,* William Cook, Wen-Qing Cao, Nobuteru Usuda, Anjana V. Yeldandi, M. Sambasiva Rao, Yashpal S. Kanwar, and Janardan K. Reddy

Department of Pathology, Northwestern University Medical School, Chicago, IL 60611

We have previously reported the cloning of rat deoxyuridine triphosphate nucleotidohydrolase (dUTPase) cDNA and demonstrated that the full-length protein as well as the N-terminal 62-amino acid peptide interacts with peroxisome proliferator-activated receptor a (PPARa). We now report the cloning of mouse dUTPase cDNA and show that it contains a 162-amino acid open reading frame, encoding a protein with a predicted Mr of 17,400 and differs from rat cDNA, which contains additional 43 amino acids at the N-terminal end. Unlike rat dUTPase, mouse dUTPase failed to bind PPARa. An evaluation of 205 amino acid containing rat dUTPase cDNA revealed that the N-terminal 43 extra amino acid segment contains an LXXLL signature motif, considered necessary and sufficient for the binding of several cofactors with nuclear receptors, and its absence in murine dUTPase possibly accounts for the differential binding of these enzymes to PPARa. In situ hybridization and immunohistochemical studies revealed that, in the adult mouse, dUTPase is expressed at high levels in proliferating cells of colonic mucosa, and of germinal epithelium in testis. At 9.5-day mouse embryonic development, dUTPase expression is predominantly in developing neural epithelium, and hepatic primordium, and in later developmental stages (11.5-, 13.5-, and 15.5-day embryo), the expression began to be localized to the liver, kidney, gut epithelium, thymus, granular layer of the cerebellum, and olfactory epithelium. We also show that the murine dUTPase gene comprises 6 exons and the 5´-flanking region of -1479 to ­27, which exhibited high promoter activity, contains a typical TATA box and multiple cis-elements such as Sp-1, AP2, AP3, AP4, Ker1, RREB, and CREB binding sites. These observations suggest the existence of variants of dUTPase, some of which may influence nuclear receptor function during development and differentiation, in addition to catalyzing the hydrolysis of dUTP to dUMP.

Key Words: dUTPase; PPAR; LXXLL motif; Embryonic development

*These authors contributed equally to the work.
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: jkreddy@nwu.edu

Gene Expression, Vol. 8, pp. 247-261, 1999
1052-2166/99 $20.00 + .00
Copyright © 1999 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Myogenic Regulatory Circuit That Controls Cardiac/Slow Twitch Troponin C Gene Transcription in Skeletal Muscle Involves E-box, MEF-2, and MEF-3 Motifs

Thorkil H. Christensen and Larry Kedes

Institute for Genetic Medicine, Department of Biochemistry and Molecular Biology and Department of Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033

We have characterized the specific DNA regulatory elements responsible for the function of the human cardiac troponin C gene (cTnC) muscle-specific enhancer in myogenic cells. We used functional transient transfection assays with deletional and site-specific mutagenesis to evaluate the role of the conserved sequence elements. Gel electrophoresis mobility shift assays (EMSA) demonstrated the ability of the functional sites to interact with nuclear proteins. We demonstrate that three distinct transcription activator binding sites commonly found in muscle-specific enhancers (a MEF-2 site, a MEF-3 site, and at least four redundant E-box sites) all contribute to full enhancer activity but a CArG box does not. Mutation of either the MEF-2 or MEF-3 sites or deletion of the E-boxes reduces expression by 70% or more. Furthermore, the MEF-2 site and the E-boxes specifically bind, respectively, to MEF-2 and myogenic determination factors derived from nuclear extracts. EMSA assays using a MEF-3 containing oligonucleotide revealed indistinguishable separation patterns with extracts from myogenic cells and nonmyogenic cells. These data suggest that expression of the cTnC gene in slow-twitch skeletal muscle is sustained through complex interactions at the 3'I1e enhancer between muscle-specific and nontissue-specific transcription factors: either a myogenic bHLH complex or MEF-2 can activate transcription but only in the presence of a third transcriptional activator that appears not to be muscle specific. We conclude from these observations that the cTnC 3'I1e element is a composite enhancer that functions through the combined interactions of at least five regulatory elements and their cognate binding factors: three or four E-boxes, a MEF-2 site, and a MEF-3 site. The data support the notion that all of these sites contribute to enhancer function in cell systems in an additive way but that none are absolutely required for enhancer activity. The data imply that the levels of transcription of cTnC in myogenic tissues in which the activities of one of the transcriptional factors is lacking would be partially but not wholly suppressed. Our data support the critical role of E-box sites in conjunction with the adjacent elements. Hence, we assign CTnC gene regulation to the "ordinary" rather than to the "novel" category of transcriptional regulation during skeletal myogenesis.

Key Words: Transcription; Muscle; Troponin C gene; E-box; MEF-2; MEF-3

Address correspondence to Larry Kedes, Institute for Genetic Medicine, USC School of Medicine, 2050 Alcazar Street, Los Angeles, CA 90033. Tel: (323) 442-1144; Fax: (323) 442-2764; E-mail: kedes@hsc.usc.edu