Enhancement of Glycoprotein Hormone Alpha Subunit Promoter Reporter Gene Activity in Co-transfection Studies – A Cautionary Reminder

Abstract

Reporter constructs have a wide range of application in determining eucaryotic gene regulation and expression. In vitro models frequently necessitate co-transfection and there have been reports of interference, predominantly inhibition, between promoters. Studies investigating the biological activity of a mutant thyrotropin receptor involved co-transfection with receptor constructs and a cAMP responsive luciferase reporter driven by the glycoprotein hormone alpha subunit promoter. We observed considerable enhancement of basal luciferase activity by co-transfecting the empty expression vector, which contained the SV40 late promoter. We have investigated the mechanism using different concentrations and several viral promoters in COS cells, following transient co-transfection. The increase was dose dependent but plateaued at 50 ng of vector DNA. This was not due to an adjuvant effect since luciferase activity was unchanged by adding increasing amounts of a promoterless plasmid. Enhancement was maintained when truncating the promoter to −346, but eliminated in the promoter truncated upstream of −244, indicating a binding site for a putative repressor of glycoprotein hormone alpha expression between −244 and −346. Enhancement was maintained with the addition of a second constitutive reporter (bos β-gal) to correct for transfection efficiency, although this was the consequence of enhanced luciferase activity by the bos β-gal, which in itself was inhibited by the SV40 promoter. Artefactual enhancement of reporter activity can occur and highlights the need for careful choice of controls when performing transient co-transfection experiments. In silico analysis of the promoters identified a possible shared forkhead transcription factor binding site.

Key words

reporter assays - transcription - squelching

References

• 1 Schenborn E, Groskreutz D. Reporter gene vectors and assays.  Mol Biotechnol. 1999;  13 29-44
• 2 Yamasaki T, Nakajima H, Kono N, Hotta K, Yamada K, Imai E, Kuwajima M, Noguchi T, Tanaka T, Tarui S. Structure of the entire human muscle phosphofructokinase-encoding gene - A 2-promotersystem.  Gene. 1991;  104 277-282
• 3 Han K, Levine MS, Manley JL. Synergistic activation and repression of transcription by Drosophila homeobox proteins.  Cell. 1989;  56 573-583
• 4 Fernandez Rachubinski FA, Weiner JH, Blajchman MA. Regions flanking exon 1 regulate constitutive expression of the human antithrombin gene.  J Biol Chem. 1996;  271 29502-29512
• 5 Benzakour O, Kanthou C, Dennehy U, Al Haq A, Berg LP, Kakkar VV, Cooper DN. Evaluation of the use of the luciferase-reporter-gene system for gene regulation studies involving cyclic AMP-elevating agents.  Biochem J. 1995;  309 385-387
• 6 Naylor LH. Reporter gene technology: the future looks bright.  Biochem Pharmacol. 1999;  58 749-757
• 7 Hollon T, Yoshimura FK. Variation in enzymatic transient gene expression assays.  Anal Biochem. 1989;  182 411-418
• 8 Bergeron D, Barbeau B, Léger C, Rassart E. Experimental bias in the evaluation of the cellular transient expression in DNA co-transfection experiments.  Cell Mol Biol Res. 1995;  41 155-159
• 9 Farr A, Roman A. A pitfall of using a second plasmid to determine transfection efficiency.  Nucleic Acids Res. 1992;  20 920
• 10 Gill G, Ptashne M. Negative effect of the transcriptional activator GAL4.  Nature. 1988;  334 721-724
• 11 Zhu H, Joliot V, Prywes R. Role of transcription factor TFIIF in serum response factor-activated transcription.  J Biol Chem. 1994;  269 3489-3497
• 12 Natesan S, Rivera VM, Molinari E, Gilman M. Transcriptional squelching re-examined.  Nature. 1997;  390 349-350
• 13 Fuhrer D, Warner J, Sequeira M, Paschke R, Gregory J, Ludgate M. Novel TSHR germline mutation (met463val) masquerading as Graves’ disease in a large welsh kindred with hyperthyroidism.  Thyroid. 2000;  10 1035-1041
• 14 Burrin JM, Jameson JL. Regulation of transfected glycoprotein hormone alpha-gene expression in primary pituitary cell-cultures.  Mol Endocrinol. 1989;  31 643-1651
• 15 Maurer RA, Kim KE, Schoderbek WE, Roberson MS, Glenn DJ. Regulation of glycoprotein hormone alpha-subunit gene expression.  Recent Prog Horm Res. 1999;  54 455-484
• 16 Jameson JL, Powers AC, Gallagher GD, Habener JF. Enhancer and promoter element interactions dictate cyclic adenosine monophosphate mediated and cell-specific expression of the glycoprotein hormone α gene.  Mol Endocrinol. 1989;  3 763-772
• 17 Pittman RH, Clay CM, Farmerie TA, Nilson JH. Functional-analysis of the placenta-specific enhancer of the human glycoprotein hormone alpha-subunit gene – emergence of a new element.  J Biol Chem. 1994;  269 19360-19368
• 18 Hamernik DL, Keri RA, Clay CM, Clay JN, Sherman GB, Sawyer HR, Nett TM, Nilson JH. Gonadotrope-specific and thyrotrope-specific expression of the human and bovine glycoprotein hormone alpha-subunit genes is regulated by distinct cis-acting elements.  Mol Endocrinol. 1992;  6 1745-1755
• 19 Burrin JM, Aylwin JB, Holdstock JG, Sahye U. Mechanism of action of pituitary adenylate cyclase-activating polypeptide on human glycoprotein hormone α-subunit transcription in ααT3-1 gonadotropes.  Endocrinology. 1989;  139 1731-1737
• 20 Maxwell IA, Harrison GS, Wood WM, Maxwell F. A DNA cassette containing a trimerized SV40 polyadenylation signal which efficiently blocks spurious plasmid-initiated transcription.  Biotechniques. 1989;  7 276-280
• 21 Holdstock JG, Aylwin SJB, Burrin JM. Calcium and glycoprotein hormone α-subunit gene expression and secretion in αT3-1 gonoadotropes.  Mol Endocrinol. 1996;  10 1308-1317
• 22 Mizushima S, Nagata S. pEF-BOS, a powerful mammalian expression vector.  Nucleic Acids Res. 1990;  18 5322-5332
• 23 Matys V, Fricke E, Geffers R, Gossling E, Haubrock M, Hehl R, Hornischer K, Karas D, Kel AE, Kel-Margoulis OV, Kloos DU, Land S, Lewicki-Potapov B, Michael H, Munch R, Reuter I, Rotert S, Saxel H, Scheer M, Thiele S, Wingender E. TRANSFAC (R): transcriptional regulation, from patterns to profiles.  Nucleic Acids Res. 2003;  31 374-378
• 24 Gruda MC, Alwine JC. Simian virus 40 (SV40) T-antigen transcriptional activation mediated through the Oct Sph region of the SV40 promoter.  J Virol. 1991;  65 3553-3558
• 25 Cazap P, Sundseth R, Hansen U. Transactivation of the Simian Virus-40 late promoter by large T-antigen requires binding sites for the cellular transcription factor TEF-1.  J Virol. 1991;  65 6535-6543
• 26 Chen HF, Campisis J, Padmanabhan R. SV40 large T antigen transactivates the human cdc2 promoter by inducing a CCAAT box binding factor.  J Biol Chem. 1996;  271 13959-13967
• 27 Grimaldi P, Geremia R, Albanesi C, Rossi P. The same sequence mediates activation of the human urokinase promoter by cAMP in mouse Sertoli cells and by SV40 large T antigen in COS cells.  Mol Cell Endocrinol. 1996;  117 167-173
• 28 Scofield MA, Xiong W, Haas MJ, Zeng Y, Cox GS. Sequence analysis of the human glycoprotein hormone α-subunit gene 5′-flanking DNA and identification of a potential regulatory element as an Alu repetitive sequence.  Biochem Biophys Acta. 2000;  1493 302-318
• 29 Hacker U, Kaufmann E, Hartmann C, Jurgens G, Knochel W, Jackle H. The drosophila fork head domain protein crocodile is required for the establishment of head structures.  Eur Mol Biol Organ J. 1995;  14 5306-5317
• 30 Berry FB, Saleem RA, Walter MA. FOXC1transcriptional regulation is mediated by N- and C-terminal activation domains and contains a phosphorylated transcriptional inhibitory domain.  J Biol Chem. 2002;  277 10292-10297

Correspondence